A novel fucosylation pathway in the cytosol of Dictyostelium Discoideum

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Title:
A novel fucosylation pathway in the cytosol of Dictyostelium Discoideum
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Gonzalez-Yanes, Beatriz, 1964-
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Fucose   ( mesh )
Dictyostelium   ( mesh )
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Thesis:
Thesis (Ph. D.)--University of Florida, 1991.
Bibliography:
Includes bibliographical references (leaves 156-165).
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Also available online.
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by Beatriz Gonzalez-Yanes.
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Typescript.
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Vita.

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Full Text











A NOVEL FUCOSYLATION PATHWAY IN THE CYTOSOL
OF DICTYOSTELIUM DISCOIDEUM

















By

BEATRIZ GONZALEZ-YANES


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1991































A mis padres
(To my parents)














ACKNOWLEDGEMENTS

First of all I would like to acknowledge my advisor,

Dr. Christopher M. West, for his time, training, and example

throughout these studies. I would also like to thank the

members of my committee, Drs. Robert Cohen, William Dunn,

and Carl Feldherr, for their time and guidance regarding

this project.

I would like to acknowledge and thank Dr. Michael Ross

for his interest and support of my graduate education.

Part of the work presented in Chapter II is reproduced

from the journal of Developmental Biology, 1989, volume 133,

pages 576-587 by copyright permission of Academic Press,

Inc.

I am very grateful to the following researchers and the

members of their laboratories for sharing equipment and/or

reagents with us: Drs. Gudrun Bennett, Ross Brown and Lina

Gritzali, William Dunn, Carl Feldherr, Michael Humphreys-

Beher, K.J. Kao, and Gillian Small.

In general, I would like to express my gratitude to the

faculty and staff in the Department of Anatomy and Cell

Biology for their support and interest in my advancement as

a scientific investigator. I would like to acknowledge Kari

Eissinger for her help in photographing and printing some of


iii








the figures contained herein. Particularly, I would like to

thank Drs. Kelly Selman and Gillian Small for the advice and

friendship they have provided during these years. I am also

grateful to have been in contact with other students, past

and present, which have made the time spent in the

department more enjoyable. I am grateful to Dan Tuttle for

reviewing my dissertation. I would also like to acknowledge

all the people that have worked in the laboratory of Dr.

West, including Scherwin Henry, for making the time spent in

the laboratory more rewarding and Mel Fields for his

assistance in some experiments.

I would like to acknowledge my professors at the

University of Puerto Rico, who encouraged me to pursue a

career in biological research.

I appreciate all the friends I have made in Gainesville

in the past years and would like to acknowledge their

importance in my education. Especially, I thank Hans van

Oostrom for his moral support, encouragement, and for

teaching me about the wonders of the electronic world and

those rare creatures called computers.

Finally, and most importantly, I would like to thank my

family. I thank my grandparents, especially Abuela Emma and

Abuela Yuya, for the faith they have showed in me and for

their prayers, which have helped me through my graduate

education. My brothers German, Omar, and Carlos have been

great sources of happiness and pride. Lastly, I thank my








parents, Beatriz and German, for everything. I firmly

believe that the education received during my first twenty-

one years of life in their company is ultimately responsible

for every achievement in my life.















TABLE OF CONTENTS


ACKNOWLEDGEMENTS . . . . . . . . . iii

LIST OF TABLES . . . . . ........ ...... .. viii

LIST OF FIGURES . . . . . . . . . .. ix

LIST OF ABBREVIATIONS . . . . . . . .. xi

ABSTRACT . . . . . . . . . . . xiv

CHAPTERS

I HISTORICAL REVIEW AND BACKGROUND . . . . 1

Introduction . . . . . . . . . 1
Fucosylated Macromolecules . . . . . . 3
Fucose-Binding Proteins . . . . . .. 13

II CHARACTERIZATION OF A FUCOSYLATION MUTANT . . 18

Introduction . . . . . . . . .. 18
Materials and Methods . . . . . . .. 19
Results . . . . . . . . . . 24
Discussion . . . . . . . . . . 40

III IDENTIFICATION OF A CYTOSOLIC FUCOPROTEIN . . 45

Introduction . . . . . . . . .. 45
Materials and Methods . . . . . . .. 46
Results . . . . . . . . . .. 54
Discussion . . . . . . . . . .. 83

IV EVIDENCE FOR A CYTOSOLIC FUCOSYLTRANSFERASE . 90

Introduction . . . . . . . . .. 90
Materials and Methods . . . . . . .. 92
Results . . . . . . . . . .. 98
Discussion . . . . . . . . .. 135

V SUMMARY AND CONCLUSIONS . . . . . .. 144

Summary of Results . . . . . . .. 144
Future Studies . . . . . . . . . 149

vi








REFERENCES . . . . . . . . . .. .156

BIOGRAPHICAL SKETCH . . . . . . . .. .166


vii















LIST OF TABLES


Table Page

2-1. Fucose content of normal and mutant spores and
vegetative cells . . . . . . . .. 29

2-2. Effect of time on conversion of GDP-mannose to
GDP-fucose . . . . . . . . . .. 34

2-3. Specific activities of fucose . . . . .. .39

3-1. Distribution of protein and radioactivity in S100
and P100 fractions . . . . . . . . 56

3-2. Radioactivity recovered in the second S100 after
different P100 treatments . . . . .. .. 65

3-3. Distribution of markers among S100 and P100
fractions . . . . . . . . . .. 66

4-1. Effect of different treatments on the cytosolic
fucosyltransferase activity . . . . . .. .102

4-2. Failure to sediment S100 fucosyltransferase
activity . . . . . . . . . .. 104

4-3. Comparison between the S100 and P100
fucosyltransferase activities . . . . .. .112

4-4. Fucosyltransferase activity in Ax3 S100 fraction 122

4-5. Utilization of 8-methoxycarbonyloctyl synthetic
acceptors by cytosolic fucosyltransferase activity
from Ax3 and HL250 . . . . . . . .. 128

4-6. Evaluation of the suitability of p-nitro-phenyl
glycosides as acceptors for cytosolic fucosyl-
transferase activities in HL250 and Ax3 S100 . 132

4-7. Reduction of fucosylation of acceptor type I analog
by purified FP21 . . . . . . . .. 134

4-8. In vitro fucosyltransferase activity of HL250 and
Ax3 slug extracts . . . . . . . .. 136


viii















LIST OF FIGURES


Figure Page

2-1. Localization of SP96 in prespore and spore cells 27

2-2. Biosynthesis of GDP-L-fucose . . . . .. .32

2-3. Conversion of GDP-mannose to GDP-fucose by normal
and mutant strains . . . . . . . .. 37

3-1. Incorporation of [3H]fucose into macromolecular
species of the S100 and P100 . . . . .. .58

3-2. Proteinaceous nature of FP21 . . . . .. 60

3-3. Comparison of S100 and releasable P100 components 63

3-4. Gel filtration chromatography of FP21
glycopeptides . . . . . . . . . . 71

3-5. Gel filtration chromatography of PNGase F digests 74

3-6. Gel filtration chromatography of FP21
oligosaccharides . . . . . . . .. 77

3-7. Gel filtration chromatography of P100
glycopeptides . . . . . . . . . 81

3-8. Incorporation of [3H]fucose into macromolecular
species of slug stage cells . . . . . .. .85

4-1. Fucosylation of endogenous acceptors by S100
fraction . . . . . . . . . .. 101

4-2. SDS-PAGE profile of endogenous acceptors
fucosylated in vitro . . . . . . .. 107

4-3. BioGel P-4 gel filtration chromatography of in
vitro labelled FP21 oligosaccharide . . . .. .110

4-4. Effect of pH on S100 and P100 fucosyltransferase
activities in the presence of Tween-20 . . . 115








4-5. Effect of GDP-fucose concentration on S100 and P100
fucosyltransferase activities in the presence of
Tween-20 . . . . . . . . . .. 117
4-6. Effect of GDP-fucose concentration on intact S100
and P100 fucosyltransferase activities . . .. .120

4-7. Fucosylation of mutant FP21 by Ax3 S100 fraction 126

4-8. Haworth projections of the structures of the synthetic
glycolipid acceptors . . . . . . .. 130















LIST OF ABBREVIATIONS

APA Asparagus Pea Agglutinin

ATP Adenosine-5'-triphosphate

BSA Bovine Serum Albumin

14C Radioactive Carbon

cDNA Complementary Deoxyribonucleic Acid

cm Centimeter(s)

Con A Concanavalin Agglutinin

cpm Counts per Minute

CHO Chinese Hamster Ovary

dH2O Distilled Water

dpm Disintegrations per Minute

EDTA Ethylenediaminetetraacetic Acid

EtOH Ethanol

GlcNAc N-acetyl Glucosamine

GDP Guanosine-5'-diphosphate

h Hour(s)
3H Tritiated

HMG High Mobility Group

HPLC High Performance Liquid Chromatography

kD Kilodaltons

K Michaelis Constant
1 Liter(s)
1 Liter(s)









M

mCi

mAb

MES

mg

ml

min

mm

mM

MW

pCi

PM

nm

p

PAGE

pmol

PMSF

PNGase F

Rev

RNAse B

SDS

TCA

Tris

U

UEA-I

Ve


Molar Concentration

Millicurie(s)

Monoclonal Antibody

2-(N-Morpholino)ethanesulfonic Acid

Milligram(s)

Milliliter(s)

Minutes)

Millimeter(s)

Millimolar

Molecular Weight

Microcurie(s)

Micromolar

Nanometers

probability

Polyacrylamide Gel Electrophoresis

Picomole(s)

Phenylmethylsulfonyl Fluoride

Peptide N-glycosidase F

Relative Elution Coefficient

Ribonuclease B

Sodium Dodecyl Sulphate

Trichloroacetic Acid

Tris(hydroxymethyl)aminomethane

Unit(s)

Ulex europaeus Agglutinin

Elution Volume


xii









Vi

V
max

vo

v/v

WGA

w/v


xiii


Inclusion Volume

Maximal Velocity

Void volume

Volume per Volume

Wheat Germ Agglutinin

Weight per Volume















Abstract of Dissertation Presented to the Graduate School
of the University Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

A NOVEL FUCOSYLATION PATHWAY IN THE CYTOSOL
OF DICTYOSTELIUM DISCOIDEUM

By

Beatriz GonzAlez-Yanes

December 1991

Chairman: Dr. Christopher M. West
Major Department: Anatomy and Cell Biology

The existence of a fucosylation pathway in the cytosol

of Dictyostelium discoideum was investigated in the

glycosylation mutant strain, HL250, and the normal parental

strain, Ax3. HL250 was characterized as a conditional

mutant that cannot convert GDP(guanosine-5'-diphosphate)-

mannose to GDP-fucose, resulting in a lack of macromolecular

fucosylation unless grown in the presence of extracellular

fucose. HL250 or Ax3 cells were metabolically labelled with

[3 H]fucose, filter-lysed, and fractionated by high speed

centrifugation into sedimentable (P100) and soluble (S100)

fractions. The fractions exhibited unique profiles of

fucoconjugates as analyzed by gel electrophoresis. The

major acceptor in the S100 was a 21 kilodalton molecular

weight protein, FP21. Analysis of FP21 oligosaccharide by

mild alkaline hydrolysis and gel filtration chromatography

xiv








revealed that fucose was incorporated into an 0-linked

oligosaccharide with an average size of 4.8 glucose units.

FP21 appeared to be endogenous to the cytosol, based on the

failure to release FP21 from P100 vesicles by sonication,

and the absence of FP21-like glycopeptides derived by

pronase digestion of the P100.

To determine if FP21 was fucosylated in the cytosol,

S100 and P100 fractions from HL250 were assayed for

fucosyltransferase activity, measured as ability to transfer

[14C] from GDP-[14C]fucose to endogenous acceptors.

Fucosylation of FP21 by the S100 was time- and protein

concentration-dependent. The cytosolic activity was

distinguished from the bulk P100 activity by its absolute

divalent cation dependence, alkaline pH sensitivity, and

very low apparent K for GDP-fucose. Fucosyltransferase

activity was not detectable in Ax3 cytosol. However,

activity was reconstituted by addition of purified mutant

FP21, suggesting FP21 was already fucosylated in living

cells, and Ax3 possessed a cytosolic fucosyltransferase

equivalent to the HL250 enzyme. S100 fractions from Ax3 and

HL250 were able to fucosylate an al,4fucosyltransferase-

specific acceptor glycolipid analog, but not analogs capable

of being modified by al,2 and al,3 fucosyltransferases.

Since fucosylation of the analog was reduced by addition of

purified FP21, the same enzyme appears to be responsible for

fucosylation of both molecules. Thus it is proposed that









FP21 is synthesized and fucosylated in the cytosol by an

al,4fucosyltransferase.


xvi















CHAPTER I
HISTORICAL REVIEW AND BACKGROUND


Introduction

Glycoprotein synthesis and localization have been

subjects of intense study in the last few decades. Much has

been learned about glycosylation and some excellent reviews

are available (Kornfeld and Kornfeld, 1985; Hirschberg and

Snider, 1987). Carbohydrate moieties are usually

categorized as N and/or 0-linked depending on whether the

carbohydrate moiety is attached to an asparagine by an amide

glycosidic linkage or to a serine or threonine,

respectively. Glycosylation is generally considered to be a

modification restricted to macromolecules that pass through

the secretary pathway, starting in the rough endoplasmic

reticulum where N-linked glycosylation is initiated

(Kornfeld and Kornfeld, 1985), but in some instances 0-

linked glycosylation also takes occurs (Spielman et al.,

1988). Glycosylation then continues in the Golgi apparatus

where further processing of N-linked oligosaccharides may

occur and 0-linked glycosylation takes place (Abeijon and

Hirschberg, 1987). In the case of fucosylation, L-fucose

has been shown to be added as a terminal modification to

either N-linked or 0-linked oligosaccharides in the Golgi








2

apparatus utilizing GDP-fucose as the sugar nucleotide donor

(Bennett et al., 1974; Hirschberg and Snider, 1987).

However, there are a few exceptions to these remarks, since

fucose has been found to be attached directly to serine and

threonine, although the site for this modification has not

been identified (Hallgreen et al., 1975), and to be present

in homopolymers, as in fucoidans (Flowers, 1981). It has

been suggested that fucosylation also occurs in the

endoplasmic reticulum as well as in the Golgi apparatus in

thyrotrophs under different physiological conditions (Magner

et al., 1986). There are two pathways for the biosynthesis

of GDP-fucose. The main source of GDP-fucose is the

conversion pathway of GDP-mannose to GDP-fucose (Yurchenco

et al., 1978; Flowers, 1985). Alternatively, synthesis of

GDP-fucose may occur by the fucose salvage pathway in the

presence of extracellular L-fucose (Yurchenco et al., 1978;

Flowers, 1985; Ripka and Stanley, 1986; Reitman et al.,

1980).

Glycosylation is carried out by diverse specific

glycosyltransferases which have been located in the

endoplasmic reticulum and Golgi apparatus. One underlying

assumption is that the acceptor macromolecule must

colocalize with the transferase enzyme in order to be

glycosylated. However, there have been occasional reports

of glycoproteins in compartments topologically discontinuous

with the lumen of the rough endoplasmic reticulum and Golgi








3

apparatus. These findings contradict the dogma that

glycosylation is strictly an endoplasmic reticulum- and

Golgi apparatus-dependent event. Two models could account

for the existence of glycoproteins outside the realms of the

secretary pathway; one postulates that lumenal glycosylated

proteins translocate across the membrane back to the

cytosolic space, the other that glycosyltransferases are

localized outside of the lumen of the endoplasmic reticulum

or Golgi apparatus. Evidence has been accumulating in the

past decade that supports the latter model. In this chapter

I shall be concerned with the presence of fucosylated

proteins in compartments topologically discontinuous with

the lumen of the endoplasmic reticulum and Golgi apparatus.

Subsequently, I shall analyze some of the studies that

suggest the presence of fucosyltransferases that would co-

compartmentalize with such fucoproteins. In light of the

results presented in this dissertation, a review focussing

on the presence of fucoproteins and fucosyltransferases

outside the secretary pathway will be useful. An excellent

review is available that examines nuclear and cytosolic

glycosylation in general (Hart et al., 1989a).



Fucosylated Macromolecules

The presence of fucosylated proteins in compartments

discontinuous with the secretary pathway has been documented

since the 1970's. Various techniques have been employed in








4

these reports, including the direct analysis of carbohydrate

content, use of lectins, use of radiolabelled fucose and/or

a combination of biochemical and morphological techniques.



Nuclear Fucosylated Macromolecules

In contrast to the prevailing dogma, fucosylated

macromolecules have been detected in the nucleus. Such

studies demonstrated lectin binding to nuclear membranes,

chromatin, nuclear proteins, and the nuclear matrix. These

studies generally involved localization by binding of

labelled lectins (fluorescent, radiolabelled, ferritin-

conjugated, etc.) to isolated nuclei. Binding specificity

was assessed by competition of labelling with hapten

inhibitors. Occasionally, these studies were supplemented

by metabolic labelling experiments with radioactive fucose

which is primarily incorporated into fucoproteins with

little metabolism into other molecular species in eukaryotic

cells (Yurchenko et al., 1978).



Nuclear membranes. One early report suggested the

presence of fucose-containing structures on the cytoplasmic

face of isolated bovine nuclei (Nicolson et al., 1972).

Nicolson et al. (1972) found that purified nuclei were

agglutinated by the L-fucose-specific lectin UEA-I from Ulex

europaeus (Lis and Sharon, 1986), and agglutination was

inhibited by incubation in the presence of L-fucose,








5

suggesting that there were fucose-containing membrane-bound

oligosaccharides on the outer nuclear membrane. Similar

results were obtained with the mannose- and glucose-specific

lectin concanavalin A (Con A), which was also found to

agglutinate purified nuclei (Nicolson et al., 1972).

However, subsequent work by another group revealed that, as

evidenced by electron microscopic examination, ferritin-Con

A appeared to stain only damaged nuclei (Virtanen and

Wartiovaara, 1976) raising some concerns about the studies

by Nicolson et al. (1972). Likewise, in the aforementioned

studies (Nicolson et al., 1972), integrity of the isolated

nuclei was not determined, allowing for the possibility that

the lectin was interacting with lumenal fucoproteins that

escaped organelles during nuclei isolation or with

contaminating fucoprotein-containing membranes, such as

plasma membrane.



Chromatin. The existence of chromatin associated

fucose-containing proteins has been suggested by several

laboratories. Early on, Stein et al. (1975) reported the

presence of [3H] labelled glycoconjugates in purified

chromatin from HeLa cells grown in the presence of

[3 H]fucose. Although the label was not examined to

corroborate its presence as fucose, in the case of

radiolabelling macromolecules with radioactive sugar

precursors, fucose is an excellent candidate because it has








6

been shown to be incorporated as such, with minimal

metabolizing of the label (Yurchenko et al., 1978). In

these studies the authors examined a very pure chromatin

preparation, with essentially no contamination from nuclear

or plasma membranes. The authors argued against plasma

membrane contamination based on mixing experiments, in which

radiolabeled cell-surface trypsinates were combined with

unlabeled nuclear preparations prior to chromatin isolation.

The fucosylated chromatin-associated macromolecules were

deemed to be fucoproteins based on their sensitivity to

pronase digestion. Unfortunately, these studies have not

been pursued further, and many questions remain unanswered

with respect to their structure and biosynthesis.

Chromatin-associated fucoproteins were also detected in

normal rat liver and Novikoff hepatoma ascites cells

(Goldberg et al., 1978) using the L-fucose-specific lectin

asparagus pea agglutinin (APA). The authors reported a

strongly basic fucoprotein, that was sensitive to pronase

digestion. Based on the reactivity with the lectin, they

calculated that the protein was three times more

concentrated in tumor chromatin than in normal liver cells.

However, the method for chromatin purification did not

eliminate adequately nuclear inner membrane contamination,

and since binding to the lectin was assayed by

affinoelectrophoresis a molecular weight for the protein was

not determined (Goldberg et al., 1978).








7

In a more recent study on duodenal columnar cells, Kan

and Pinto da Silva (1986) used UEA-I conjugated to colloidal

gold in freeze-fracture electron microscopy of cross-

fractured nuclei. They compared binding of the conjugated

lectin to euchromatin, heterochromatin, and nucleolus;

compartments which can be differentiated ultrastructurally.

Binding of UEA-I showed that colloidal gold particles were

almost exclusively confined to cross-fractured areas where

euchromatin was exposed. Labelling was abolished by

pretreatment and incubation in the presence of L-fucose, as

expected for a specific label. Pre-digestion of the

fractions with trypsin also abolished labelling, suggesting

the receptors for UEA-I binding were glycoproteins. The

preferential binding to euchromatin may be of importance,

because replication and transcription take place at

euchromatin regions. Although the results reported are

intriguing, the authors did not identify the type of fucose-

containing proteins detected. DNA-associated proteins can

be classified as either histone or non-histone proteins, and

as summarized below, both types of proteins appear to be

fucosylated.



Histones. The histones are the most abundant proteins

associated with DNA. Histones are very basic proteins and

are found in all nuclei (Darnell et al., 1986). Based on

the specific binding to UEA-I, Levy-Wilson (1983) presented








8

evidence that histones isolated from the macronucleus of

Tetrahymena thermophila appear to contain fucose. These

results were strengthened by metabolic incorporation of

[3H]fucose into histones, which showed that all five

Tetrahymena histones, HI, H2A, H2B, H3, and H4, appear to

contain fucose, with H2A incorporating the highest amount

(Levy-Wilson, 1983). In these studies macronuclei were

isolated to high purity, and highly pure histone

preparations were obtained after extensive washing in high

salt to remove nonhistone proteins. Using Con A, the author

showed specific binding to histones, which was interpreted

as evidence for the presence of mannose residues. However,

Con A has previously been shown to also recognize D-glucose

residues. Based on the extent of fucose incorporation and

its specific radioactivity, the author estimated, as the

lowest estimate, that one in a thousand nucleosomes

contained a fucosylated H2A molecule. To date, the

glycosylation pathway of histones and the oligosaccharide

structures) present in histones remain unknown.



Nonhistone proteins. High mobility group (HMG)

proteins are fairly abundant nonhistone chromosomal proteins

classified according to their relative electrophoretic

mobilities (Darnell et al., 1986). HMG proteins undergo a

variety of posttranslational modifications, including

glycosylation. Since they appear to be preferentially








9

associated with actively transcribed DNA, it has been

speculated that glycosylation may influence gene activity.

Highly purified HMG14 and HMG17 from mouse Friend

erythroleukemic cells were found to contain fucose, among

other sugars, by direct composition analysis (Reeves et al.,

1981). These proteins bound UEA-I specifically and could be

metabolically labelled with [3H]fucose. The

oligosaccharides were largely insensitive to B-elimination,

suggesting an N-linkage to protein (Reeves et al., 1981).

When purified HMG14 and HMG17 were digested with mixed

glycosidases, the binding of HMG14 and HMG17 to the nuclear

matrix was abolished. Even though they did not employ a

fucosidase, making it difficult to ascertain the role of

fucose in binding, it was evident that glycosylation

influenced binding to the nuclear matrix (Reeves and Chang,

1983). These studies presented convincing evidence that HMG

14 and 17 are fucosylated, although they did not explore the

site of modification or the composition of the

oligosaccharide(s).



Nuclear fucoproteins and transcriptional activity.

Since some glycoproteins have been found associated with DNA

there has been speculation about a possible correlation

between the state of nuclear glycosylation and

transcriptional activity (Hart et al., 1989a). There are

examples in the literature of glycosylated transcription








10

factors and, at least in one case, glycosylation may have

influenced transcriptional activity (Lichtsteiner and

Schibler, 1989; Jackson and Tjian, 1989). As mentioned

earlier, in the case of fucosylated macromolecules, it was

found that in Novikoff hepatoma cells chromatin had three

times the amount of a fucoprotein of normal liver cells

based on APA binding (Goldberg et al., 1978). However, the

identity, size, or number of such proteins were not well

documented. Putative fucoproteins were found preferentially

associated with euchromatin (Kan and Pinto da Silva, 1986).

Fucosylated histones were found in the macronucleus of

Tetrahymena, where transcriptionally active chromatin is

compartmentalized (Levy-Wilson, 1983). Nevertheless,

histones from non-active chromatin were not studied, so no

comparisons can be made with heterochromatin. Levy-Wilson

(1983) suggested that the reason why other investigators

have failed to detect fucosylation in mammalian histones is

due to the low proportion of transcriptionally active genome

which would imply a low concentration of fucosylated

histones.

Although the data gathered in these reports are

interesting, they are the result of isolated studies from

diverse organisms and it is difficult to draw generalized

conclusions. In addition, virtually nothing is known about

the structure or biosynthesis of the fucose-containing

moieties. Even though the fractions in all these reports








11

appeared to have almost no contamination, structural studies

showing a different glycoconjugate from those found in other

organelles would convincingly argue against contamination.

Nevertheless, these provocative studies are encouraging and

deserve to be pursued further.



Cytosolic Fucoconjugate

The existence of glycoproteins in the cytosol has been

documented in the past and reviewed recently (Hart et al.,

1989a). Studies suggesting the existence of glycoproteins

in the cytosol were based on determinations of lectin

binding sites or biochemical compositional analyses (Hart et

al., 1989a). Some of the negative results reported by those

studies, that relied on lectin binding as confirmation for

the presence of a sugar residue, may be misleading since

lack of binding may reflect a poor choice of lectins to

probe with and not necessarily the absence of a fucose

residue. While there are several fucose-binding lectins

available that serve as useful biochemical tools, they do

not recognize every possible fucose-containing structure.

Lectin binding is dependent on a specific array of sugar

residues, and does not depend solely on the presence of

fucose (Lis and Sharon, 1986). However, as will be

described below, there is one biochemical study that reports

the existence of a cytosolic fucoconjugate. In many cell

fractionation experiments, the cytosolic compartment is









12

defined by the lack of sedimentation during high-speed

centrifugation. However, this criterion alone is not

sufficient, since cytosolic glycans might arise from

contamination by other organelles. The most convincing

evidence describes fucoconjugates that appear to be

preferentially enriched in the cytosol in relation to other

compartments.

In studies in rat brain, a soluble proteoglycan that

contains novel 0-linked mannose-containing oligosaccharides

was recovered in the cytosolic fraction (Margolis et al.,

1976; Finne et al., 1979). The proteoglycan was

characterized as a soluble chondroitin sulfate proteoglycan,

that contained neutral oligosaccharides releasable by mild

alkaline borohydride treatment. The oligosaccharides

contained mannose at their proximal ends, and one

oligosaccharide was proposed to be composed of mannose,

GlcNAc, fucose, and galactose (Finne et al., 1979).

However, the possibility remains that the oligosaccharides

are not integral components of the proteoglycan, but were

associated with other co-purified material. Nevertheless,

the oligosaccharides appeared to be endogenous to the

cytosol and not the result of contamination from other

fractions, since they were present in only trace amounts in

the microsomal or synaptosomal membrane fractions (Finne et

al., 1979). Unfortunately, the function and biosynthetic

pathway of these oligosaccharides remain unknown.








13

Fucose-Bindinq Proteins

The presence of fucoproteins in the nucleus and cytosol

prompted the idea that there might be specific proteins

inside the cell that bind, and/or modify these fucoproteins,

as is the case with the fucoproteins in the secretary

pathway. This would include fucose-binding proteins,

fucosyltransferases responsible for fucosylation, and

fucosidases responsible for fucose removal. There are some

studies suggesting the existence of fucose-binding lectins

and fucosyltransferases. However, there is no evidence for

a nuclear or cytosolic fucosidase.



Endogenous Lectins

In light of evidence for glycoproteins in the cytosolic

and nuclear compartments, investigators sought the existence

of carbohydrate-binding proteins that would colocalize with

such glycoproteins. Endogenous lectins have been detected

in preparations of nucleoplasmic and/or cytosolic fractions

of a variety of cells (Hart et al., 1989a).

Aided by fluorescein-labeled neoglycoproteins, Sbve et

al. (1986) have postulated the presence of endogenous

fucose-specific lectins. Baby hamster kidney cell nuclei

were isolated by two different procedures, cell lysis and

enucleation in Ficoll, to argue against contamination by

cytoplasmic or membrane-derived components. Using

fluorescein-labelled BSA conjugated to fucose in the order








14

of 205 sugar units per molecule, fluorescence microscopy

experiments suggested that the majority of the binding

appeared to be associated with nucleoli and nucleoplasmic

ribonucleoprotein elements (Seve et al., 1986).

Interestingly, it was shown by quantitative flow

microfluorometry that nuclei from exponentially growing

cells bound one order of magnitude more fucose-BSA than

nuclei from contact-inhibited cells. In spite of these

results, the authors acknowledge that it is impossible based

on the data to ascribe biological roles to the nuclear

fucose-binding sites or to conclude that they influence

cellular physiology (Seve et al., 1986).

In Dictyostelium discoideum a family of lectins, the

discoidins, has been identified, and discoidin I has been

the most extensively studied isoform. In erythrocyte

agglutination assays, agglutination by discoidin may be

inhibited by galactose, modified galactose residues, L-

fucose, D-fucose, and other sugars, suggesting the existence

of cell-surface sugar-dependent epitopes on erythrocytes

recognized by Discoidin (Barondes and Haywood, 1979).

Although discoidin I was originally thought to be a cell

surface protein involved in cell-cell adhesion, it has since

been established that it is primarily present in the cytosol

(Erdos and Whitaker, 1983). Although galactose and modified

galactose residues are the ligands bound by discoidin I with

highest affinity, it is possible that a fucose-containing








15

macromolecule may serve as a ligand for it or that another

lectin, possibly from the discoidin family, will be present

in the cytosol with fucose-binding capability.



Fucosyltransferases

Intracellular fucosyltransferases identified to date,

are localized in the lumen of the Golgi or possibly,

endoplasmic reticulum. Thus the presence of fucoproteins in

the nucleus and cytosolic compartments poses questions

pertaining to the mode of synthesis and/or intracellular

transport of such glycoproteins. Early on, Kawasaki and

Yamashina (1972) theorized, based on metabolic labelling,

that nuclear membrane glycoproteins were not synthesized in

and transported from microsomes, but were made in the

nuclear membrane or its vicinity. A few reports have

suggested the presence of glycosyltransferases in the

nucleus, nuclear membranes, or cytosol that may be involved

in the modification of several glycoproteins; however, none

of these glycoproteins was reported to contain fucose

(Richard et al., 1975; Galland et al., 1988, Haltiwanger et

al., 1990).

Though there is evidence for nuclear and cytosolic

fucosylated macromolecules, to my knowledge there are no

reports of nuclear or cytosolic fucosyltransferases.

Louisot and collaborators reported the purification and

separation of two soluble fucosyltransferase activities from








16

rat small intestinal mucosa (Martin et al., 1987). They

report the isolation of al,2 and al,3/1,4fucosyltransferases

that could be candidates for cytosolic fucosyltransferases

based on their inability to sediment after homogenization of

cells in 0.25 M sucrose, followed by 90 min, 200k x g

centrifugation (Martin et al., 1987). They compare the

al,2fucosyltransferase with the a2,6sialyltransferase, which

is normally a Golgi enzyme converted to a soluble form by

cleavage of the amino terminal signal anchor to allow for

secretion (Weinstein et al., 1987). In the case of the

fucosyltransferases, the authors did not report using

protease inhibitors during isolation, nor did they document

the partitioning of cellular markers in the different

fractions (Martin et al., 1987). The reasons the enzymes

localize to the high speed supernatant, which would usually

be considered as the cytosolic fraction, may include the

breakage of the microsomal vesicles or the fact that they

were initially present extracellularly in the body fluids of

the mucosa. In another report these investigators tested

for the presence of glycosyltransferases in the nucleus. In

highly purified rat liver nuclei there was a total absence

of fucosyltransferase activity when endogenous

macromolecules or asialofetuin were used as acceptors

(Richard et al., 1975).

One explanation for the lack of evidence of nuclear

and/or cytosolic fucosyltransferases activity in any








17

organism may be that there indeed are no

fucosyltransferases. Alternatively, if there are no

cytosolic fucosyltransferases, the presence of

fucoconjugates in the cytosol or nucleus may be explained by

membrane-associated enzymes that face the cytosolic

compartment, as has been reported for other

glycosyltransferases (Haltiwanger et al., 1990). In

addition, the lack of adequate acceptors, unfavorable

conditions for in vitro activity, and the scarcity of

sustained interest in the field, could account for the lack

of evidence for such fucosyltransferase(s) that, just as is

the case with other glycosyltransferases, are not of lumenal

origin.

Evidence for fucosylation, and glycosylation in

general, has been accumulating in the past decades. Until

recently, only sporadic reports about fucoproteins appeared

in the literature. With the identification of newly

reported glycoconjugates (Hart et al, 1989b), interest has

been revived in this area of research and currently there

appears to be much interest in the field. However, a

careful examination and sustained interest will be necessary

in order to elucidate the structures, and modes of

biosynthesis and functions of nuclear and cytosolic

fucoconjugates.














CHAPTER II
CHARACTERIZATION OF A FUCOSYLATION MUTANT


Introduction

Since fucosylation comprises numerous steps which are

potential sites for mutations, many fucosylation mutants

have been obtained. Fucosylation consists of the synthesis

of the sugar nucleotide donor GDP-fucose and the transfer of

fucose from the GDP-fucose to an acceptor (Kornfeld and

Kornfeld, 1985). The main source of GDP-fucose is the

conversion pathway of GDP-mannose to GDP-fucose (Yurchenco

et al., 1978; Flowers, 1985). Alternatively, synthesis of

GDP-fucose by the fucose salvage pathway can occur in the

presence of extracellular L-fucose, allowing cells defective

in the conversion pathway to phenotypically revert (Ripka

and Stanley, 1986; Reitman et al., 1980). Lesions may

affect the formation of GDP-fucose, the transport of GDP-

fucose to the fucosylation compartment, the transferases

responsible for fucosylation and/or the biosynthesis or

transport of the acceptor species to the fucosylation

compartment. Two mutants deficient in protein-associated

fucose were shown to be defective in the formation of GDP-

fucose (Reitman et al., 1980; Ripka et al., 1986). A

chinese hamster ovary (CHO) cell line that showed a marked








19

reduction of incorporation of fucose into macromolecules was

unable to synthesize complex-type N-linked oligosaccharides,

resulting in a deficiency of acceptors (Hirschberg et al.,

1982). In another case, two glycosylation mutants were

shown to express a fucosyltransferase activity absent in the

parental cell line with the concomitant expression of a

novel linkage (Campbell and Stanley, 1984).

There are several putative glycosylation mutants. One

of these mutants, HL250, was selected after mutagenesis of

the parental normal strain Ax3 for the inability to bind

anti-SP96 antiserum (Loomis, 1987). This mutant also failed

to express a fucose-dependent epitope recognized by the

monoclonal antibody 83.5 (West et al., 1986). The focus of

my initial investigation was to characterize the mutation in

HL250 with the help of biochemical and morphological

techniques. We determined that HL250 is a fucosylation

mutant that lacked cellular fucose when grown in the absence

of fucose and that the defect is probably a result of a

lesion detectable in vitro in the conversion pathway that

forms GDP-fucose from GDP-mannose.



Materials and Methods

Materials

GDP-[l-3Hjmannose (9.1 Ci/mmol) was purchased from New

England Nuclear and L-[(5,6)-3H]-fucose (60 Ci/mmol) from

American Radiochemical Corporation. KC1 and MgCl2 were








20

obtained from Mallinckrodt; formic acid from Fisher; ATP

(disodium salt, catalog number A-5394), niacinamide, NAD4,

NADPH, Trizma base, phenylmethylsulfonyl fluoride, Dowex-1

(1x8, minus 400, chloride form), hexokinase, GDP-mannose,

and Amberlite MB-3 were obtained from Sigma. ATP was stored

frozen at a concentration of 500 mM in 1 mM Tris-HCl (pH

7.4), resulting in a final pH of approximately 5.5. Dowex-1

format form was made as follows: 1) The column was washed

with 1 M HC1 until pH of eluate was below 2 (as determined

by pH paper). 2) The column was washed with water until pH

was higher than 4.5. 3) Subsequently, the column was washed

with 1 M NaOH until pH was higher than 13. 4) The column

was washed with water as described in step 2. 5) The column

was washed with 3 volumes of 1 M formic acid until pH of

eluate was below 2. 6) Lastly, the column was washed with

water as described in step 2. This procedure was also

followed for regeneration of column.



Strains and Conditions of Growth and Development

Dictyostelium discoideum amoebae were grown on HL-5, a

medium that contains glucose, yeast extract, and proteose

peptone (Loomis, 1971). The axenic strains Ax3 (from F.

Rothman) and HL250 (from W.F. Loomis) were maintained by

passage during logarithmic growth phase. Ax3 is the normal

strain and HL250 is a mutant obtained from Ax3 by N-methyl-

N'-nitro-N-nitrosoguanidine mutagenesis. For metabolic








21

labelling experiments, cells were grown for 4-6 doublings in

8-20 pCi/ml (0.10-0.26 MM) of [3H]fucose in FM medium, a

minimal defined medium that lacks fucose (Franke and Kessin,

1977). When appropriate, the medium was supplemented with

L-fucose (Sigma). For development, cells were plated in PDF

buffer (20 mM KC1, 45 mM sodium phosphate, 6 mM MgSO4, pH

5.8) on filters as previously described (West and Erdos,

1988).



Cell Lysis and Fractionation

Logarithmically growing amoebas were harvested and

washed in 50 mM Tris-HCl (pH 7.5) and resuspended to a

concentration of 2x108 cells/ml in the lysis buffer

consisting of 0.25 M sucrose, 50 mM Tris-HCl buffer (pH 7.5)

supplemented with 1 mM PMSF. All operations were carried

out at 0-4C. Cells were immediately lysed by forced

passage through a 5 Mm nuclepore polycarbonate filter, with

pore diameter slightly smaller than the diameter of the

cells (Das and Henderson, 1986). This method routinely

yields more than 99% cell breakage. The lysate was

centrifuged at 100k xg for 1 hour. The supernatant (S100)

was made 2.5% (v/v) in glycerol by addition of 100% glycerol

and either used immediately or saved at -80 without

significant loss of activity for four weeks.








22

Localization by Immunofluorescence

Prespore and spore cells were examined as described

previously (West and Loomis, 1985; GonzAlez-Yanes et al.,

1989). The monoclonal antibodies utilized have been

described previously elsewhere (West et al., 1986; GonzAlez-

Yanes et al., 1989).



Determination of Fucose Content and Specific Activity

The method is a modification of the protocol developed

by Yurchenco and Atkinson (1975) for HeLa cells. Spores

were harvested from sori not more than a day old and

resuspended in water without washing, since it has been

determined that a significant amount of spore coat protein

may be lost after washing spores in water (West and Erdos,

1990). Vegetative cells were grown in FM (in the presence

or absence of extracellular L-fucose) for determination of

sugar content. For determination of specific activity,

cells were grown in FM media supplemented with [3H]fucose.

After harvesting, cells were washed twice in PDF followed by

EtOH precipitation. The ethanol supernatant was reextracted

with EtOH and the resulting pellet pooled with the previous

precipitate. EtOH was evaporated under a stream of air.

Alternate methods such as TCA precipitation of the ethanol

supernatant, did not significantly increase the yield.

Samples were then hydrolyzed in a reacti-vial (Pierce) in

0.1 N HCl for 45 min at 100 on a heating block.








23

Macromolecules were EtOH precipitated and the remaining

supernatant dried down, resuspended in water, desalted on an

Amberlite MB-3 column and dried by vacuum centrifugation.

The samples were redissolved in water and analyzed using a

Dionex Bio-LC ion chromatograph by the method of Hardy et

al. (1988). Standards and modifications to the

chromatography procedure have been published elsewhere

(Gonzdlez-Yanes et al., 1989). Specific activity, when

applicable, was determined by counting elution fractions and

was expressed as radioactivity present in the fucose peak

divided by the amount of fucose detected by the pulsed

amperometric detector coupled to the HPLC, with reference to

previously established calibration curves for L-fucose

(Hardy et al., 1988). Greater than 95% of the eluted

radioactivity was recovered from the column eluate at the

elution position of fucose.



Assay for Conversion of GDP-mannose to GDP-fucose

The conversion assay was carried out essentially as in

Ripka et al. (1986). In short, the standard assay mixture

contained in a final volume of 1 ml 600-750 pg S100 protein,

10 mM niacinamide, 5 mM ATP, 0.2 mM NAD, 0.2 mM NADPH, 7.5

pM GDP-[ H]mannose (approximately 105 cpm) in 50 mM Tris-

HC1, pH 7.5. After incubation at 37 the reaction was

stopped with 50 pi of 2 N HC1, the reaction mixture boiled

for 20 min and subsequently neutralized with 55 pl of 2 N








24

NaOH. Quantitation of the conversion of GDP-mannose to GDP-

fucose was achieved by determining the amount of fucose

present after acid hydrolysis. Free mannose, released from

GDP-mannose, was phosphorylated by adding 4 units of

hexokinase in the presence of 5 mM ATP and 5 mM MgCl2 at 37

for 1 hour. Hexokinase catalyzes the transfer of one

phosphate to C-6 of any acceptor hexose. Since fucose lacks

a hydroxyl group at position C-6, it cannot be

phosphorylated. Parallel controls with no enzyme were used

to correct for losses. The mixture was passed over a Dowex-

1 formatt) column (0.6 x 5 cm) and eluted with water. 1 ml

fractions were collected an aliquot of 100 p1 from the

eluate was counted using ScintiVerse LC (Fisher); fucose-

associated radioactivity usually eluted by the first 2 ml.

Calculation of K and V was done by the Lineweaver-Burk
m max

double reciprocal plot (1/v vs. 1/[S]) as discussed by

Henderson (1985).



Results

Phenotypic Description of a Fucosylation Mutant

The normal strain Ax3 was mutagenized with

nitrosoguanidine and surviving clones were screened with

anti-SP96 antiserum (Loomis, 1987). One of the clones,

HL250, was selected due to its inability to bind anti-SP96

antiserum which recognizes carbohydrate and peptide

epitopes. HL250 has been found to have a more permeable








25

spore coat, lower germination efficiency in older spores,

and a longer doubling time when compared to the parental

strain Ax3 (Gonzalez-Yanes et al., 1989; West et al.,

manuscript in preparation).



Absence of a fucose-dependent epitope. The failure of

HL250 to react with anti-SP96 antiserum suggested that the

mutant might be deficient in a form of protein

glycosylation. This hypothesis was confirmed by finding

that HL250 did not react at appreciable levels with the

fucose-dependent monoclonal antibody (mAb) 83.5 (West et

al., 1986; Gonzalez-Yanes et al., 1989). Spores from mutant

and the parental normal strains were subjected to SDS-PAGE

and Western blotting and probed with mAbs 83.5 and A6.2

(West et al., 1986; Gonzalez-Yanes et al., 1989). The

latter monoclonal is specific for SP96. Consistent with the

supposition that glycosylation is affected in HL250, SP96

was reduced in apparent molecular weight compared to SP96

from Ax3 spores (West et al., 1986). The absence of the

fucose-dependent epitope was further examined in developing

cells and spores. Cells were plated for development and

slugs dissociated by shearing in the presence of EDTA.

Cells and spores were then processed for indirect

immunofluorescence. Figure 2-1 shows the localization of

SP96 in spores and prespore cells. Ax3 exhibits peripheral

labelling of spores and a punctate labelling from prespore

































Figure 2-1. Localization of SP96 in prespore and spore cells.

Slugs cells were dissociated, placed onto polylysine-coated
glass slides, fixed, permeabilized, and processed for
indirect immunofluorescence using mAbs 83.5 or A6.2.





















PRESPORE CELLS


83.5


A6-2


SPORES


83.5


A6-2









28

vesicles using both mAb. However, HL250 shows the same

pattern only when A6.2 is used, in agreement with the

results observed by Western blotting. The fact that

labelling of spores with A6.2 is similar in both strains

indicates that spore coat localization of SP96 is not

affected by the mutation.



Fucose content of normal and mutant strains. Epitope

recognition by mAb 83.5 was inhibited by L-fucose (West et

al., 1986), so the possibility of a defect in fucosylation

in strain HL250 was investigated. Initially, the

macromolecular fucose content of vegetative cells grown in

fucose-free media, and of spores was investigated. Ethanol

insoluble macromolecules were acid hydrolyzed, ethanol

precipitated, and the supernatant deionized and

chromatographed on an alkaline anion-exchange column

equipped with a pulsed amperometric detector. When

authentic [3H]fucose was fractionated in this manner, more

than 95% of the radioactivity eluted at the fucose position

(not shown). Fucose content was found to be negligible in

the mutant, HL250, both in spores and vegetative cells when

compared to the normal strain, Ax3 (table 2-1). However,

when HL250 cells were grown in FM supplemented with 1 mM L-

fucose, they possessed detectable amounts of fucose.

Previous investigators have found by autoradiography that

[3H]fucose incorporation is highest in prespore cells
















0,-I

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30

compared to prestalk and vegetative cells, so higher levels

of fucose would be expected in spores (Lam and Siu, 1981;

Gregg and Karp, 1978). The levels of glucose and mannose

were measured for comparison to determine if there was a

difference in the amounts of other sugars in the mutant.

None of the contents of the other sugars were reduced in the

mutant when grown in FM. Thus it seems that the lesion in

HL250 is selectively affecting fucose metabolism, relative

to that of other sugars. This is consistent with the fact

that fucosylation is usually a terminal modification of

oligosaccharides, so its addition is not a prerequisite for

the addition of other sugars (Kornfeld and Kornfeld, 1985).



Characterization of the Mutant Lesion

Earlier investigations have described mutants with a

fucose minus phenotype that are the result of a defect in

GDP-fucose biosynthesis. The main source of GDP-fucose is

the conversion pathway of GDP-mannose to GDP-fucose

(Yurchenco et al., 1978; Flowers, 1985). It consists of the

reactions presented in figure 2-2. Alternatively, synthesis

of GDP-fucose by the fucose salvage pathway can occur in the

presence of extracellular L-fucose (figure 2-2), allowing

cells defective in the conversion pathway to phenotypically

revert (Ripka and Stanley, 1986; Reitman et al., 1980).

HL250 cells grown and developed in the presence of 1 mM L-

fucose reexpressed the fucose epitope (Gonzdlez-Yanes et

































Figure 2-2. Biosynthesis of GDP-fucose.

Diagrams of the conversion pathway elucidated in bacteria
and mammalian cells (adapted from Flowers, 1981) and the
salvage pathway as described in HeLa cells (adapted from
Yurchenko et al., 1978).




























CH20H CH3 H
0 'H O-0 lH C 0 4 .c~ H 0---0\
4,6-ehydrete C ,5-.pm.r 4- renuctm

O H. O' 0O-DP 0- N 11 1 L O-GDP

H H H H ON M OH H


A CONVERSION PATHWAY



H 0H
H N H

/ CH3 3 J fucm / CHI pyroplsphoryIloe |/CH \
1\ ON k~, nose N OMN ---- > H M y
I\LH OH"
OO N ON 0- p OH O-GDP
OH H OH H OH H


B. SALVAGE PATHWAY








33

al., 1989), and vegetative cells grown in 1 mM L-fucose had

detectable amounts of macromolecular-associated fucose

(table 2-2). These results suggested that HL250 had a

normal salvage pathway, but a defect in the GDP-mannose to

GDP-fucose conversion pathway.



In vitro conversion of GDP-mannose to GDP-fucose.

Conversion of GDP-mannose to GDP-fucose has been reported in

bacteria, in higher plant cells, and mammalian cells

(Kornfeld and Ginsburg, 1966; Liao and Barber, 1971; Ripka

et al., 1986). It was assumed that Dictyostelium would

share this ability with other species, so I assayed if cell

extracts in vitro were able to convert GDP-mannose to GDP-

fucose. High speed supernatants from Ax3 and HL250 were

assayed in vitro for their ability to convert GDP-

[ 3H]mannose into GDP-[3H]fucose. Cells were homogenized and

a 100k x g soluble fraction (Si00) assayed as described by

Ripka et al. (1986) and the effects of time, varying protein

and GDP-mannose concentrations were examined. Table 2-2

shows that the conversion activity in Ax3 was proportional

to the time of incubation. In contrast, mutant extracts

showed negligible activity. Ax3 and HL250 cytosols were

mixed to determine if a soluble inhibitor of GDP-mannose to

GDP-fucose conversion activity was present. When equal

amounts of extracts were mixed, activity was commensurate

with the Ax3 contribution (table 2-2) indicating HL250 does




























Table 2-2.
GDP-fucose.


Effect of time on conversion of GDP-mannose to


nmol fucose/mg protein


time (min)


7.5
15


Ax3

0.57
2.5
2.9
9.2


HL250

0
0
0.15
0


Ax3+HL250 (0.5:0.5)


0.21
1.3
2.2
4.3


Protein (600 pg total) from a 100,000 xg supernatant of a
vegetative cell-free extract was assayed for ability to
convert GDP-[ 14C]mannose (7.5 pM initial concentration) to GDP-
[14C]fucose; data are the result of the average of two
determinations








35

not contain an inhibitor for the activity. Conversion was

linear with respect to protein through 800 Mg (figure 2-3,

panel A). In agreement with the results from the time

dependence experiment, HL250 expressed less than 1% of the

activity possessed by Ax3 at all concentrations of protein

assayed (figure 2-3, panel A). Conversion by Ax3 S100 was

also dependent on the amount of GDP-mannose present while

the activity present in HL250 was insignificant (figure 2-3,

panel B). The Ax3 GDP-mannose to GDP-fucose conversion

activity showed an apparent KM of 14.1 yM and V of 18.3

nmol fucose/mg protein/30 min (fig. 2-3, panel C). Previous

reported values for the apparent KM of the conversion

pathway range from 2 MM in CHO cells (Ripka et al., 1986) to

160 pM in the higher plant Phaseolus vulgaris (Liao and

Barber, 1971).

The conversion activity is absent in mutant extracts in

vitro, if absent in vivo, this would explain the lack of

fucose in living cells and the correction by exogenous

fucose. Taken together, all these results point to the

conversion pathway as the site of the lesion in HL250.

Furthermore, the defect seems to be in the first step of the

conversion of GDP-mannose to GDP-fucose because no

radioactivity was recovered after hexokinase treatment. If

the 4,6-dehydratase was active, the product would have

eluted from the ion-exchange column after hydrolysis and

phosphorylation by hexokinase (see figure 2-2).




























Figure 2-3. Conversion of GDP-mannose to GDP-fucose by normal
and mutant strains. Closed circles, Ax3; open circles,
HL250. Results are the average of two determinations.

Panel A. Effect of protein concentration on conversion. 30
min. assay, 7.5 pM GDP-[3H]mannose.

Panel B. Effect of GDP-mannose concentration on conversion.
30 min assay, 750 pg protein for each strain.

Panel C. Apparent Michaelis constant for GDP-[3H]mannose,
determined for Ax3 conversion. Apparent K was determined
to be 14.1 pM and apparent V 18.3 nmol/mg protein/30 min
by the Lineweaver-Burk double reciprocal plot method.


























0 200 400 600 800
ig protein


0 5 10 15 20 25
GDP-mannose (uiM)


0.3--
c



0.2



0.1 -



0.0-"^
-0.09


-0.00 0.09 0.18 0.27
1/[S]








38

Specific activity of fucose pools. A lesion in the

GDP-mannose to GDP-fucose conversion pathway would render

the mutant defective in macromolecular fucosylation when

grown in the absence of fucose, as was shown earlier. Such

a scenario would require that the GDP-fucose inside the cell

be derived from the salvage pathway fed exclusively by

fucose from the extracellular media. In the case of the

Ax3, however, there would be a contribution of GDP-fucose

derived from the conversion pathway. If the mutation in

HL250 was indeed in the GDP-mannose to GDP-fucose

conversion, macromolecular fucose of cells grown in the

presence of L-[3H]fucose would have the same specific

activity as the fucose present in the media. To test this

hypothesis, mutant and normal cells were grown in fucose-

free defined media supplemented with [3H]fucose. Whole cell

preparations were then assayed for fucose content as

described above and the specific activity expressed as the

radioactivity present in the fucose peak divided by the

amount of fucose detected. As expected, the macromolecular

pool of the mutant had essentially the same specific

activity as the fucose in the medium. In contrast, the

specific activity in Ax3 was diluted approximately 400-fold

compared to the medium (table 2-3). These results confirmed

that HL250 derived its intracellular fucose from the salvage

pathway. Meanwhile it appears that in Ax3 the contribution






























Table 2-3. Specific activities of fucose.


fucose concentration


50 *M
0.1 PM


cpm/nmol fucose
medium macromolecular


1. 1x105
1.8x107


9.5x10'
4.7xlO4


Cells were grown for 3 days in FM media in the presence of
6x106 dpm/ml of [3H]fucose supplemented with non-radioactive
fucose to yield the noted concentration of fucose in the
media. Specific activity was determined as described in
Materials and Methods.


strain

HL250
Ax3









40

of fucose by the salvage pathway is minimal, 1/400 of the

total content. These studies agree with earlier reports on

fucose metabolism, where the majority of fucose in mammalian

cells is derived from the conversion of GDP-mannose to GDP-

fucose (Yurchenko et al., 1978).



Discussion

HL250 failed to express a fucose-dependent epitope

recognized by the mAb 83.5. However, it expressed SP96, one

of the polypeptides that bears the carbohydrate epitope

recognized by 83.5. These results were reproduced by

Western blot (GonzAlez-Yanes et al., 1989) and indirect

immunofluorescence. Interestingly, the immunofluorescence

microscopy studies showed that the mutant is not defective

in its ability to package SP96 in vesicles or in the

targeting of the glycoprotein to the spore coat. Similar

results were reported for other proteins in another

Dictyostelium glycosylation mutant (Aparicio et al., 1990;

West and Loomis, 1985). Measurements of fucose content of

cells and spores demonstrated that the mutant contained

almost undetectable amounts of fucose, in contrast to Ax3

which contained macromolecular-associated fucose in both

cell types.

Once HL250 was identified as having a mutation that

resulted in decreased macromolecular fucose, I tried to

identify the nature of the lesion. It was speculated that








41

the mutant HL250 may have a defect in (1) fucosyltransferase

activities, (2) endogenous acceptors for

fucosyltransferases, (3) transport of GDP-fucose into

microsomal vesicles, and/or (4) synthesis of GDP-fucose. In

vitro microsomal extracts of normal and mutant cells were

active in the transfer of [14C]fucose from GDP-[14C]fucose to

endogenous acceptors and the activity was latent (see

Chapter IV), so it was reasoned that fucosyltransferases may

be normal and probably the uptake of GDP-fucose by vesicles

was not impaired, so the lesion might be at another point in

the fucosylation pathway. Since fucose is normally added as

a terminal modification, the fucose minus phenotype could be

the result of a lack of formation of acceptors for the

fucosyltransferases (Stanley, 1984; Hirschberg et al.,

1982). HL250 expresses levels comparable with Ax3 of other

carbohydrate epitopes and has normal neutral monosaccharide

composition (Gonzdlez-Yanes et al., 1989; West et al.,

1986), for these reasons it was speculated that the defect

was not in an earlier step of glycosylation but it involved

the fucosylation pathway directly.

The glycosylation defect in HL250 appears to result

from an inability to produce GDP-fucose. I have found that

the GDP-mannose to GDP-fucose conversion activity in vitro

is reduced to undetectable levels in mutant cell extracts.

The fact that there is partial rescue when the cells are

grown in the presence of fucose, suggests that the cells are








42

producing GDP-fucose via the salvage pathway and that the

rest of the fucosylation machinery is probably normal.

Earlier studies have reported the phenotypic reversion of

mammalian mutants with a defective GDP-mannose to GDP-fucose

conversion pathway when the cells were supplied with

extracellular fucose (Ripka and Stanley, 1986; Reitman et

al., 1980). However, Ripka and Stanley (1986) used the

recovery of lectin sensitivity as a marker for phenotypic

reversion, but did not report measuring the fucose content

of the cells or show the data for lectin binding compared to

the parental strain. Reitman et al. (1980) showed that a

mouse lymphoma cell line which has a defect in the

conversion of GDP-mannose to GDP-fucose was defective in pea

lectin binding compared to the parental cell line. The

ability to bind pea lectin was restored to wild type

parental cell line levels after culturing in 10 mM fucose.

Fucose is an important determinant in the carbohydrate-

binding specificity of pea lectin (Kornfeld et al., 1981).

It is important to note that the mutant mouse lymphoma cell

line had approximately one fifth the amount of fucose and

one third the number of high affinity lectin-binding sites

as the parental line, indicating that mutant cells were

salvaging fucose from the medium, or that the mutation was

only partial. The evaluation for phenotypic reversion of

HL250 is more rigorous since it demands the expression of a








43

carbohydrate epitope and measures total levels of fucose

from a cell that was grown in fucose-free media.

The specific activity of the medium was compared to the

specific activity of the intracellular macromolecular

fucose. Consistent with a lesion in the GDP-mannose to GDP-

fucose conversion pathway, the mutant cells relied on

extracellular fucose as their only fucose source. In

contrast, extracellular fucose only contributed to a small

fraction of the total Ax3 fucose pool. This is useful

because it means that radioactivity from cells grown in

[3H]fucose can be used as a direct measure of fucosylation

in HL250.

In conclusion, even though HL250 has a severe

glycosylation lesion which renders it unable to carry out

fucosylation when grown in the absence of fucose, the strain

is able to grow, develop, and form spores. To my knowledge,

there are no previous reports in the literature of

eukaryotic cells defective in the GDP-mannose to GDP-fucose

conversion pathway that can survive in fucose-free media.

The fucosylation mutants reported are cell lines that have a

functional salvage pathway maintained in culture in the

presence of animal serum, so they can synthesize GDP-fucose

from the fucose present in the cell culture media (Ripka and

Stanley, 1986; Reitman et al., 1980). For this reason,

these investigators were unable to totally deprive the

mutants of fucose, as I am able to do with Dictyostelium.








44

Other lower eukaryotes, such as yeast, do not carry out

fucosylation (Kukuruzinska et al., 1987) so the existence of

a Dictyostelium fucosylation mutant could be very important

to study fucosylation. In any event, HL250 has already

served as a very useful tool in which to study fucosylation

events, as will be evident in the following chapters.















CHAPTER III
IDENTIFICATION OF A CYTOSOLIC FUCOPROTEIN


Introduction

There is some evidence that fucoconjugates are present

in the nucleus and cytosol (Hart et al., 1989a; Chapter I).

However, virtually nothing is known about the structure or

biosynthesis of these fucosylated macromolecules. Most

studies have limited themselves to reporting the existence

of evidence for nuclear or cytoplasmic glycoproteins, but

have not gone further to characterize the sugar-peptide

linkage or compare it with material derived from the

secretary pathway. As discussed in Chapter II, there is a

conditional fucosylation mutant, HL250, that can be readily

labelled when grown in radioactive fucose. Using this

strain, I have identified a fucoprotein that fractionated

with the cytosol and appeared to be the major fucosylated

species in the cytosol. The oligosaccharide-peptide linkage

was characterized and the fucoprotein was compared with

fucosylated material derived from vesicles, and

differentiated based upon several criteria.








46

Materials and Methods

Materials

L-[(5,6)-3H]-fucose (60 Ci/mmol) was obtained from

American Radiochemical Corporation and D-[(2,3)-3H]mannose

(24 Ci/mmol) from New England Nuclear. TS-1 was purchased

from Research Product International; POPOP was from

Mallinckrodt; SDS, leupeptin, aprotinin, phenyl

methylsulfonyl fluoride, Triton X-100,

dimethyldichlorosilane, MES, all nitro-phenyl substrates,

mannose-6-phosphate, bovine serum albumin (fraction V), blue

dextran, bromo phenol blue, and trypsin were from Sigma;

glycine, benzene, ammonium acetate, PPO, and toluene were

from Fisher. The concentrations of Triton X-100 and NP-40

are expressed as v/v, all others are expressed as w/v,

unless specified otherwise.



Strains and Conditions of Growth

Dictyostelium discoideum strains Ax3 (from S. Free) and

HL250 (from W.F. Loomis) were grown on HL-5, a complete

medium that contains glucose, yeast extract, and proteose

peptone (Loomis, 1971). Ax3 is the normal strain and HL250

is a mutant obtained from Ax3 by N-methyl-N'-nitro-N-

nitrosoguanidine mutagenesis (Loomis, 1987). HL250 lacks

the enzyme activity that converts GDP-mannose into GDP-

fucose which results in a lack of cell fucose (Gonzalez-

Yanes et al., 1989; also see Chapter II). In all








47

experiments cells were collected at the logarithmic growth

phase, with a cell density of 1 to 9 x 106 cells/ml. For

metabolic labelling experiments, cells were grown for 4-6

doublings in 2-20 pCi/ml (0.03-0.26 pM) of L-[3H]-fucose in

FM medium, a minimal defined medium that lacks fucose

(Franke and Kessin, 1977).



Cell Lysis and Fractionation

Logarithmically growing amoebae were harvested and

washed in 50 mM MES (pH 7.4) and resuspended to a

concentration of 2x108 cells/ml in the lysis buffer

consisting of 0.25 M sucrose, 50 mM MES buffer (pH 7.4)

supplemented with the protease inhibitors leupeptin (10

pg/ml), aprotinin (10 pg/ml), and PMSF (1 mM). When

specified, cells were fractionated in the presence of a

comprehensive cocktail of protease inhibitors which have

been developed for the isolation of various proteolitically

sensitive proteins in Dictyostelium (Goodloe-Holland & Luna,

1987; Stone, et al., 1987). All steps were carried out at

0-4C. At once, the cells were gently lysed by forced

passage through a 5 pm nuclepore polycarbonate filter, with

a pore diameter slightly smaller than the diameter of the

cells (Das and Henderson, 1986). This method routinely

yielded more than 99% cell breakage as assessed by contrast

phase microscopy. The lysate was clarified from unbroken

cells and nuclei by a 2k xg centrifugation for 5 min, then








48

it was centrifuged at 100k xg for 1 hour, unless otherwise

specified. The pellet (P100) was resuspended in lysis

buffer by pipetting to the same volume as the supernatant

(S100). For lysing vesicles, the P100 was sonicated using a

Branson Sonifier Cell Disrupter 185.

Slug cells were plated for development as described

earlier (West and Erdos, 1988), and harvested in buffer of

Berger and Clark (as described in West and Brownstein, 1987)

supplemented with 20 mM EDTA. Cells were dissociated in

this buffer by passing 20 times through a long, 9 inches,

pasteur pipette, followed by passing 20 times through a 23-

gauge needle. EDTA was washed by resuspending cells in 50

mM MES, pH 7.4, titrated with NaOH. Cells were resuspended

in lysis buffer and immediately lysed by passage through a 3

pm nuclepore polycarbonate filter (Das and Henderson, 1986).

Cell lysates were then treated as described above for

vegetative cells.



Gel Electrophoresis and Western Blotting

SDS-PAGE was carried out under reducing conditions

essentially as described in West & Loomis (1985). Samples

were resolved by 7-20% acrylamide linear gradient gels or

15% acrylamide gels and, initially, molecular weight

assigned using low MW markers kit (Sigma). In later

experiments, trypsin was used as a molecular weight marker.

Following electrophoresis, the gels were cut immediately








49

and/or stained and destined and then cut into either 2.2 mm

or 0.5 cm slices. Gel pieces were shaken and swollen

overnight in a scintillation cocktail composed of 111.1 ml

of tissue solubilizer (TS-1), 6.0 g PPO, 0.15 g POPOP, and

20 ml dH2O to 1 1 of toluene. Gel slices were counted and

recounted until dpm were determined to be stable, usually 1-

2 days later. For gel-purified material, the sample was run

in a 7-20% linear gradient gel and the 21 kD area

(approximately 1 cm below trypsin) cut out and electroeluted

overnight using a Bio-Rad electroeluter following

manufacturer's directions, except the Laemmli

electrophoresis buffer used for SDS-PAGE (West and Loomis,

1985) was used instead of the recommended volatile buffer to

avoid alkaline hydrolysis. Western blotting was carried out

as previously described (West and Loomis, 1985).


Partial Purification of FP21 by Anion Exchange
Chromatography

In preliminary studies, FP21 was partially purified by

fractionation of metabolically labelled S100 fraction on a

TSK DEAE-5PW 8 x 75-mm HPLC anion-exchange column (LKB)

preequilibrated with 10 mM NH4Ac, pH 7.0. Sample was

dialyzed against 2 1 of 10 mM NH4Ac, pH 7.0, for several

hours, and clarified by centrifugation at 10k x g for 10 min

prior to injection. Protein was eluted using an increasing

linear gradient (10 mM to 1 M NH4Ac, pH 7.0) for 40 min at a

rate of 0.75 ml per min, and the majority of FP21 was found









50

to elute at 0.5 M input buffer concentration. Fractions

were analyzed by SDS-PAGE and counting of the gel slices.



Protein Concentration Assay

The Bio-Rad protein assay was used for determination of

protein concentration, and bovine serum albumin used as

standard.



Enzyme Assays

a-glucosidase-2 assays contained 100-300 yg protein,

8.6 mM p-nitrophenyl-a-D-glucoside, 0.1% Triton X-100, in 21

mM citrate-phosphate buffer (pH 7.5) at 37 (Borts and

Dimond, 1981). Reaction was stopped after 1 hr by addition

of Na2CO3 to a concentration of 0.5 M and the absorbance

read at 420 nm. Glucose-6-phosphatase and mannose-6-

phosphatase were measured by release of phosphate from

mannose-6-phosphate, which has been previously shown to be a

suitable substrate for both enzymes (Arion et al., 1976).

Reaction mixtures contained 100-300 pg protein, 1 mM MgCl2,

2 mM mannose-6-phosphate, 0.1% Triton X-100 in 10 mM MES

titratedd with NaOH to a pH of 7.4) in a volume of 200 p1.

After 20 min incubations at 30 reactions were stopped by

adding 200 M1 20% ice-cold TCA (Snider et al., 1980). Tubes

were centrifuged at 14k rpm, for 10 min in an Eppendorf

table top microfuge and aliquots of the supernatants were

assayed for Pi by the method of Chen et al. (1956). Acid









51

phosphatase was assayed as described in McMahon et al.

(1977) except that Triton X-100 was included at a

concentration of 0.1% and absorbance was measured at 420 nm,

instead of 400 nm.



PNGase F Digestion

Gel-purified FP21 (3000-8000 dpm) was boiled in 0.5%

SDS in water for 3 min. For digestion, the protocol of

Tarentino et al. (1985) was followed. The sample was

incubated in 104 mM sodium phosphate, pH 8.6, 10 mM EDTA, 10

mM 1,10-phenanthroline (stock solution of 100 mM in

methanol), 2% NP-40, 0.21% SDS, and 20 U/ml PNGase F

(Boehringer-Mannheim) in 300 pi for 22 h at 37. Fetuin (B-

grade, Calbiochem) and RNAse B (Sigma) were treated

identically as controls and digestion was quantitative, or

nearly quantitative, as determined by shifts in molecular

weight in SDS-PAGE. In one trial FP21 was digested with

trypsin prior to PNGase F digestion by incubating gel

purified FP21 in 0.8 mg/ml trypsin, 1 mM CaCI2 for 1 hr at

37. The reaction was stopped by boiling in the presence of

1 mM PMSF and 10 pg/ml aprotinin for 3 min.



Pronase Digestion

Glycopeptides were prepared by exhaustive pronase

digestion as described (Ivatt et al, 1984). In short, after

gel purification and electroelution, the samples were dried








52

down and resuspended in water to a volume of 200 il

containing at least 5x103 dpm. In other experiments, 200 il

of the entire P100 fraction were left intact or made 0.1%

Triton X-100. 200 il of freshly dissolved 1% pronase

(CalBiochem) in 0.1 M Tris-HCl (pH 8.0), 1 mM CaCI2, were

added at 0, 24, and 48 hours. Incubation was at 50 and a

few drops of toluene were added to prevent microbial growth.

At 72 hours the reaction was stopped by incubating in a

boiling water bath for 3 min.



Oligosaccharide Release by Alkaline-Borohydride Treatment

The oligosaccharide in FP21 was released by mild

alkaline hydrolysis under reducing conditions, also known as

B-elimination. To approximately 5x103 dpm of gel purified

protein, freshly made NaOH and NaBH4 concentrated solutions

were added in that order to yield a final concentration of

0.1 M and 1 M, respectively. Samples were incubated for 15

hours in a water bath at 45. The reaction was stopped by

the addition of acetic acid to a final concentration of 1 M.

The samples were dried down by vacuum centrifugation,

resuspended once in 1 ml 100 mM HAc, dried again, and

resuspended twice in methanol and stored dry at -80 until

ready to use.

The oligosaccharide was also released by strong

alkaline-borohydride treatment. The method is that of Zinn

et al. (1978) and very similar to the procedure for 3-









53

elimination, with some exceptions: NaOH and NaBH4 were

present at a final concentration of 1 and 4 M, respectively,

and samples were incubated at 80 for 24 h. Reaction was

terminated by diluting the sample twofold with water and

adding acetic acid to a final concentration of 4 M. Borate

salts were removed by methanol evaporation as described

above.



P-4 Column Fractionation

Dry samples from B-elimination and strong alkaline-

borohydride treatment were resuspended in 800 4I of 50 mM

pyridinium acetate (pH 5.5). Pyridinium acetate was made in

the hood by mixing in water, to a final volume of 2 liters,

8.06 ml pyridine, 2.17 ml glacial acetic acid. After the

previous reagents were dissolved, 0.4 g of sodium azide was

added. The solution had a final pH of approximately 5.5.

The buffer was degassed and stored under chloroform

atmosphere. Samples from pronase digestion were centrifuged

for 5 min at 14k rpm on a Eppendorf table top microfuge and

the supernatant taken for P-4 chromatography.

Oligosaccharides and glycopeptides were fractionated in a

0.9 cm x 1 m BioGel P-4 column (-400 mesh) equilibrated with

50 mM pyridinium acetate (pH 5.5) as the mobile phase.

Prior to pouring, the column was acid washed overnight and

siliconized by coating with 1% (v/v) dimethyldichlorosilane

in benzene for 10 min. The column was calibrated with








54

glucose oligomers used as standards (Yamashita et al., 1982)

that were derived from a dextran hydrolysate which was

reduced with NaB3 H4 (kindly provided by J. Baezinger).

Twelve drop fractions (approximately 250 pi) were collected

and counted using ScintiVerse LC (Fisher Scientific). All

runs were performed at 37. Recovery varied somewhat

between runs, but it was between 30-80% of the loaded

radioactivity. The void volume (Vo) was determined with

blue dextran (at a concentration of 0.2%), and the inclusion

volume (Vi) with either bromo phenol blue (0.2%) or

[3H]mannose (approximately 2,000 dpm). The relative elution

coefficient (Rev) for each component was determined from the

elution volume (Ve): Rev = (Ve-Vo)/(Vi-Vo).



Results

Analysis of Cellular Fucoproteins by SDS-PAGE

HL250 amoebae were grown in minimal defined media

supplemented with L-[ 3H]fucose. During logarithmic growth

phase, cells were harvested, washed, and resuspended in 0.25

M sucrose buffer supplemented with protease inhibitors.

Immediately, the cells were lysed and the lysate was

clarified from unbroken cells and nuclei by centrifugation

at 2k x g for 5 min. The resulting supernatant from this

spin was centrifuged at 100k x g for 1 hour. Both fractions

were analyzed by SDS-PAGE and the gels stained, cut into 2.2

mm slices, and counted. While total protein distributed in








55

a ratio of almost 1:1 (P100:S100), the specific activity

(expressed as dpm/mg of protein) distributed roughly in a

6:1 ratio (P100:S100) (table 3-1). The majority of the

radioactivity fractionated with the P100. However, the

amounts of radioactivity recovered in the S100 were

unexpected; 36% of the radioactivity in the S100 (this value

varied in individual experiments from 30-68%) migrated as

one peak at the 21 kD position, slightly ahead of trypsin,

which was used as a molecular weight marker. On the other

hand, the P100 showed two main broad peaks, one at 10-25 kD

and another at 58-84 kD with 10% of the total radioactivity

in the P100 migrating at the 21 kD level (figure 3-1). The

proteinaceous nature of the S100 fucoprotein was confirmed

by digestion with the proteases trypsin and pronase with

quantitative recovery of radioactivity at lower MW positions

in unfixed gels (results from pronase digestion shown in

figure 3-2). The S100 fucosylated protein has been called

FP21 for fucoprotein of 21 kD molecular weight.

The abundance of FP21 was estimated based on the

specific activity of fucose and determined to be 103

molecules in FP21, assuming one fucose molecule per molecule

of FP21. Based on the dilution of fucose specific activity

in Ax3 (from table 2-3, Chapter II), there would be 4 x 105

molecules in Ax3. If there is one fucose per copy of FP21,

and all FP21 molecules are fucosylated (evidence for

quantitative FP21 fucosylation in Ax3 will be presented in



























Table 3-1. Distribution of protein and radioactivity in S100
and P100 fractions.


S100


total protein
(equivalents)


P100


0.95


specific activity
3 days
5 days


14 dpm/pg
13 dpm/pg


88 dpm/Mg
225 dpm/pg


HL250 amoebae were grown in the presence of 2 pCi/ml of
[13H]fucose in FM media for the indicated period of time. Data
are from one representative experiment. Cells were harvested,
filter lysed, and fractionated into an S100 and P100.





























Figure 3-1. Incorporation of [ 3H]fucose into macromolecular
species of the S100 and P100.

HL250 amoebae were metabolically labelled with 2 pCi/ml of
[3Hjfucose, lysed, fractionated into an S100 and P100, and
subjected to 7-20% linear gradient SDS-PAGE; the gel was
sliced into 2.2 mm pieces and counted. 100 pg of protein
were electrophoresed for the S100 and P100, respectively.
Open circles, P100; closed circles, S100; arrow, migration
of trypsin.























2000 600


P~- 500
1600- 500

] '-400
0n 1200- n
o 0
S-300

800n 2
n 1I200


400 1 0-10


0 -
0 10 20 30 40 50

gel slice
































Figure 3-2. Proteinaceous nature of FP21.

Approximately 1,500 dpm of metabolically [3H]fucose-labelled
FP21 from HL250 was gel purified, as described in Materials
and Methods, and subjected to either a mock or pronase
digestion. Resulting digests were electrophoresed on a 15%
SDS polyacrylamide gel, which was then sliced into 0.5 cm
pieces and counted. Open circles, FP21; closed circles,
FP21 digested with pronase.








60











160
trypsin dye front



120




o_ 80




40-


0 I I I I I I I I
0 2 4 6 8 10 12 14 16

gel slice









61

Chapter IV), and the number of copies of FP21 per cell is

not affected by the mutation, then there may be a maximum of

4 x 105 copies of FP21 per cell.



Evidence that FP21 is Endogenous to the Cytosol

Fucosylation has been shown to occur in the Golgi

apparatus in other organisms (Hirschberg and Snider, 1987;

Kornfeld and Kornfeld, 1985), so I assessed the possibility

that FP21 was a lumenal microsomal protein that leaked

during the P100 and S100 isolation procedure. To guard the

P100 from chemical lysis, the vesicles were prepared in a

cocktail of protease inhibitors that contained additional

inhibitors from those utilized in the standard fractionation

protocol, and which have been developed for the isolation of

various proteolitically sensitive proteins in Dictyostelium

(Goodloe-Holland & Luna, 1987; Stone, et al., 1987). To

address the possibility of mechanical disruption of the

vesicles, the P100 was disrupted by sonication and

recentrifuged at 100k x g for 1 h. Approximately 11% of the

radioactivity was released and the supernatant of this

centrifugation was analyzed by SDS-PAGE as described above

(figure 3-3). Although several radioactive peaks were

present in the supernatant of the second centrifugation, the

radioactivity profile was different from the S100 suggesting

that FP21 is not a protein released by disruption of the

vesicle fraction. Although radioactivity which comigrated





























Figure 3-3. Comparison of S100 and releasable P100 components.

HL250 amoebae were metabolically labelled with 2 pCi/ml of
[13H]fucose, lysed, fractionated into an S100 and P100. The
P100 was sonicated and recentrifuged. After centrifugation,
the resulting supernatant was examined by slicing 7-20%
linear gradient SDS-PAGE into 2.2 mm and counting the gel
pieces (panel B). Included for comparison, is the profile
from the S100 radiolabelled species from the same
preparation run on the same gel (panel A). 50 Mg of protein
were electrophoresed for the S100 and P100, respectively.
Arrow, migration of trypsin.































0 10 20 30 40 50
gel slice



200
B

150 -

0.
0 100


50


0"
0 10 20 30 40 50
gel slice








64

with FP21 was observed in the P100-derived supernatant, this

material was a minority of the radioactivity released, and

probably reflected the general heterogeneity of the P100

vesicle contents. Less than 1% of the total cell

radioactivity that migrated at the 21 kD molecular weight

position was released from the P100 by sonication,

indicating that the remaining FP21 is recovered in the S100.

In a different approach, P100 from in vivo [3H]fucose

labelled cells was mixed with unlabelled post-nuclear

supernatant and recentrifuged (table 3-2). More than 98% of

the radioactivity sedimented with the P100, suggesting that

once associated with the P100, radioactivity is not lost,

unless vesicles are purposely disrupted, as in sonication.

The enzymes a-glucosidase-2, glucose-6-phosphatase, and

acid phosphatase have been used as markers of the

endoplasmic reticulum, Golgi apparatus and endoplasmic

reticulum, and lysosomes, respectively (Borts and Dimond,

1981; McMahon et al., 1977). To examine the distribution of

these enzyme markers in the high speed fractions, HL250

amoebae were harvested, homogenized, fractionated into an

S100 and P100 and assayed for activity of the different

marker enzymes. As seen in table 3-3, the majority of the

activity was recovered in the P100, suggesting minimal

contamination of the S100 by vesicles containing these

enzymes. Less than 7% of the total a-glucosidase-2 activity



























Table 3-2. Radioactivity recovered in the second S100 after
different P100 treatments.

condition % radioactivity recovered

untreated 2.2%
sonicated 11.3%
mixed* 1.4%


Cells were labelled in vivo by growing in 2 MCi/ml of
[3H]fucose in FM media for 3 days, lysed, and fractionated
into an S100 and P100. The P100 was then subjected to
different treatments and recentrifuged at 100k x g for 1 hr.
The S100 from this second centrifugation was analysed for
radioactivity. *P100 from metabolically labelled cells was
mixed with unlabelled post-nuclear supernatant from cells
grown in FM media and recentrifuged at 100k x g for 1 hr.






























Table 3-3.
fractions.


Distribution of markers among S100 and P100


a-glucosidase-2
acid phosphatase
glucose-6-phosphatase


S100

6.5%
12.8%
n.d.


P100

93.5%
87.2%
100%


HL250 amoebae grown in HL-5 were fractionated into S100 and
P100, and assayed for activity as described in Materials and
Methods. Activity expressed as percentage of total activity
detected in both fractions, n.d., not detectable.








67

was found in the S100; mannose-6-phosphatase was only

detectable in the P100. More than 87% of the activity of

the lysosomal enzyme acid phosphatase was detected in the

P100. In Dictyostelium, this enzyme has been shown to be a

soluble lumenal lysosomal protein (Dimond, et.el., 1981).

Further evidence that P100 vesicles are stable, closed

structures comes from earlier studies from the laboratory.

Prespore proteins SP75 and SP96 sediment at 100k x g unless

cells are sonicated (West and Erdos, 1988). These spore

coat proteins are contained in secretary vesicles (Erdos and

West, 1989; West and Erdos, 1988), which appear to be intact

since they are resistant to proteolysis by Proteinase K

unless they are treated with 0.1% Triton X-100 (West et al.,

1986; Q.H. Yang and C.M. West, unpublished data). In other

studies, N-acetyl glucosaminyltransferase activity was

inhibited by EDTA in the P100 fraction only when assayed in

the presence of detergent, suggesting that Golgi-like

vesicles in the P100 were closed (R.B. Mandell and C.M.

West, unpublished observations)



FP21 is Unrelated to Other Known Cytoplasmic Proteins

The possible relationship of FP21 with other known

Dictyostelium proteins was investigated. I examined the

reactivity of antisera raised against discoidin I and II

(Erdos and Whitaker, 1983) and against gp24 (Knecht et al.,

1987) for FP21. On Western blots, antiserum against gp24








68

recognized a band that migrated with a slower mobility than

metabolically labelled FP21 (not shown). The possibility of

FP21 being the lectin discoidin was examined, since it is

primarily present in the cytosol and exhibits weak affinity

for L-fucose (Erdos and Whitaker, 1983; Bartles and Frazier,

1980). Metabolically labelled FP21 from HL250 was

electrophoresed in a 15% SDS polyacrylamide gel, while a

replicate lane was blotted onto nitrocellulose paper and

immunoprobed using an anti-discoidin antiserum. The

antiserum recognized a band of higher MW than FP21 with

mobility slower than trypsin, reproducing results reported

by others where discoidin I was shown to have a slower

mobility than trypsin in 15% SDS polyacrylamide gels

(Kohnken and Berger, 1987). Migration of purified discoidin

in SDS-PAGE differed upon boiling of the sample, migrating

as a tetramer (ca. 100 kD) when samples were not boiled,

whereas metabolically labelled FP21 was found to migrate as

a discrete peak of radioactivity ahead of trypsin regardless

of boiling (Q.H. Yang and C.M. West, unpublished data).

FP21 could be partially purified by HPLC DEAE

chromatography. Metabolically labelled S100 was

fractionated on an anion exchange column and FP21 recovery

monitored by counting of SDS-PAGE slices. FP21 eluted at

0.5 M NH4Ac with an increase of 24-fold the specific

activity relative to the starting sample (data not shown).

Discoidin eluted earlier than FP21 from the HPLC DEAE column








69

and no radioactivity was found associated with discoidin,

suggesting it does not bind to discoidin during

purification. I conclude that FP21 is not related to

discoidin or any discoidin isoforms, or gp24, and does not

bind to any discoidin isoforms.



Oligosaccharide Studies
FP21 from Ax3 and HL250 yield similar size
glycopeptides after pronase digestion. Pronase digested

gel-purified FP21 that had been metabolically labelled from

Ax3 and HL250 were compared (figure 3-4). More than 50% of

the radioactivity from both sources eluted as a major peak

with a relative elution coefficient (Rev) of 0.42 and 0.43,

respectively (ca. 5.5 glucose units). In the case of FP21

derived from Ax3, the rest of the radioactivity eluted

earlier in the void volume and distributed into minor peaks.

The digestion products from HL250 FP21 yielded a major peak,

with the rest of the radioactivity eluting earlier, and less

than 10% eluting after the major peak, although some

variability was observed in different runs regarding the

minor peaks. Radioactivity eluting at an earlier position

than the major peak may be explained by incomplete

digestion. The minor amount of radioactivity that eluted at

a later position may be due to breakdown of the

oligosaccharide. These results indicate that the main

glycopeptides derived from Ax3 and HL250 have the same




























Figure 3-4. Gel filtration chromatography of FP21 glycopeptides.

Ax3 and HL250 vegetative cells were metabolically labelled
with 2 pCi/ml of [3H]fucose. Cells were lysed, fractionated
into an S100 and P100, and FP21 isolated by SDS-PAGE and
electroelution from the S100. The samples were exhaustively
digested with pronase and analyzed by BioGel P-4 gel
filtration. Data obtained from one representative
experiment.

Panel A. Glycopeptides derived from Ax3 FP21, arrow
identifies major peak with a Rev of 0.42. Vo, 32; Vi, 142.

Panel B. Glycopeptides derived from HL250 FP21, arrow
identifies major peak with a Rev of 0.43. Vo, 26; Vi, 166.



















600-O


450


n 300 -
Q Vo jVi

150 V



0 20 40 60 80 100 120 140
fraction



240 -
B

180-


120

Vo Vi

60-


0'1
0 20 40 60 80 100 120 140 160
fraction








72

sizes, and suggest that both strains form in vivo the same

oligosaccharide when grown in fucose-containing media.



Oligosaccharide in FP21 is 0-linked. An enzymatic and

a chemical approach were used to determine whether the

fucose-containing oligosaccharide in FP21 is N-linked or 0-

linked. I first tried the enzyme PNGase F. The minimum

requirement of PNGase F is a di-N-acetylchitobiose core

(Chu, 1986; Tarentino et al., 1985). This enzyme has a

broad specificity and can cleave most asparagine linked N-

glycans (including high mannose and complex multibranched

oligosaccharides) provided they are not located at the amino

or carboxy termini (Chu, 1986). Gel-purified FP21 from

metabolically labelled Ax3 was digested with PNGase F. As

shown in figure 3-5, the fucose label eluted in the void

volume, while control substrates were quantitatively

digested as determined by SDS-PAGE (not shown). FP21

trypsinized prior to digestion with PNGase F also eluted in

the void volume (not shown). The inability of PNGase F to

release radioactivity from FP21 suggested that either the

fucose-containing oligosaccharide was not N-linked or the

oligosaccharide was insensitive to the enzyme.

I then considered the possibility that the

oligosaccharide in FP21 was 0-linked. Metabolically

labelled gel purified FP21 was subjected to mild alkaline,

reducing conditions, to release intact 0-linked































Figure 3-5. Gel filtration chromatography of PNGase F digests.

Ax3 vegetative cells were metabolically labelled with 2
PCi/ml of [3H]fucose, an S100 was prepared, and FP21 was
isolated by SDS-PAGE and electroelution. FP21 digested with
PNGase F as described in Materials and Methods, and analyzed
by BioGel P-4 gel filtration. Nine drops fraction were
collected, instead of the usual 12 drops; [3H]mannose was
used to determine Vi.





















1200

Vo

900




600- i




300-



0-
0 20 40 60 80 100 120 140 160 180

fraction








75

oligosaccharides (B-elimination). Under the conditions

employed, N-linked oligosaccharides are insensitive to

chemical release (Biermann, 1988). More than 95% of the

radioactivity was released from Ax3 (Rev 0.48) and HL250

(Rev 0.49) in vivo labelled FP21 and it was resolved as one

peak with a size of 4.8 glucose unit (figure 3-6, panels A

and B). The oligosaccharide appeared to have been released

by B-elimination and thus is concluded to be 0-linked.

Consistent with the pronase digestion studies reported

above, Ax3 and HL250 yielded a similar size oligosaccharide,

supporting the idea that both strains produced the same

oligosaccharide. The slightly smaller size of the

oligosaccharide compared to the glycopeptide is consistent

with the notion that the glycopeptide consisted of one or

more amino acids and the oligosaccharide chain. If the

glycopeptide resulting from pronase digestion consisted of

the oligosaccharide attached to two or more amino acids, it

was possible that mild alkaline hydrolysis under reducing

conditions resulted in further hydrolysis of the remaining

polypeptide backbone yielding one amino acid and the

oligosaccharide. This would yield a smaller radioactive

species, with concomitant increase in Rev. To investigate

this possibility, I employed harsher chemical conditions.

In vivo labelled FP21 from Ax3 was gel purified and

subjected to strong alkaline hydrolysis in the presence of

sodium borohydride, which cleaves N- and 0-linked



























Figure 3-6. Gel filtration chromatography of FP21
oligosaccharides.

Vegetative cells were metabolically labelled with 2 yCi/ml
of [3H]fucose, an S100 was prepared, and FP21 was isolated
by SDS-PAGE and electroelution. Gel purified FP21 from Ax3
and HL250 were subjected to B-elimination or strong alkaline
hydrolysis followed by fractionation by gel filtration
chromatography. Data obtained from one representative
experiment.

Panel A. B-elimination of Ax3 FP21. Vo, 36; Vi, 141.

Panel B. B-elimination of HL250 FP21. Vo, 30; Vi, 140.

Panel C. Alkaline hydrolysis of Ax3 FP21. Vo, 38; Vi, 134.








6000
A


4000

0
Vo I Vi






0 20 40 60 80 100 120 140
fraction

800-
B

600-


o 400

Vo vi





0 20 40 60 80 100 120 140
fraction

2500
C
2000


1500
0-
SVo Vi
1000


500 -

0*
0 20 40 60 80 100 120 140
fraction











oligosaccharides (Zinn et al., 1978; Biermann, 1988). The

results of this reaction are seen in figure 3-6, panel C.

More than 75% of the radioactivity was released, eluting

with a Rev of 0.49. Most of the remainder of the

radioactivity eluted in the void volume. I expected to see

a change in Rev by strong alkaline hydrolysis compared to

mild alkaline hydrolysis if mild alkaline hydrolysis did not

release the oligosaccharide, but hydrolysed the protein.

The fact that similar results are obtained by mild and

strong conditions suggested that B-elimination occurred to

release the oligosaccharide. Since mild alkaline hydrolysis

had been shown earlier not to cleave N-linked sugars

(Biermann, 1988), I conclude that the oligosaccharide in

FP21 is linked via an 0-linkage. The released

oligosaccharide eluted as an asymmetrical peak, both by mild

and strong alkaline hydrolysis. These results suggest that

there is more than one type of oligosaccharide in FP21 that

differ slightly in size. These results also suggest that

the slight heterogeneity seen in the glycopeptide size may

reflect amino acid heterogeneity.


Comparison of glycopeptides derived from vesicular and
cytosolic material. To further address the possibility of

FP21 arising by contamination of the S100 from the P100

fraction, glycopeptides derived from the Ax3 P100 were

compared to gel purified pronase digested FP21 from Ax3.

P100 derived glycopeptides were obtained in three different








79

manners. Metabolically labelled P100 was subjected to SDS-

PAGE and the material that comigrated with FP21 was gel

purified, pronase digested, and fractionated by gel

filtration chromatography. Entire P100 from metabolically

labelled cells was digested in the presence or absence of

detergent, and analyzed by BioGel P-4 gel filtration. As

seen earlier in figure 3-4, panel A, the S100 digest eluted

mainly as a single peak with a Rev of 0.42; on the other

hand, the digest from the FP21-comigrating P100 material,

fractionated as a major peak of 0.50 Rev (figure 3-7, panel

A). An analysis of glycopeptides from the entire P100

digestion showed a different elution profile from the FP21

digestion (figure 3-7, panel B). Note than in this

chromatograph there is a minor peak of 0.51 Rev, consistent

with the idea that the major peak seen in panel A is a minor

component of the entire P100 glycopeptide repertoire. Also

approximately 40% of the radioactivity eluted with the void

volume, suggesting it may be resistant to pronase digestion.

Digestion of the entire P100 fraction was carried out in the

presence of Triton X-100, to determine if solubilization of

the sample yielded a different digestion profile, by

facilitating accessibility of the enzyme to the substrates

(figure 3-7, panel C). The overall profile is similar, with

more than 30% of the radioactivity eluting at the void

volume, and a peak with a Rev of 0.51 is still a minor

component of the glycopeptides released. West et al. (1986)


























Figure 3-7. Gel filtration chromatography of P100 glycopeptides.

Vegetative Ax3 cells were metabolically labelled with 2
pCi/ml of [3H]fucose. A P100 was prepared and the 21 kD MW
material that comigrated with FP21 on SDS-PAGE was
electroeluted and pronase digested. In another assay,
entire P100 was pronase digested in the absence or presence
of 0.1% Triton X-100. After pronase digestion, samples were
subjected to BioGel P-4 fractionation. Arrow identifies the
position with a Rev of 0.51.

Panel A. Glycopeptides from 21 kD MW P100-derived material.
Vo, 34; Vi, 162.

Panel B. Glycopeptides from entire pronase-digested P100.
Vo, 36; Vi, 142.

Panel C. Glycopeptides from entire P100 digested with
pronase in the presence of Triton X-100. Vo, 32; Vi, 146.











2000
A.


1500


S1000
Q Vo Vi

500 -



0 20 40 60 80 100 120 140 160
fraction


2500


2000


1500
2
a-
S1000


500


0


0 20 40 60 80 100 120 140 160
fraction


C. v




Vvi







I I I R^


I I I I a a 1
20 40 60 80 100 120 140 160
fraction








82

have reported the existence of pronase-resistant material in

the particulate fraction of vegetative cells, so it seems

possible that the pronase-resistant material eluting in the

void volume is, or is related to, the smear previously

described, although SDS-PAGE analysis will be needed to

confirm this supposition.



Fucose is Covalently Bound to FP21

The fact that the radioactivity in FP21 was not

released by boiling in SDS/ B-mercaptoethanol, nor after

boiling in SDS under reducing conditions followed by SDS-

PAGE, suggested that 3H was covalently bound to in vivo

labelled FP21. Nevertheless, there have been reports of

covalent-bound enzymatic intermediates in the literature

(Scrimgeour, 1977). Although ES (enzyme-substrate)

intermediates are very reactive and usually cannot be

isolated without some sort of stabilization or chemical

trapping technique, the possibility that FP21 is really a

cytosolic fucosyltransferase that binds GDP-fucose or other

fucose metabolites covalently was considered. The Rev of

the radioactivity released by mild alkaline hydrolysis

suggests that it is not related to GDP-fucose or fucose

because it elutes with a different Rev than GDP-fucose or

fucose. Additional evidence that 3H is present as fucose

was presented in Chapter II, where it was shown that more








83

than 95% of the macromolecular-associated radioactivity from

metabolically labelled cells migrated as authentic fucose.



FP21 is Present in Migrating Slug Stage Cells

To investigate whether FP21 was present also in

developing cells, HL250 amoebae were plated for development

on nuclepore filters and 9 hours after plating the filters

were lifted and cells were metabolically labelled by

placement on 100 pCi of [3H]fucose. After 5 hours of

exposure to [3H]fucose, filters were lifted again, carefully

washed from H label, and cells were allowed to continue

development for two more hours. Cells were then harvested,

disaggregated, and fractionated into an S100 and P100

fractions, run in an SDS-PAGE, and the gel cut and the

pieces counted. As shown in figure 3-8, a fucosylated

macromolecule is preferentially fucosylated in the cytosol

and it has the same mobility in SDS-PAGE as FP21. Thus, it

seems that FP21 fucosylation is not restricted to the growth

phase of Dictyostelium.



Discussion

In this chapter I have presented evidence for the

existence of a fucosylated single molecular weight species,

which has been termed FP21 based on its mobility as

determined by SDS-PAGE. This protein cofractionates with





























Figure 3-8. Incorporation of [ 3H]fucose into macromolecular
species of slug stage cells.

HL250 cells were harvested, plated on filters for
development, and exposed to [3H]fucose for 5 h as described
in the text. Cells were then collected, disaggregated,
filter-lysed, and S100 and P100 fractions were prepared and
analyzed by 7-20% linear gradient SDS-PAGE. 70 pg and 24.6
pg of protein were electrophoresed for the S100 and P100,
respectively. Open circles, P100; closed circles, S100;
arrow, migration position of trypsin.