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CHARACTERIZATION OF THE HUMAN CYT19 GENE PRODUCT: AN ARSENIC
ALEX J. MCNALLY
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
Alex J. McNally
I would like to thank my mentor, Dr. David S. Barber, for all his help, guidance,
and knowledge he has given me. I would also like to thank my committee members, Dr.
Nancy Denslow, Dr. Lena Ma, and Dr. Steve Roberts, for their suggestions and advise. I
thank my fiance and my parents for their support and encouragement throughout my
TABLE OF CONTENTS
A C K N O W L E D G M E N T S ......... .............. .................................................................... iii
L IS T O F T A B L E S .................. ........ .............................................................. v i
LIST OF FIGURE S ......... ..................................... ........... vii
ABSTRACT ........ .............. ............. ...... ...................... ix
1 IN TR OD U CTION ............................................... .. ......................... ..
Sources of A rsenic .................. ............................. .. ...... ................... .
N atu ral S ou rces ............................................................... 1
A nthropogenic Sources ................................................................................... 1
Exposure and H health Effects ............................................................................ 2
E x p o su re ................................................................................. 2
H health E effects ....................................................... 3
M echanism s of Toxicity ......................................................... ...............
A rsenic B iotransform ation................................................... ............................. 6
Reduction of Pentavalent Arsenicals ............... .............................................6
M ethylation of Trivalent Arsenicals................................... ....................... 7
V ariation in A rsenic M ethylation ....................................................... .... ........... 8
Role of M ethylation in Arsenic Toxicity ............... ............................................ 10
Specific A im s of R esearch......................................................................................... 11
2 MOLECULAR CLONING AND CHARACTERIZATION OF HUMAN CYT19,
AN S-ADENOSYL-L-METHIONINE:AS-METHYLTRANSFERASE FROM
H E P G 2 C E L L S .............................................. .............................................. .. 13
Introduction ............... ........... ........................ ............................13
M materials and M methods ....................................................................... .................. 14
M molecular Cloning ................................ .... ........ ...... .. ................. 14
RACE PCR .................. ....... ....... ...............15
Expression of Recombinant cytl9 ....... ......... ................... .................15
Characterization ........ ............ ........ ... ..................... 16
Confirmation of Methylated Arsenicals .................. ....... ..................17
R results and D iscu ssion ................... .................................................. .............. 18
3 IDENTIFICATION OF A SPLICE VARIANT OF HUMAN CYT19 ARSENIC
M ETH YLTRAN SFERA SE............................................... ............................. 33
Introduction .............. ... .... ............. .......... .......... ............ 33
M materials and M methods ..................................................................... ....................34
Molecular Cloning of cytl9 Splice Variants.............. .... ..................34
H um an L iv er Sam ples ..............................................................................35
qPCR of cytl9 Splice Variants............................ ..................................... 35
R results .............. ......... ............ .... ............................................36
Discussion ............ .............................................. ....... ... ...... .. 37
4 GENERAL CONCLUSIONS............................ .................................. 45
ROLE OF CYT19 IN ACUTE ARSENIC TOXICITY. IS CYT19 THE ONLY
HUMAN ARSENIC METHYLTRANSFERASE?.............. ...............49
M materials and M methods ....................................................................... ..................49
R results and D iscu ssion ..................................................................... ................ .. 50
cytl9 mRNA Knockdown by siRNA.....................................................50
Antibody Specificity and Purification............... ................................................51
F uture E xperim ents ......................... ...................... .. .. ...... .......... 5 1
L IST O F R EFER EN CE S ........................................................................... ...............57
B IO G R A PH IC A L SK E TCH ..................................................................... ..................67
LIST OF TABLES
2-1. Primers used in the PCR amplification ofcytl9 .................................................23
2-2. Kinetic analysis of the methylation activity of cytl9-WT and cytl9S81R..............23
3-1. The individual information and Shapiro's score of cytl9 exon 2 and exon 3. ........44
3-2. The amount of cytl9 and cytl9AE2 in different human liver samples and HepG2
c e lls ........................................................................... 4 4
A-1. Sequence of double stranded siRNA ............................ ........ ............. .................. 53
LIST OF FIGURES
2-1. Sequence alignment of cytl9t and Genbank sequence (accession AF226730) ......24
2-2. Sequence alignment of cytl9S81R and Genbank sequence (accession
A K 057833) ....................................................................... 25
2-3. Sequence alignment of cytl9-W T and cytl9t .....................................................26
2-4. Sequence alignments of the 5' & 3'RACE-PCR products and the Genbank
sequences (accession AK057833 and AF226730)..................... .................27
2-5. Purification of recombinant human cytl9.................................... .................... 28
2-6. The effects of As"' & MINAV concentrations ..................................................29
2-7. The effect of pH on activity ...................................... ............... 30
2-8. The effects of reductants on methylation activity ..............................................30
2-9. The effect of SAM concentration on activity ............................... .....................31
2-10. Arsenical metabolites formed after incubation with [3H]SAM and cytl9 for 30
m in at 3 7 C ........................................................................... 3 2
3-1. The hypothesized scheme of iAs methylation proposed by Cullen, McBride et
al. 1984 ....................................................................40
3-2. PCR products of cytl9 amplification................................. ...............40
3-3. Alignment of the reference cytl9 nucleotide sequence and cytl9AE2 ..................41
3-4. cytl9 isoforms ......... ......... .......... ...............42
3-5. Alignment of the reference cytl9 amino acid sequence and product of cytl9AE2
u O R F ...............................................................................4 3
A-1. Determination of the most efficient siRNA in the knockdown of cytl9 mRNA
levels assayed by qP C R ............................................................................. .... .... 54
A-2. The siRNA pool knockdown of cytl9 mRNA relative to control............................54
A-3. Determination of cell viability after siRNA knockdown by the XTT assay............55
A-4. Western blot of the specificity of the crude antisera to the antigen, purified cytl9
p ro te in ...................................... ................................................... 5 5
A-5. Western blot of the specificity of the crude antisera to cytl9 in human liver
cytosolic preparations.................. ......................... .. ......55
A-6. Western blot of the specificity of the purified antibody to the antigen, purified
cy tl9 p rotein ........................................................................................... 5 6
A-7. Western blot of the specificity of the crude antisera to cytl9 in human liver
cytosolic preparations.................. ......................... .. ......56
Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
CHARACTERIZATION OF THE HUMAN CYT19 GENE PRODUCT: AN ARSENIC
Alex J. McNally
Chair: David S. Barber
Major Department: Veterinary Medicine
Chronic arsenic exposure poses a threat to millions of people throughout the world
due to arsenic in drinking water; however the mechanisms underlying arsenic
carcinogenicity and individual susceptibility are unknown. Methylation has been
considered the primary detoxification pathway of inorganic arsenic in many species but
there is evidence that methylation may increase arsenic toxicity. It has been shown that
methylated arsenicals that contain As"' are more cytotoxic and genotoxic than either
arsenate or arsenite. Rat liver S-adenosyl-L-methionine: arsenic"'-methyltransferase has
been identified and is homologous to human cytl9, but there are species specific
differences in arsenic biotransformation and toxicity. Additionally, there is considerable
variation among humans in the rate of methylation of inorganic arsenic leading to
measurable differences in toxicity. Therefore, it is important to better understand the
enzymes that catalyze the methylation of arsenic in humans. In this study, we PCR
amplified and cloned cytl9, a putative arsenic methyltransferase from human HepG2
hepatoma cells. The PCR product was ligated into an E. coli pET expression vector with
a polyhistidine tag at the amino-terminal residue. The recombinant human cytl9 was
successfully expressed in BL21 (DE3) and purified using a nickel-nitrilotriacetic acid
metal-affinity chromatography. The recombinant protein catalyzes the methylation of
arsenite as well as monomethylarsonic acid (MMA). The specific activity of arsenite
methylation was 597 pmol/mg protein/min in a reaction mixture containing 5mM GSH, 1
mM DTT, 1 mM MgC12, 100 iM S-adenosyl-L-methionine, 50M sodium m-arsenite,
and 5 .g of S-adenosyl-L-methionine: arsenic methyltransferase in 100mM tris/100mM
sodium phosphate buffer pH 7.4 at 37 C for 30 minutes. The results suggest that the
human cytl9 gene, in fact, is translated to an S-adenosyl-L-methionine: arsenic
methyltransferase which methylates both arsenite and MMA.
Studies have shown that humans exposed to arsenic excrete variable amounts of
methylated arsenicals in the urine which may be due to differences in arsenic
methyltransferase activity. While polymorphisms in the coding region of cytl9 may
account for some of the observed variation in arsenic methylation, other mechanisms are
likely to be involved. In this study we identified an alternative splice variant of the
human cytl9 (cytl9AE2), in which exon 2 is removed creating a bicistronic transcript
that is unlikely to produce an active protein. This variant was expressed in 7 out of 7
male Caucasian human liver samples tested and in HepG2 cells. The human cytl9
appears to be alternatively spliced in many individuals and may play a role in the
observed variation in arsenic methylation seen in individuals.
Sources of Arsenic
Arsenic (As) is a member of the nitrogen group in the periodic table and is
classified as a metalloid. This metalloid is a naturally occurring element and is the 20th
most abundant element in the earth's crust . Arsenic is found in the environment as
sulfides, and complex sulfides of iron, nickel, and cobalt. The natural weathering of
rocks and soils containing various forms of arsenic contribute to its levels in the
environment. Arsenic is present in the atmosphere, aquatic environments, soils &
sediments, and in organisms. This metalloid is found naturally in rocks, geothermal
wells, minerals, and metal ores such as copper and lead. Arsenic is present in the
environment in both organic and inorganic forms and exists in four valence states, -3, 0
(elemental), +3 (trivalent), and +5 pentavalentt arsenic), however it exists mainly in the
latter two valence states. Many marine plant and animal species have naturally high
levels of As, but in organic forms that appear to cause little toxicity. The main species of
arsenic in marine animals is the arsenosugar, arsenobetaine . In general, organic forms
of arsenic are less toxic than inorganic forms of arsenic and the pentavalent inorganic
forms are less toxic than trivalent inorganic arsenic compounds.
Anthropogenic sources of arsenic stem from its use in pesticides and wood
preservatives as well as mining and smelting wastes. In the U.S., 2,200 tons of arsenic
was produced in 1985 . Since 1985, the domestic production of arsenic in the US has
ceased. However, arsenic is still used domestically and therefore is imported from
countries such as China, Japan and Mexico. In 2003, the United States was world's
largest consumer of arsenic, demanding 21,600 metric tons  Arsenic has also been
used in the production of dessicants and as growth stimulants for plants and animals.
Organoarsenicals have been shown to have both therapeutic and growth promoting
properties in poultry and swine. Arsanilic acid and its sodium salts, such as 4-
nitrophenylarsonic acid are added to pet feed . Most of this arsenic passes through
the animal and becomes part of the waste stream resulting from animal production. In
addition, arsenic has been used for therapeutic purposes and of course as a poison.
Arsenic has been used to treat syphilis, tropical diseases such as trypanosomiasis (African
sleeping sickness), yaws, amoebic dysentery and recently as an anticancer. Paul Ehrlich
developed an organic arsenical, arsphenamine, also known as Salvarsan, which was used
to treat syphilis . Arsphenamine was also believed to be effective in treating
trypanosomiasis . Arsenic trioxide has been shown to be highly effective in the
treatment of various cancers especially of acute promyelocytic leukaemia .
Exposure and Health Effects
Arsenic is present throughout our environment, in the air we breathe, the water we
drink, and the food we eat. Water contributes more to iAs exposure than food or air. On
average Americans are exposed to 50 .g per day of arsenic of which 10 .g is in the
inorganic form . People are exposed to higher than average arsenic due to living or
working around higher exposure sources. For example living or working near a
hazardous waste site can lead to exposure via the air, ingestion, or the food chain.
Arsenic is present in 47% of all sites on the National Priorities List (NPL) sites or
Superfund sites making it second only to lead as the most common contaminant of
concern . Occupational settings such as workers who use or produce arsenic
compounds: vineyards, ceramics, glass-making, smelting, pharmaceuticals, refining of
metallic ores, pesticide manufacturing & application, wood preservation, or
semiconductor manufacturing, have the potential to be exposed to higher than average
arsenic levels .
Currently, people in Taiwan, Mexico, western United States, western South
America, China, West Bengal and Bangladesh are exposed to high levels of arsenic due
to anthropogenic and/or natural contamination of potable water. It has been estimated
that 200 million people worldwide are at risk from health effects associated with this
exposure . The two most affected areas in the world are Bangladesh and West
Bengal, India; it has been estimated that around 122 million people in these areas are
exposed to groundwater arsenic concentrations above the World Health Organization
maximum permissible limit of 50g/L . The study showed that in West Bengal,
26.4% (n=10,991 tube wells) of the water samples had arsenic ranging in 100-299 [g/L.
In the U.S. the arsenic maximum contaminant level (MCL) was decreased to 10g/L by
the US Environmental Protection Agency (EPA) in January 2001. Frost et al. 
identified 33 counties in 11 states in the western United States with mean arsenic
concentrations of 10g/L or greater. In addition, from 1950-1990 there were over 60
million people in the US exposed to arsenic contaminated water exceeding 10g/L .
Ingestion of arsenic is a widespread human health problem. There are different
symptoms associated with acute and chronic arsenic exposure. Acute exposure to arsenic
can result in acute paralytic syndrome, acute GI syndrome, and even death. Oral
exposure above 60 ppm in food or water can result in death . Chronic arsenic
exposure can result in skin disorders such as hyper and hypo pigmentation, and
hyperkeratosis . It was found that even at 0.005-0.01mg/L of arsenic in water there is
an increase in the prevalence of skin lesions . In addition, chronic iAs exposure can
affect the circulatory and nervous systems leading to diseases such as Blackfoot disease.
Major organs such as the liver, kidneys, lung, bladder, and heart can be affected as a
result of arsenic cytotoxicity. There is an increase of cancer, death from cancer, and
diabetes mellitus associated with chronic arsenic exposure. Epidemiological studies
show that there is dose-response relationship between exposure to iAs and skin cancer
. A study in Taiwan and Japan demonstrated a significant association
between long-term arsenic exposure in drinking water with lung and bladder cancer. In
northern Chile an increase in mortality from bladder, lung, kidney, and skin cancer is
associated with As exposure; bladder and lung cancer showing highest increase in
mortality . A follow up study in Taiwan compared the incidence of diabetes mellitus
in an arsenic exposed population to two control areas showed an association between As
exposure and diabetes mellitus .
Mechanisms of Toxicity
The mechanism of arsenic toxicity is dependent on oxidation state. Trivalent
arsenicals, including methylated arsenicals produce toxicity by enzyme inhibition by
interactions with sulfhydryl groups in proteins  and the generation of reactive oxygen
species (ROS). For example, in vitro studies have shown that MMA111 and arsenite are
capable of inhibiting pyruvate dehydrogenase (PDH) activity in hamster kidney and
purified porcine heart PDH resulting in the subsequent blockage of adeonosine
triphosphate (ATP) production because of the disruption of the citric acid due to the
depletion of cellular citrate . Inorganic arsenicals, arsenite and arsenate, have been
shown to induce ROS and reactive nitrogen species (RNS) which result in DNA and
protein oxidative damage . Another study demonstrated that MMA"' and DMA"'
induced ROS result in DNA damage . Arsenate has been shown to interfere with
ATP production by substituting for phosphate leading to production of an unstable ADP-
arsenate complex which spontaneously hydrolyzes . This process leads to a depletion
of cellular energy due to this futile cycle.
Although there is strong evidence of the carcinogenicity of arsenic in humans, the
mechanism by which tumors are produced is unknown. Studies of arsenic carcinogenesis
have been hampered because there are very few animal models in which arsenic induces
carcinogenesis . DMA concentrations of 50 and 200 ppm have been shown to be
carcinogenic in F344 rats urinary bladder . Recent work has demonstrated the
promotion effects of inorganic arsenicals and methylated arsenicals. Inorganic arsenic
(42.5 and 85 ppm)has been shown to be a transplacental carcinogen in mice .
Organic arsenicals, such as MMA, DMA, and TMAO have been shown to act as
promoters in carcinogenesis of several rat organs . However, some of these studies
have received much criticism due to the high arsenical exposure levels used ranging from
about 50 to 400 ppm and the use of several initiators such as diethylnitrosamine, N-butyl-
N-(4-hydroxybutyl)nitrosamine, N-methyl-N-nitrosourea, dihydroxy-di-N-
propylnitrosamine and N-N'-dimethylhydrazine, prior to arsenic exposure.
Several possibilities for mechanisms of arsenic induced malignancies have been
hypothesized such as chromosomal abnormality, oxidative stress, and the promotion of
tumorigenesis. Zhao et al. , have shown that chronic low level arsenic (0-0.5[iM)
exposure will result in the malignant transformation of epithelial cells associated with
DNA hypomethylation due to depletion of SAM and aberrant gene expression. An in
vivo long-term arsenic exposure study to mice demonstrated that arsenic in potable water
can induce aberrant gene expression, global DNA hypomethylation, and hypomethylation
of the gene for the estrogen receptor-a resulting in enhanced transcription, which
cumulatively could lead to arsenic hepatocarcinogenesis . Arsenate was shown to
have a dose-dependent transcriptional induction of several different signal transduction
pathways, including the dose-response induction of several promoters and/or response
elements responsive to oxidative damage and DNA damage  which may help
understand the mechanisms of carcinogenicity for arsenic. Binet et al.  has shown
that arsenic induced apoptosis via reactive oxygen species (ROS) production occurs but,
the ROS is not produced from nicotinamide adenine dinucleotide phosphate
dehydrogenase activation. Trivalent methylated arsenicals have been shown to indirectly
cause DNA damage by ROS. One study showed that DMA"' promotes tumorigenesis
and gentoxicity via dimethylated arsenic peroxides 
Reduction of Pentavalent Arsenicals
The biotransformation of iAs alternates between the reduction of arsenate (iAsV) to
arsenite (iAs"') followed by oxidative methylation. The hypothesized scheme of iAs
methylation involves oxidative methylation and reduction:
AsVO43- + 2e As"133- + CH3 CH3As 032- + 2e CH3As"122- + CH3+
(CH3)2AsVO2- + 2e (CH3)2As"'O
Arsenic reduction must occur first before it can be methylated. Several enzymes
have been shown capable to reduce arsenic. Purine nucleoside phosphorylase (PNP) has
the ability to reduce iAsV to iAs"' in the presence of a dithiol and a purine nucleoside
guanosinee or inosine) in vitro [33, 34]. However, studies performed by Nemeti et al.
2003 showed that PNP does not play a role in iAsv reduction in vivo .
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the presence of glutathione
(GSH) and NAD has the ability to reduce iAsV to iAs"' in human red blood cells and rat
liver cytosol [36, 37]. A human arsenate reductase was discovered capable of reducing
arsenate but not methylarsonic acid (MMAV) . Zakharyan et al 1999 presented an
enzyme from rabbit liver capable of reducing MMAV, arsenate, and dimethylarsinic acid
(DMAv) in the presence of GSH; this enzyme was also present in human liver .
MMAV reductase was sequenced and 92% of the sequences were identical to human
glutathione-S-transferase Omega class (hGSTO-1) . This hGSTO-1 catalyzes the
reduction of iAsV, MMAV, and DMAv [39, 41]. There is evidence that pentavalent
arsenicals can be reduced nonenzymatically. Glutathione (GSH) has been shown to
reduce pentavalent arsenicals [42, 43].
Methylation of Trivalent Arsenicals
Following the first reduction step, arsenite is enzymatically oxidatively methylated
to MMAV. In this reaction, a high energy methyl group from S-adenosyl-L-methione
(SAM) is transferred to a trivalent arsenical in an oxidative process that produces a
pentavalent methylated arsenical. The resulting monomethylarsonic acid (MMA) can be
reduced a second time and methylated again to form dimethylarsinic acid (DMA). In
West Bengal, MMA1" and DMA1" were detected in the urine of exposed humans in 48%
and 72% respectively out of the 428 subjects . In some animals, including humans, a
third methylation can occur resulting in formation of trimethylarsine oxide (TMAO). It
was shown that a single dose of arsenic trioxide in hamsters resulted in a very small
amount of TMAO production in the liver. Yoshida et al 1997 demonstrated that rats
excrete TMAO in their urine after a single oral administration of DMA . Urine
excretion of TMAO in man has been observed after ingestion of an arsenosugar and
DMA [47, 48].
Arsenic methyltransferases (AS3MT) have been isolated from many mammalian
species. An AS3MT has been purified 2000-fold from rabbit liver by DEAE
chromatography to a single band . The rabbit liver AS3MT was capable of
performing both methylation steps. The Golden Syrian hamster liver was used to purify
AS3MT and was shown to have similar activities as the rabbit AS3MT . Rat liver S-
adenosyl-L-methionine:arsenic"'-methyltransferase has been identified and is
homologous to human cytl9 . The rat arsenic methyltransferase has been shown to
perform both mono and dimethylation of arsenic. Arsenic methyltransferase activity has
been determined in mice and primates, including humans [52-55].
Variation in Arsenic Methylation
There is significant variation in the arsenic methylation rate and arsenic metabolite
production among mammalian species. The variability of arsenic methylation is apparent
in the amounts of methylated arsenic metabolites seen in the urine of exposed mammals
such as the rat, rabbit, hamster, dog, and mouse. For example, mice quickly excrete
about 90% of the dose in two days of which 80% is DMA . The rat efficiently
methylates arsenic, but it accumulates DMA in red blood cells resulting in subsequent
lower DMA excretion levels making it a poor model for human metabolic studies .
Healy et al. , purified arsenic methyltransferases from livers of rabbit, hamster, and
rhesus monkey and found different rates of methylation which may affect arsenic
elimination and toxicity.
However, these mammals with arsenic methylation capacity have something in
common which distinguishes them from humans. On average, humans exposed to
arsenic excrete more MMA in the urine than other mammals, specifically 10-30% iAs,
10-20% MMA, and 60-80% DMA . This suggests that non-human mammals are
more efficient at catalyzing the second methylation step which produces MMA. This
may relate to their lower susceptibility to iAs carcinogenesis following exposure versus
man. Additionally, there is significant variation in human susceptibility to As induced
toxicity, which may be related to differences in arsenic biotransformation between
individuals. Epidemiological studies have shown differences in the amount of MMA and
DMA excreted in the urine of exposed populations which may be associated with
differences in arsenic methyltransferase activity. Several studies on the urinary excretion
of arsenic metabolites in native Andean people and mixed ethnicities in northeastern
Argentina exposed to arsenic in potable water revealed low excretion of MMA [60-63].
One study revealed higher than normal MMA in urine, on average 27%, in people
exposed to arsenic in drinking water on the northeast coast of Taiwan .
Only a few polymorphisms have been found in the coding region of cytl9 to date.
The Met287Thr mutation has been reported on three different occasions [65-67]. In two
of these studies, the methylation activity of this allozyme was determined and showed to
have a higher methylation capacity than the wild-type [65, 67]. However, the activity of
this allozyme was determined either from a cytosol preparation or by analysis of cells and
culture media of exposed human hepatocytes. This type of analysis does not take into
account the possible different expression levels of the arsenic methyltransferase. In fact
one study shows that the allozyme Met287Thr was expressed at higher levels than the
wild-type arsenic methyltransferase , making unclear as to whether the increase in
activity results from the mutation or the increased expression levels. There are
differences in arsenic methylation capacities among individuals, which cannot be
supported alone by polymorphisms within the cytl9 coding region. All of the single
nucleotide polymorphisms (SNPs) in cytl9 available as seen in the NCBI SNP database
on January 12, 2006 are within the intron or untranslated region (UTR). In addition, two
separate studies which examined the frequency of polymorphisms within cytl9 found one
nonsynonymous SNPs (nsSNPs) out of 58 SNPs  and the other study found 3
nsSPNPs out of 26 SNPs , the remainder of the SNPs occurring in introns or UTRs.
There is another mechanism which may help explain the differences seen in
arsenic methylation, alternative splicing. Alternative splicing is frequently used to
regulate gene expression and to generate tissue-specific mRNA and protein isoforms [68,
69]. Introns contain sequence elements in which splicesome assembly occurs .
Mutations within these sequence elements could alter the constitutive splicing of a gene
which may affect the methylation capacity within and among different population groups.
Role of Methylation in Arsenic Toxicity
Arsenic methylation has traditionally been thought to be a detoxification pathway.
Pentavalent methylated arsenic metabolites are less reactive and are readily excreted in
the urine compared to iAs . Pentavalent methylated arsenicals have also been shown
to be less cytotoxic and genotoxic compared to arsenite . Mure et al. ,
demonstrated that arsenite induces delayed mutagenesis and transformation in human
osteosarcoma cells but MMA1" showed no significant increase in mutagenesis or
transformation. While As methylation has been viewed as a detoxification pathway,
recent studies have shown monomethylarsonous acid (MMA"') and dimethylarsinous
acid (DMA"') to be more toxic than inorganic arsenicals. In vitro and in vivo studies
have shown that MMA1" is more cytotoxic than As"'. Petrick et al , revealed that the
order of toxicity in Chang human hepatocytes is as follows: MMA11 "> arsenite > arsenate
> MMAV = DMAv. In vivo studies performed in hamsters demonstrated that MMA"11 is
more lethal than arsenite . In addition, Hirano et al. , has shown that
monomethylarsonous acid diglutathione is more acutely toxic than other arsenicals. It is
important to point out that not all mammals methylate arsenic such as the marmoset
monkey, tamarin, chimpanzee, and the guinea pig [57, 75-78]. These mammals have not
been shown to be more susceptible to acute arsenic intoxication. One study showed no
correlation between the induction of micronuclei and the ability to methylate arsenic in
the leukocytes of four mammalian species, humans, mice, rats, and guinea pigs .
Another factor that questions the role of methylation is the fact that arsenic is a known
human carcinogen, but very few animals exist in which arsenic initiates carcinogenesis.
The debate over whether As methylation is a detoxification or bioactivation pathway
leads to confusion over the role of methylation in toxicity.
Specific Aims of Research
The proposed study will address the role of arsenic methylation in human toxicity
by better understanding the kinetics of the human arsenic methyltransferase. The overall
hypothesis is the following: Human arsenic methyltransferase, cytl9, activity is the
determining factor in the rate of arsenic methylation and toxicity. To test this hypothesis,
I examined two specific aims: 1) clone and characterize the human arsenic
methyltransferase (cytl9) and 2) determine cytl9s role in toxicity and arsenic
methylation variability. Specific aim one was addressed by determining rate of arsenic
methylation by an in vitro assay. The optimum conditions required for human cytl9
activity were determined such as pH optimum, substrate specificity and concentration,
and thiol requirements. Specific aim two was addressed by examining the role of
polymorphisms and splice variants on arsenic methylation variability. The determination
of cytl9s role in toxicity was addressed by As toxicity in the presence and absence of
methylation activity. In order to determine if cytl9 is the only arsenic methyltransferase
in humans, the mRNA levels, protein concentration, and activity, in the presence and
absence of siRNA knockdown was determined.
MOLECULAR CLONING AND CHARACTERIZATION OF HUMAN CYT19, AN S-
ADENOSYL-L-METHIONINE:AS-METHYLTRANSFERASE FROM HEPG2 CELLS
Chronic arsenic exposure is a threat to millions of people throughout the world.
Exposure to arsenic has been linked to various types of cancers such as skin cancer, lung
cancer, and cancer of other internal organs . Methylation has been considered the
major route of biotransformation and excretion of inorganic arsenic (iAs) in many species
including humans. The hypothesized scheme of iAs methylation involves reduction
followed by oxidative methylation :
AsVO43- + 2e -> As"33- + CH3+ CH3As 032- + 2e CH3As"O22- + CH3+
(CH3)2Asv02- + 2e > (CH3)2As"IO
While traditionally thought to be a detoxification pathway, recent studies have
shown monomethylarsonous acid (MMA1") and dimethylarsonous acid (DMA"') to be
more toxic than inorganic arsenicals . In addition, Hirano et al., has shown that
monomethylarsonous acid diglutathione is more acutely toxic than other arsenicals. The
debate over whether As methylation is a detoxification or bioactivation pathway leads to
confusion over the role of methylation in toxicity. Rat liver S-adenosyl-L-
methionine:arsenic"'-methyltransferase has been identified and is homologous to human
cytl9 . While this enzyme can be used as a model for human arsenic
biotransformation, the rat is considered a poor model for metabolic studies due to its
accumulation in red blood cells and subsequent lower DMA excretion levels . There
are also other species specific differences in arsenic biotransformation and toxicity.
Healy et al. (1999) , purified arsenic methyltransferases from livers of rabbit,
hamster, and rhesus monkey and found different rates of methylation which may affect
arsenic elimination and toxicity. Humans excrete greater amounts of monomethylarsonic
acid (MMAV) compared to most other mammals . Additionally, there is considerable
variation among humans in the rate of methylation of inorganic arsenic possibly leading
to measurable differences in toxicity . Therefore it is important to better understand
the arsenic methylation capacity in human. To date, human cytl9 has been expressed,
but it has not been fully characterized . In this study, we cloned, expressed, and
characterized cytl9, an arsenic methyltransferase from human HepG2 hepatoma cells.
Materials and Methods
Two separate sequences available from Genbank (accession number AK057833
and AF226730) were used to design primers to amplify the open reading frame (ORF) of
cytl9, an arsenic methyltransferase (Table 1). Total RNA was isolated from HepG2 cells
using Trizol reagent (Invitrogen, Carlsbad, USA)). Total RNA was treated with DNAase
(DNA-free kit, Ambion, Austin, USA), and reverse transcribed (RETROscript for RT-
PCR, Ambion) using 2 tg of RNA. HepG2 cDNA was polymerase chain reaction
(PCR)-amplified, the PCR product was ligated in pET100/D-TOPO (Invitrogen) and
transformed into chemically competent Escherichia coli One Shot TOP 10 chemically
competent cells (Invitrogen). The PCR reaction consisted of 2.5 U of Pfu DNA
polymerase, 0.4 pM each primer, 5tl of the RT reaction, 0.2 mM dNTP mix, 5Pl of 10X
PCR Buffer, and nuclease-free water to 50pl. The PCR conditions were as follows: an
initial denaturation at 940C for 2min, followed by 35 cycles of denaturation at 940C for
Imin, annealing at 600C for Imin, extension for 720C for 2min 30s and a final extension
at 720C for 7min. The PCR products, the complete open reading frame of cytl9 (cytl9-
WT), the mutated cytl9 (cytl9S81R), and the truncated cytl9 (cytl9t) were then ligated
and transformed. Ampicillin resistant colonies were analyzed by PCR and visualized by
agarose gel electrophoresis. Once a correct clone was identified it was sent for
sequencing to the DNA Sequencing Core Laboratory at the University of Florida. Each
clone was sequenced several times and the consensus sequence determined.
Rapid amplification of cDNA ends (RACE) was performed to determine which of
the two separate sequences available in Genbank was actually expressed in HepG2 cells.
The RACE-PCR was performed using the FirstChoice RLM-RACE kit from Ambion.
Primers were designed (Table 1) according to the instruction manual and the PCR
reaction consisted of 1.25 U of Taq DNA polymerase, 0.4 VM each primer, 1 l of the RT
reaction, 0.2 mM dNTP mix, 5pl of 10X PCR Buffer, and nuclease-free water to 50al.
The PCR conditions were as follows: an initial denaturation at 940C for 3min, followed
by 35 cycles of denaturation at 940C for 30s, annealing at 600C for 30s, extension for
720C for Imin and a final extension at 720C for 7min. The PCR product was ligated in
pGEM-T Easy Vector (Promega) and transformed into chemically competent
Escherichia coli JM109 chemically competent cells (Promega).
Expression of Recombinant cytl9
The pET100/D-TOPO constructs (cytl9-WT, cytl9S81R, and cytl9t) were
transformed into BL21 Star (DE3) E. coli strain for expression (Invitrogen). First, 10ml
of Luria-Bertani (LB) broth containing ampicillin (100[tg/ml) and 1% glucose were
inoculated with the transformed bacteria and the cultures were grown overnight. The
next day, 5ml of the overnight culture was used to inoculate 250ml of LB broth
containing ampicillin and 1% glucose and grown to an OD600 of 0.5. Expression was
induced by the addition of ImM isopropyl-1-thio-P-D-galactoside. The culture was
allowed to grow for one hour.The pET100/D-TOPO construct was then transformed into
BL21 Star (DE3) E. coli strain for expression (Invitrogen).
The cells were harvested by centrifugation at 5,000g for 15 minutes at 40C. The
pellet was resuspended in binding buffer (50mM NaH2PO4, 300mM NaC1, 10mM
imidazole, pH 8.0). The cells were lysed by addition oflysozyme to a final concentration
of lmg/ml and incubated on ice for 30 minutes followed by further incubation for 10
minutes at 40C on a rocking platform. Triton X-100 was added to a final concentration of
1% and the incubation continued for another 10 minutes at 40C with rocking. The
cellular debris was removed by centrifugation of the lysate at 3000g for 30 minutes at
4C The recombinant 6xHis-tagged protein was purified using a nickel-nitrilotriacetic
acid (Ni-NTA) metal-affinity chromatography according to the manufacturer's
instructions (QIAGEN, Valencia, USA).
Activity of the expressed proteins was determined by the rate of arsenic
methylation. All incubations were carried out at 370C for 30 minutes in a final volume of
250 il, unless otherwise noted. The reaction mixtures contained 5mM glutathione
(GSH), 1 mM dithiothreitol (DTT), 1 mM MgC12, 100 [M S-adenosyl-L-methionine
(SAM), 13pM (3H-methyl)-SAM (S.A.), 50KM sodium m-arsenite, and 5 pg of S-
adenosyl-L-methionine: arsenic methyltransferase in 100mM tris/100mM sodium
phosphate buffer pH 7.4. The pH optimum was determined using the above conditions
but at different pHs (6.0 11). The substrate specificity and optimum substrate
concentrations were also determined by addition of various concentrations of sodium m-
arsenite or MMA, ranging from 1 iM to 200 iM or 10 iM to 1000 iM respectively. The
requirements of SAM and reductants by cytl9 were determined by addition of various
concentrations of SAM, and the reductants GSH and tris(2-carboxyethyl)-phosphine
(TCEP). The methylation reactions were stopped by placing on ice. The standard
extraction procedure described by Zakharyan et al.  was used to separate radioactive
SAM from radioactive MMA and DMA. Briefly, the reaction mixture (250 il) was
treated with 10 il of 40% KI, 20 [l of 1.5% potassium dichromate, 750 [l of
concentrated HC1 and 750 pl of chloroform. The mixture was then mixed on a vortex for
3 min followed by centrifugation at 1500g for 3 min. The upper aqueous phase contained
SAM and was discarded. The lower organic phase was washed twice with 250 pl of
water, 5 il of 40% KI, and 750 pl of concentrated HC1. The mixture was mixed on a
vortex and centrifuged and the upper aqueous phase was discarded each time. The
methylated arsenicals contained in the organic phase were back extracted with 1 mL of
water, vortexed for 3min and centrifuged at 1500g for 5 min. Half a milliliter from the
final aqueous phase after back extraction was counted in a liquid scintillation counter.
The activity was calculated from the dpm 3H transferred from SAM to arsenic.
Confirmation of Methylated Arsenicals
Methylated arsenicals were separated from each other and contaminating species
using the ion exchange method described previously by Zakharyan et al. (1995) . A
10 mL glass pipette was filled to 2 mL with Bio-Rad AG 50W-X4 cation exchange resin
(100-200 iM mesh). The column was equilibrated by addition of 0.5N HC1 (30 mL),
followed by water until the pH of the effluent was 5.5, 0.5N NaOH (30 mL), water until
the pH of the effluent was 5.5, 0.5N HC1 (30 mL), and 0.05N HC1 (50 mL). After
equilibrating the column, 0.5 mL of the final aqueous phase extract from above was
applied to the column. The columns were eluted by 6 mL of 0.05M HC1 to obtain MMA
and 10 mL of 0.5M NaOH for DMA elution. One milliliter of the these fractions were
counted in a liquid scintillation counter.
Results and Discussion
Both cytl9 and cytl9t transcripts were amplified by PCR from HepG2 cells and
human liver samples. The sequencing results of cytl9t showed 4 point mutations, 3
transverions and 1 transition, (Figure 2-1A) resulting in 3 missense mutations (Figure 2-
1B) compared to the Genbank sequence (accession AF226730). The wild-type cytl9 was
also amplified, cloned, and sequenced from two different populations of HepG2 cells.
One of the clones, designated cytl9-WT, was aligned to the Genbank sequence,
accession AK057833, and showed a 100% homology (data not shown). The other clone
designated cytl9S81R contained a nonsynonymous single nucleotide polymorphism
(nsSNPs) when compared to the Genbank sequence, accession AK057833 (Figure 2-2A),
which results in a change from serine to arginine at residue 81 in the peptide sequence
(Figure 2-2B). This change occurs in the SAM-binding site, however SAM-dependent
methyltransferases have poor conservation of SAM-binding residues. SAM-dependent
methyltransferases contain 3 regions of sequence similarity (motif I, II, and III) which are
thought to be important in SAM binding. The only highly conserved residues in the
SAM-binding N-terminal region appear to be the glycine-rich sequence E/DXGXGXG
found at residues 76 to 82 . Therefore, the amino acid change may not have a
significant effect on the activity of the recombinant protein.
The cytl9t clone and the cytl9-WT clone are identical except for the deletion of a
nucleotide at position 997 resulting in a premature stop codon (Figure 2-3A). Further
analysis of the protein sequences revealed 6 missense mutations including a cysteine to
valine mutation and deletions of the final 37 amino acids from the C-terminus including 4
cysteines due to the deletion in the nucleotide sequence (Figure 2-3B). In other SAM-
dependent methyltransferases, the C-terminus is important in substrate binding . The
cytl9t protein showed no arsenic methylation activity. This indicates that the cysteine
rich C-terminus is important for As binding and critical for activity.
RACE-PCR was performed on both the 5' and 3' ends. The sequencing results
revealed that the 5'end of the cDNA was identical to the ORF of both sequences
available in Genbank (accession AK057833 and AF226730). The 5'-untranslated region
(UTR) is different from the Genbank sequences containing 18 mutations (Figure 2-4A).
The 3'RACE-PCR revealed that HepG2 cells expressed mRNA identical to the 3'end of
the Genbank sequence, accession AK057833. In particular, the sequencing showed that
HepG2 cells cytl9 mRNA does not have a nucleotide deletion resulting in a premature
stop codon (Figure 2-4B).
The recombinant human cytl9s (cytl9-WT, cytl9t, and cytl9S81R) were
successfully expressed in BL21 (DE3) and purified to homogeneity using a nickel-
nitrilotriacetic acid metal-affinity chromatography (Figure 5). The recombinant proteins,
cytl9S81R and cytl9-WT, catalyze the transfer of a methyl group from SAM to As"' as
well as MMA1", which is consistent with previous studies (Figure 2-6) [51, 52].
However, the different arsenite methyltransferase activity profiles between cytl9-WT and
cytl9S81R are apparent. Arsenite concentrations above 50 uiM appears to have an
inhibitory effect on cytl9S81R activity, which is similar to what is seen in the rabbit .
This inhibitory effect is not seen in the cytl9-WT arsenite methylation activity. The
apparent Km and Vmax of cytl9 As"' methyltransferase (AS3MT) activity for cytl9-WT
and cytl9S81R are 251.6 riM, 3505 pmole/mg/min, 6.176 riM, and 804.9 pmole/mg/min
respectively (Table 2). The Km and Vmax values of cytl9-WT MMA methylation is 164.6
IM and 926.8 pmole/mg/min (Table 2). The MMA methylation profile is very similar to
that seen in the rabbit. These enzymes seem to saturate at MMA concentrations of 1000
The Km values can be used to interpret the affinity of an enzyme for its substrate (a
larger Km implies a weak affinity). Other kinetic analysis of the human arsenic
methyltransferase had very low Km [67, 84] values compared to cytl9-WT but, the Km
value of cytl9S81R was very similar to the other kinetic analysis. However, these other
studies did not use purified enzymes which may explain the difference, especially the
difference seen with cytl9-WT. The kinetic analysis suggests that the cytl9S81R has a
higher affinity for arsenite than cytl9-WT. However, cytl9-WT has a considerably
higher Vmax value compared to cytl9S81R. The Vmax values of both cytl9-WT and
cytl9S81R are considerably higher than that seen among other mammals such as the
hamster, rabbit, and rhesus monkey . Kinetic analysis of MMA methylation
demonstrates that the Vmsx and Km values for cytl9-WT are much higher than the values
seen in the hamster, rabbit, and rhesus monkey. The higher Vmax values of arsenite
methyltransferase compared to MMA methyltransferase in cytl9-WT may explain the
higher MMA urine excretion levels seen in humans compared to other mammals. The
rabbit, which excretes higher amounts of MMA than most other mammals, has a higher
MMA than arsenite methyltransferase Km . Possibly, arsenite is converted very
quickly to MMA, allowing it to accumulate before the dimethylation resulting in the
higher excretion of MMA seen in humans.
The optimum pH of As"' methylation for cytl9-WT and cytl9S81R was found to
be about 8 and about 9 respectively (Figure 2-7). This is similar to previous results
which show that at basic pHs, methylation activity of rat cytl9, and As"'
methyltransferase & MMA1" methyltransferase activity from rabbit liver increase [49,
51]. This may be due to the deprotonation of cysteines at higher pHs, which increases the
rate of binding between arsenic and cysteines in the substrate binding domain. The
reductant requirements were examined and it was determined that GSH is not required
for cytl9-WT to methylate arsenic (Figure 2-8). In addition, it was determined that
cytl9S81R does not require GSH. Previous study suggests that the substrates for cytl9
are arsenic triglutathione and monomethylarsonic glutathione . Our results
demonstrate that only a strong reductant such as TCEP is necessary for methylation of
arsenic by cytl9, however, the addition of GSH appears to increase the activity above the
reductant alone. Finally, the effect that different SAM concentrations would have on
activity was determined. It was found that above 500 riM, SAM began to have an
inhibitory effect (Figure 2-9). This differs from what is seen in rat, where SAM
concentrations above 50 |iM have an inhibitory effect .
The ion exchange method confirmed that cytl9 indeed produces MMA when
arsenite is the substrate (Figure 2-10A). When MMAV is used as the substrate, both
MMA and DMA are seen as products (Figure 2-10B). However, DMA is the major
metabolite. The MMAV used as a substrate is not 100% pure and likely contains some
iAs as contaminants. It is possible that the contaminating iAs is methylated to MMA.
In conclusion, we have shown that cytl9 is in fact an arsenic methyltransferase
methylating both arsenite and MMA. Examination of the cytl9t activity, indicates that
the cysteine rich C-terminus is important for As binding and critical for activity. The
data suggests that a mutation within the SAM-binding site of cytl9 can drastically change
the methylation capacity of the enzyme. The characterization and kinetic analysis may
explain the higher MMA urine excretion levels and increased susceptibility seen in
humans compared to other mammals. It appears that arsenite is converted very quickly to
MMA, allowing it to accumulate before the dimethylation resulting in the higher
excretion of MMA seen in humans. The apparently deficient dimethylation activity in
humans compared to other mammals is supported by the kinetic analysis and suggests
that methylation may actually be a detoxification pathway.
Table 2-1. Primers used in the PCR amplification of cytl9
5'RACE Outer: TTTCAGCCACTTCCACCTGGCCTT
3'RACE Outer: AGGACCAACCAAGAGATGCCAA
Figure 2-2. Kinetic analysis of the methylation activity of cytl9-WT and cytl9S81R.
methyltransferase Km (FM) 83.010.9 6.20.9
vm. 1585142 804.933.4
methyltransferae Km (FiM) 164.641.2 43.713.3
a. 926.875.4 365.125.7
CCAAAGGCCA GGTGGAAGTG GCTGAAAAGT ATCTTGACTA
CCAAAGGCCA GGTGGAAGTG GCTGAAAAGT ATCTTGACTA
AAATATGGCT TCCAGGCATC TAATGTGACT
AAATATGGCT TCCAGGCATC TAATGTGACT
GAAGTTGGCA GAGGCTGGAA TCAAGAATGA
GAAGTTGGGA GAGGCTGGAA TCAAGAATGA
CAAACTGTGT TATTAACCTT GTGCCTGATA AACAACAAGT
CAAACTGTGT TATTAACCTT GTGCCTGATA AACAACAAGT
AF226730 MAALRDAEIQ KDVQTYYGQV LKRSADLQTN GCVTTARPVP
cytl9t MAALRDAEIQ KDVQTYYGQV LKRSADLQTN GCVTTARPVP
CGLVIPEHLE NCWILDLGSG SGRDCYVLSQ
CGLVIPEHLE NCWILDLGSG SGRDCYVLSQ
AEKYLDYHME KYGFQASNVT FFHGNIEKLA
AEKYLDYHME KYGFQASNVT FIHGYIEKLG
VPDKQQVLQE AYRVLKHGGE LYFSDVYTSL
VPDKQQVLQE AYRVLKHGGE LYFSDVYTSL
Figure 2-1. Sequence alignment of cytl9t and Genbank sequence (accession AF226730).
(A) Nucleotide alignment of cytl9t and Genbank sequence. The four point
mutation are in red. (B) Alignment of the deduced cytl9t amino acid sequence
and Genbank sequence. The 3 resulting missense mutations are in red.
TAGCCCTAAG ATATTATGGC TGTGGTCTGG
TAGCCCTAAG ATATTATGGC TGTGGTCTGG
AACTGCTGGA TTTTGGATCT GGGTAGTGGA
AACTGCTGGA TTTTGGATCT GGGTAGTGGA
ACTTAGCCAG CTGGTTGGTG AAAAAGGACA
ACTTAGCCAG CTGGTTGGTG AAAAAGGACA
AK057833 MAALRDAEIQ KDVQTYYGQV LKRSADLQTN GCVTTARPVP KHIREALQNV
cytl9S81R MAALRDAEIQ KDVQTYYGQV LKRSADLQTN GCVTTARPVP KHIREALQNV
AK057833 HEEVALRYYG CGLVIPEHLE NCWILDLGSG SGRDCYVLSQ
cytl9S81R HEEVALRYYG CGLVIPEHLE NCWILDLGSG RGRDCYVLSQ
AK057833 IDMTKGQVEV AEKYLDYHME KYGFQASNVT FIHGYIEKLG
cytl9S81R IDMTKGQVEV AEKYLDYHME KYGFQASNVT FIHGYIEKLG
Figure 2-2. Sequence alignment of cytl9S81R and Genbank sequence (accession
AK057833). (A) Nucleotide alignment of cytl9S81R and Genbank sequence.
The tranversion is in red. (B) Alignment of deduced cytl9S81R amino acid
sequence and Genbank sequence. The resulting nsSNP is in red. The SAM-
binding N-terminal region site is underlined.
ATCAGACCAA TTGGAGAGAA GTTGCCAACA
ATCAGACCAA TTGGAGAGAA GTTGCCAACA
ACAGTATGAA GTCCAGATGT GTCCCTGATG
LYWKELAVLA QKIGFCPPRL VTANLITIQN
LYWKELAVLA QKIGFCPPRL VTANLITIQN
KHSKTGPTKR CQVIYNGGIT GHEKELMFDA NFTFKEGEIV
KHSKTGPTKR CQVIYNGGIT GHEKELMFDA NFTFKEGEIV
KNSRFAQDFL IRPIGEKLPT SGGCSALELK
KNSRFAQDFL IRPIGEKLPT SGAVLLWS*-
Figure 2-3. Sequence alignment of cytl9-WT and cytl9t. (A) Nucleotide alignment of
cytl9-WT and cytl9t sequence. The missense mutations are in red. The 5
cysteine residues which are not included in the cytl9t are highlighted in the
ACAGGAGCTG GCTGCGGGAG CCCGCCGTCC TGAGTCGCAG GCCGAGGAGA
TGCTGTGGCA CAAAGAAAAG CTGCTAAATC
TGCTGTGGCA CAAAGAAAAG CTGCTAAATC
TGCTGGAGGC TGCTGTGGCA CAAAGAAAAG CTGCTAAATC TATAGCCAAC
Figure 2-4. Sequence alignments of the 5' & 3'RACE-PCR products and the Genbank
sequences (accession AK057833 and AF226730). (A) Sequence alignment of
the 5'RACE-PCR product against the Genbank sequences. The mutations in
the 5'RACE product are in red. The start codon for all three sequences are
highlighted. (B) Sequence alignment of the 3'RACE-PCR product and the
Genbank sequences. The nucleotide deletion in the Genbank sequence,
accession AF226730, is highlighted. The stop sites for the Genbank
sequences are in red.
1 2 3
120 k .
39.2 k A.
18.3 k ,
4W.F.U if .: IBMlf
Figure 2-5. Purification of recombinant human cytl9. Fractions were electrophoresed on
a 10% polyacrylamide gel and stained. Lanel, molecular weight markers;
Lane2, cell lysate; Lane3, flowthrough, Lane4, Washl, LaneS, Wash2, Lane6,
Wash3. Lane7. Elution
0 50 100 150 200 250
Figure 2-6. The effects of As" & MMAV concentrations. All incubations were carried
out at 370C for 30 min. in a final volume of 250 [il. A) Reaction mixtures
contained 5 mM GSH, 1 mM DTT, 1 mM MgCl2, 13 pM [3H]SAM, 0.1 mM
SAM, various [As"'], and 5 tg of cytl9, in 100 mM Tris/100 mM Na
phosphate, pH 7.4 B) Same as B but with various [MMAV].
5 6 7 8 9 10 11 12
Figure 2-7. The effect of pH on activity. All incubations were carried out at 370C for 30
min. in a final volume of 250 gl. Reaction mixtures contained 5 mM GSH, 1
mM DTT, 1 mM MgC12, 13 pM [3H]SAM, 50 gM AsIII, and 5 gg of cytl9, in
100 mM Tris/100 mM Na phosphate of the appropriate pH.
1mM TCEP 1mM
Figure 2-8. The effects of reductants on methylation activity. All incubations were
carried out at 370C for 30 min. in a final volume of 250 gl. Reaction mixtures
contained 1 mM MgC12, 13 pM [3H]SAM, 0.1 mM SAM, 50 gM AsIII, and 5
gg of cytl9-WT, in 100 mM Tris/100 mM Na phosphate, pH 7.4, with
different reductants. The activity of cytl9R81S was not determined for 1 mM
TCEP + 1 mM GSH.
0 200 400 600 800 1000 1200
Figure 2-9. The effect of SAM concentration on activity. All incubations were carried
out at 370C for 30 min. in a final volume of 250 pil. Reaction mixtures
contained 5 mM GSH, 1 mM DTT, 1 mM MgCl2, 13 pM [3H]SAM, 50 ipM
AsIII, and 5 pg of cytl9-WT, in 100 mM Tris/100 mM Na phosphate pH 7.4.
with various [SAM].
0 5 10 15 20 25
0 5 10 15 20 25
Figure 2-10. Arsenical metabolites formed after incubation with [3H]SAM and cytl9 for
30 min at 37C. (A) Formation of MMA and DMA using As"' as a substrate.
(B) Formation of MMA and DMA using MMAV as the substrate.
IDENTIFICATION OF A SPLICE VARIANT OF HUMAN CYT19 ARSENIC
Arsenic (As) is a naturally occurring element and ranks 20th in abundance in the
earth's crust . Arsenic is present in the environment in both organic and inorganic
forms and exists mainly in three valence states, -3, +3, and +5. Generally, inorganic
arsenic (iAs) is the more toxic form and people are exposed to iAs primarily through food
and potable water. In Taiwan, Mexico, western United States, western South America,
China, and Bangladesh, people are exposed to high levels of arsenic due to anthropogenic
and/or natural contamination of potable water [12, 62, 86]. In these areas, chronic
exposure to arsenic is associated with various tumors occurring in skin, liver, lung,
urinary bladder, and prostate [8, 87].
Once in the body, many mammals, including humans, methylate iAs to
monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) [49, 51, 54]. The
biotransformation of iAs alternates between the reduction of arsenate (iAsV) to arsenite
(iAs"') followed by oxidative methylation (Figure 1) [88, 89]. Because pentavalent
methylated arsenicals are less toxic than inorganic arsenic, methylation has been
considered a detoxification mechanism. Recent studies indicate that trivalent methylated
arsenicals may be more acutely toxic and genotoxic than iAs suggesting that methylation
may actually be a bioactivation of iAs [20, 73, 74, 90]. For this reason the role of
methylation in acute and chronic arsenic toxicity remains unclear. There is significant
variation in human susceptibility to As induced toxicity, which may be related to
differences in arsenic biotransformation between individuals . Epidemiological
studies have shown differences in the amount of MMA and DMA excreted in the urine of
exposed populations which may be associated with genetic polymorphisms . Cytl9
has been identified as a human S-adenosyl-L-methionine:arsenic methyltransferase [52,
55] however, only a few coding region polymorphisms have been detected which may
alter the iAs methylation rate [65, 67]. In this study we identified an alternative splice
variant of human cytl9, which contains an upstream open reading frame (uORF)
followed by an internal start codon (AUG). This variant was expressed in 7 out of 7
human livers and represents another possible mechanism for regulating As methylation.
Materials and Methods
Molecular Cloning of cytl9 Splice Variants
Total RNA was isolated from HepG2 cells and human liver samples using Trizol
reagent according to the manufacturer's instruction (Invitrogen, Carlsbad, USA). Total
RNA was treated with DNase I using the DNA-freeTM kit from Ambion (Austin, TX) and
cDNA was made using the RETROscriptTM Kit for RT-PCR and 2 ig of RNA as
template (Ambion). HepG2 cDNA was polymerase chain reaction (PCR)-amplified
using the following primers: forward primer (5'-
CACCATGGCTGCACTTCGTGACGCTGAGATACAG-3') and the reverse primer (5'-
TTAACTCCAAAGCAGAACAGCTCCAGATGT-3'). The PCR reaction consisted of
2.5 U ofPfu DNA polymerase, 0.4 tM each primer, 5[l of the RT reaction, 0.2 mM
dNTP mix, 5[l of 10X PCR Buffer, and nuclease-free water to 50al. The PCR
conditions were as follows: an initial denaturation at 940C for 2min, followed by 35
cycles of denaturation at 940C for Imin, annealing at 600C for Imin, extension for 72C
for 2min 30s and a final extension at 720C for 7min. The PCR products, designated
cytl9 and cytl9AE2, were ligated into the pET100/D-TOPO vector (Invitrogen) and
transformed into competent Escherichia coli (E. coli) One Shot TOP 10 chemically
competent cells (Invitrogen). Ampicillin resistant colonies were analyzed by PCR and
visualized by agarose gel electrophoresis. Several clones containing inserts were
sequenced by the DNA Sequencing Core Laboratory at the University of Florida.
Human Liver Samples
Human liver samples were obtained from Vitron (Tucson, AZ). All the tissues
were from Caucasian males between the ages of 24 and 46. The tissues were preserved
in Viaspan after death. The tissue samples were stored at -800C until use. All procedures
using human samples were approved by the Institutional Review Board at the University
of Florida and all identifying information has been removed.
qPCR of cytl9 Splice Variants
Total RNA was isolated and cDNA synthesized as described above. Real-time
quantitative PCR (qPCR) was carried out using a Bio-Rad iCycler with the following
primers: cytl9 forward primer: (5'-TTCGTGACGCTGAGATACAGAAG-3'); reverse
primer: (5'-TGGAGGTCTGCCGATCTCTT-3'); cytl9AE2 forward primer: (5'-
GATACAGAAGGACGTGCAGATATTATG-3'); reverse primer: (5'-
CCAGATCCAAAATCCAGCAGTT-3'). Each PCR reaction consisted of 12.5 [l iTaq
SYBR Green Supermix with ROX (Bio-rad), 0.4 tM each primer, 5[l of the RT reaction,
and RNase/DNase-free water to 25 al. The PCR cycling conditions included an initial
denaturation of 95C for 3 min followed by cycling at 950C for 15s, 600C for 45s for 45
cycles. The constructs, pET100-cytl9 and pET100-cytl9AE2 were used to generate
calibration curves for quantification of cytl9 and cytl9AE2. A melting curve analysis
was performed after every run to determine product uniformity.
When the full open reading frame of cytl9 was amplified using the primers
described in Molecular Cloning of cytl9 splice variants under Materials & Methods, two
products were generated, 1132 bp (cytl9) and 1005 bp (cytl9AE2) products (Figure 3-2).
Sequencing of these two products revealed that the 1132 bp product is the reference cytl9
while the 1005 bp product is a splice variant (Figure 3-3). The reference cytl9 mRNA is
composed of 10 exons (Figure 3-4A) which encode a 375 amino acid protein with a
theoretical molecular weight of 41.747 kDa (Figure 3-5). The product, cytl9AE2, is
missing 128 bp due to a deletion of exon 2 which could result in a protein that is about
102 amino acids shorter and the creation of a short 24 amino acid peptide as a product
from an upstream open reading frame (uORF) (Figure 3-3 & Figure 3-4B). This variant
may encode a 273 amino acid polypeptide chain that is identical to the reference cytl9
but lacks the first 102 amino acids present in the reference (Figure 3-5). Analysis of the
sequences surrounding the splice revealed that the splice occurs at conserved acceptor
and donor sites (Figure 3-4C). The individual information (Ri) technique and Shapiro's
method were used to compare the splice-sites strength of exon 3 and exon 2 of cytl9
. Exon3, a constitutive exon has a stronger splice site compared to exon2, the
alternative exon (Table 3-1).
The steady state mRNA levels of each transcript were determined in 7 human liver
samples as well as in HepG2 cells by qPCR (Table 3-2). Expression of cytl9 mRNA in
the human liver samples ranged from 7.04 x 104 to 1.26 x 106 copies per microgram of
RNA. The cytl9AE2 splice variant was detected in all 7 human liver samples tested.
(Table 3-2). The amount of cytl9AE2 mRNA was much lower than cytl9 mRNA
ranging from 1.89 x 103 through 4.33 x 103 copies per microgram of RNA. The HepG2
cells had an average of 5.14 x106 copies/Gg RNA and 8.55 x104 copies/Gg RNA of cytl9
and cytl9AE2 mRNA, respectively.
In this study, we identified an alternative splice variant of cytl9, which contains an
uORF. The variant mRNA contains a short ORF followed by an internal AUG codon
beginning 106bp downstream from the uORF (Figure 3-3). While this alternative variant
may encode a 273 amino acid protein it is unlikely that expression of the cytl9AE2 splice
variant will result in production of an active protein. Studies have shown that SAM
dependent methyltransferases share 3 regions of sequence similarity (motif I, II, and III)
. These motifs are found in the same order on the polypeptide chain and separated by
similar intervals . It has been suggested that these conserved regions are important in
SAM binding . Mutations of a conserved amino acid in rat guanidinoacetate
methyltransferase near motif I have resulted in an inactive enzyme. In addition,
mutations of motif II lead to reduced Kcat/Km values for substrates . It is unlikely
that the protein translated from cytl9AE2 would result in an active protein due to the
removal of motif I (Figure 3-5).
Whether the mRNA actually is translated into protein is not clear because the
internal AUG codon contains a relatively weak Kozak sequence suggesting that
translation may not reinitiate at the internal start codon. The sequence
(GCCA/GCCATGG) is a consensus Kozak sequence for the initiation of translation in
vertebrates [94, 95]. Deviation from the consensus sequence at position -3 and +4 would
be considered a weak initiator codon. The cytl9AE2 transcript deviates from the
consensus sequence at position +4; the variant contains an A instead of a G (Figure 3-3).
It is also possible that cytl9AE2 will not be translated but that this variant is a
substrate for the nonsense mediated decay (NMD) pathway due to the premature stop
codon. NMD is a pathway that recognizes and quickly degrades mRNAs containing
premature translation termination codons (PTC) in eukaryotes . While cytl9AE2
does contain a PTC, Zhang et al. identified a sequence motif which when present 3' of a
nonsense codon promotes rapid decay of the mRNA transcript by the NMD pathway
. This sequence motif (TGYYGATGYYYYY) is not found in the cytl9AE2 mRNA
transcript and it remains unclear if this variant will undergo degradation by the NMD
The cytl9AE2 variant was present in all seven human liver samples tested,
suggesting that cytl9 mRNA exists both in the full length and alternatively spliced form
in most individuals. The cytl9AE2 variant mRNA comprised 0.2 to 3.8% of the total
cytl9 transcript. The liver samples had lower copy numbers per microgram of RNA of
both reference and cytl9AE2 variants compared to HepG2 cells. It is possible that some
degradation of cytl9 message occurred during collection and storage of the livers which
reduced apparent copy number.
Many mammalian species methylate arsenic through an enzymatic reaction that is
performed by cytl9. There are significant variations in the arsenic methylation capacity
between species and within species including humans [58, 59, 62, 81]. The reason for
this variation is unclear but has been attributed to cytl9 polymorphisms. However, only
a few polymorphisms have been found in the coding region of cytl9 to date [65, 67],
while the vast majority of mutations are found within the introns and the 5' and 3'
untranslated region (UTR). Introns contain the acceptor site, branchpoint,
polypyrimidine tract, and the donor site, which are conserved sequences in which
splicesome assembly occurs . While mutations within these sequence elements could
alter the constitutive splicing of a gene [98-100], there are differences in arsenic
methylation capacities among individuals, which are unlikely to be supported solely by
polymorphisms within the cytl9 coding region.
Alternative splicing is frequently used to regulate gene expression and to generate
tissue-specific mRNA and protein isoforms [68, 69]. Thirty-five to 60% of human genes
produce transcripts that are alternatively spliced, in addition 70-90% of these variants
alter the resulting protein products [101, 102]. Further studies should analyze the mRNA
expression levels of cytl9 splice variants in a larger number of fresh liver samples or
primary hepatocytes and correlate it to arsenic methylation activity. In addition, work to
determine if this transcript is a substrate for the NMD pathway or if a variant protein is
expressed will help clarify the role of cytl9AE2 in human arsenic metabolism. Even
though the splice variant comprises a relatively small fraction of the total cytl9 transcript
in the livers tested it is possible that different population groups have varying amounts of
the cytl9 splice variant. It is also likely that the level of cytl9AE2 in an individual will
change over time as alternative splice selection can be controlled by many variables
including developmental stage and xenobiotics [103, 104]. In conclusion, cytl9 appears
to be alternatively spliced in many individuals and may play a role in the observed
variation in arsenic methylation seen in individuals.
As043- + 2e- As"033- + CH3+ CH3ASV032-+ 2e-
CH3As' 022-+ CH3+ (CH3)2As'2 + 2e --
1 Reduction step of As biotransformation
2 Oxidative methvlation steD of As biotransformation
Figure 3-1. The hypothesized scheme of iAs methylation proposed by Cullen, McBride et
Ladder cytl9 cytl9AE2
Figure 3-2. PCR products of cytl9 amplification. 1% Agarose DNA gel of cytl9 and
TTCGTGACGC TGAGATACAG AAGGACGTGC
TTCGTGACGC TGAGATACAG AAGGACGTGC
---------- ---------- ---------- ---------- ----------
CGGGCAGGTG CTGAAGAGAT CGGCAGACCT CCAGACCAAC GGCTGTGTCA
CCACAGCCAG GCCGGTCCCC AAGCACATCC GGGAAGCCTT GCAAAATGTA
---- -- ATATTATGGC TGTGGTCTGG TGATCCCTGA
CACGAAGAAG TAGCCCTAAG ATATTATGGC TGTGGTCTGG TGATCCCTGA
GCATCTAGAA AACTGCTGGA TTTTGGATCT GGGTAGTGGA AGTGGCAGAG
GCATCTAGAA AACTGCTGGA TTTTGGATCT GGGTAGTGGA CGTGGCAGAG
cytl9AE2 ATTGCTATGT ACTTAGCCAG CTGGTTGGTG AAAAAGGACA CGTGACTGGA
cytl9 ATTGCTATGT ACTTAGCCAG CTGGTTGGTG AAAAAGGACA CGTGACTGGA 300
cytl9AE2 ATAGACATGA CCAAAGGCCA GGTGGAAGTG GCTGAAAAGT ATCTTGACTA
cytl9 ATAGACATGA CCAAAGGCCA GGTGGAAGTG GCTGAAAAGT ATCTTGACTA 350
Figure 3-3. Alignment of the reference cytl9 nucleotide sequence and cytl9AE2. $
Represents the initial start codon. *Represents the putative PTC which results
due to the removal of exon 2. + Represents the putative downstream start site.
The kozak sequence is underlined. The deviation from the kozak sequence at
position +4 is highlighted in grey.
2 3 5 6 7 8 9
/42bp 1 i2bp 1jl 1bp
1 2 3 mRNA Splicing
7 3 V ibp 7,2 2?bp
Reference 2 3
Exon 2 1 3
Exo 1 EMon 3
GCAGgt aTATATIA Exon 2A
Eam 1 ErLon 2
GCAGgt agACCTAC Reference
Figure 3-4. cytl9 isoforms. (A) Diagram representing the exonic regions of the wild-type
cytl9 mRNA. The region in which the alternative splicing occurs is
demonstrated in greater detail. (B) Schematic representation of the two
alternative splice variants, the reference or wild-type sequence, and the
deletion of exon 2. The shaded box represents the cassette exon. (C) The
donor and acceptor site of the two alternative splice variants. The exonic
nucleotides are capitalized while the acceptor and donor sites are in lower case
MAALRDAEIQ KDVQTYYGQV LKRSADLQTN GCVTTARPVP
Figure 3-5. Alignment of the reference cytl9 amino acid sequence and product of
cytl9AE2 uORF. Consensus Motif I for SAM dependent methyltransferases
CGLVIPEHLE NCWILDLGSG SGRDCYVLSQ
AEKYLDYHME KYGFQASNVT FIHGYIEKLG
AEKYLDYHME KYGFQASNVT FIHGYIEKLG
VPDKQQVLQE AYRVLKHGGE LYFSDVYTSL
VPDKQQVLQE AYRVLKHGGE LYFSDVYTSL
LYWKELAVLA QKIGFCPPRL VTANLITIQN
LYWKELAVLA QKIGFCPPRL VTANLITIQN
KHSKTGPTKR CQVIYNGGIT GHEKELMFDA
KHSKTGPTKR CQVIYNGGIT GHEKELMFDA
KNSRFAQDFL IRPIGEKLPT SGGCSALELK
KNSRFAQDFL IRPIGEKLPT SGGCSALELK
KNSREAQDFL IRPIGEKLPT SGGCSALELK
Table 3-1. The individual information and Shapiro's score of cytl9 exon 2 and exon 3.
SSequence Splice site Shapiro's method Ri, bits
Exon 2 GACGCTGGGTCAGA Acceptor (-13 to +1) 61.5 -1.9
AAGGTAGAGT Donor (-3 to +7) 72.0 5.5
Exon 3 TTCCATTTCCCAGA Acceptor (-13 to +1) 84.8 9.5
CAGGTGAGGC Donor (-3 to +7) 88.1 7.4
a. Exon 2 is the alternative exon of cytl9 and exon 3 is the constitutive exon.
Table 3-2. The amount of cytl9 and cytl9AE2 in different human liver samples and
HepG2 cells. The percentage of cyl9 AE2 in each sample.
Samples cytl9 cytl9AE2 %cyt 9AE2a
(copies/tg RNA) (copies/tg RNA)
HL-541 1.21x106 0.49x106 3.25x103 1.14x103 0.27
HL-546 1.22x105 0.35x105 3.55x103 +0.89x103 2.83
HL-611 7.04x104 +3.91x104 1.89x103 +1.45x103 2.61
HL-612 7.89x105 +1.78x105 3.63x103 +2.70x103 0.46
HL-656 1.09x105 +0.44x105 4.33x103 +0.69x103 3.84
HL-710 8.40x105 +2.64x105 3.95x103 +1.06 103 0.47
HL-714 1.26x106 +0.42x106 3.22x103 +1.23x103 0.26
HepG2 cells 5.14 x106 1.08x106 8.55x104 + 6.42 x104 1.64
a. The percentage of cytl9AE2 is calculated by dividing the copies/Gg of RNA for each
sample by the total copies of both transcripts for each sample (cytl9AE2/(cytl9 +
The first study showed that cytl9 is an arsenic methyltransferase. In addition, we
have demonstrated that this enzyme does both methylation steps involved in arsenic
biotransformation seen in humans. We also showed that the C-terminus is critical in the
activity of the protein. In other SAM-dependent methyltransferases, the C-terminus is
important in substrate binding. This indicates that the cysteine rich C-terminus is
important for As binding and critical for activity. Others have shown that mutations can
change the methylation capacity of the protein. Here, we demonstrated that a single
mutation can drastically change the activity of the protein even though we believed that
the amino acid change would not have a significant effect on the activity of the
recombinant protein. This change occurs in motif I of the SAM-binding site which might
inhibit its ability to bind SAM therefore decreasing its activity. The mutation appears to
cause a change in the substrate affinity of the proteins as well as cause different
methylation profiles specifically for arsenite. However, the Vmax values of both cytl9-
WT and cytl9R81S are considerably higher than that seen among other mammals such as
the hamster, rabbit, and rhesus monkey. The kinetic analysis of these proteins may
explain the high levels of MMA excreted in human urine. Possibly, arsenite is converted
very quickly to MMA, allowing it to accumulate before the dimethylation step resulting
in the higher excretion of MMA seen in humans. The human arsenic methyltransferase
did have some similarities with the other mammalian arsenic methyltransferases. These
arsenic methyltransferases have been shown to increase in activity at basic pHs which
may be due to the deprotonation of cysteines at higher pHs, which increases the rate of
binding between arsenic and cysteines in the substrate binding domain. Our results
demonstrate that only a strong reductant is necessary for methylation of arsenic by cytl9,
however, the addition of GSH appears to increase the activity above the reductant alone.
The second study introduced another possible explanation for the variability in
arsenic methylation capacities among individuals, alternative splicing. Alternative
splicing is frequently used to regulate gene expression and to generate tissue-specific
mRNA and protein isoforms. Thirty-five to 60% of human genes produce transcripts that
are alternatively spliced, in addition 70-90% of these variants alter the resulting protein
products. In this study we identified an alternative splice variant of the human cytl9
(cytl9AE2), in which exon 2 is removed creating a bicistronic transcript. The cytl9AE2
variant was present in all seven human liver samples tested, suggesting that cytl9 mRNA
exists both in the full length and in alternatively spliced forms in most individuals. It is
unlikely that this variant would result in expression of an active protein. Studies have
shown that SAM dependent methyltransferases share 3 regions of sequence similarity
(motif I, II, and III). It has been suggested that these conserved regions are important in
SAM binding. Therefore, it is unlikely that the protein translated from cytl9AE2 would
result in an active protein due to the removal of exon 2 which contains motif I. Whether
the mRNA actually is translated into protein has not been determined. The majority of
mutations discovered within the cytl9 gene occur within the intron or untranslated
regions [66, 67]. In fact, only 4 mutations have been found within the coding region.
Introns contain sequence elements in which splicesome assembly occurs [66, 67].
Mutations within these elements could alter the constitutive splicing of a gene.
Future studies should focus on isolating the human AS3MT from human livers.
To this date, preparations from human livers, cytosolic or homogenates have shown no
activity in arsenic methylation. Reasons for this may be due to inhibitory factors present
in the liver preparation or the process of making the preparations may render the protein
inactive. Yet another explanation may be due to the possibility that cytl9 is an inducible
protein. Perhaps the average daily exposure does not cause high levels of expression of
the protein, making it difficult to purify from liver samples. It may be beneficial to
attempt protein purification from known higher than normal arsenic exposed populations.
Perhaps populations exposed to higher than normal levels have reached a threshold of
exposure resulting in higher expression levels of cytl9. Another possibility for
purification of cytl9 from normally exposed populations may be by immunoprecipitation
from human liver preparations using an antibody which is highly selective for cytl9.
Further studies should analyze the mRNA expression levels of cytl9 splice
variants in a larger number of fresh liver samples or primary hepatocytes and correlate it
to arsenic methylation activity. In addition, work to determine if this transcript is a
substrate for the NMD pathway or if a variant protein is expressed will help clarify the
role of cytl9AE2 in human arsenic metabolism. In addition, more investigations should
look at mutations within the intron of cytl9 and determine if these alter splicing events.
Another important study would be to establish a method to test exposed populations at
both the mRNA and protein levels of cytl9. This study would help determine vulnerable
individuals in high arsenic exposed populations. Finally, the ultimate question which still
remains unanswered: is arsenic methylation a bioactivation or detoxification mechanism.
If time and funding were not a factor, I would enjoy working on solving these different
unanswered questions which may help identifying possible vulnerable populations.
ROLE OF CYT19 IN ACUTE ARSENIC TOXICITY. IS CYT19 THE ONLY HUMAN
While my work has clearly demonstrated that cytl9 is a human arsenic
methyltransferase, it is not clear that it is the only arsenic methyltransferase in humans.
In order to address this question, it is necessary to: 1) alter the expression of cytl9 and 2)
have the ability to measure cytl9 protein levels. The first challenge was addressed by the
use of small interfering RNA (siRNA) to reduce the expression of cytl9. The second
challenge was addressed by developing an antibody specific for human cytl9.
Small RNAs can theoretically be used to reduce the expression of any target gene.
There are two main categories of small RNA, microRNA (miRNA) and siRNA. These
small RNA have natural functions such as defense from viral and transposon invasion as
well as gene regulation. Scientists have used this new technology for several reasons
such as determination of gene function, validating drug targets, and treatment of diseases.
Small RNAs have two mechanisms by which protein translation is inhibited. If the small
RNA is 100% homologous to its mRNA target it results in degradation of the mRNA.
However, if the small RNA is not 100% identical to its mRNA target it results in
inhibition of translation without mRNA degradation .
Materials and Methods
Multiple siRNAs were designed and synthesized according to the SilencerTM
siRNA Construction Kit (Ambion). Forty-five thousand HepG2 cells were plated on a 24
well plate in normal growth media overnight and transfected in duplicates with the
appropriate siRNA according to SilencerTM siRNA Trasnfection Kit. Total RNA was
isolated 2 days after transfection. The most efficient siRNA was assessed by measuring
the mRNA levels of cytl9 normalized to RNA polymerase II (RPII) by quantitative PCR
(qPCR). Once the most efficient siRNA is determined, further optimization steps can be
taken such as cell plating density, transfection agent, and siRNA amount. The siRNA
pool was further optimized in a 96-well plate using 16,000 cells per well and assayed.
The mRNA levels were measured by qPCR as above, and the viability was determined by
the XTT assay. GAPDH was used as negative control.
An antibody to cytl9 was developed in rabbits by sending purified recombinant
cytl9 to Cocalico. The antibody specificity was determined from western blots of
purified protein and HepG2 cell extracts. To increase the specificity of the antibody, it
was affinity purified using the AminoLink Kit (Pierce) according the manufacturer's
instructions. Briefly, the purified cytl9 protein was coupled to the gel followed by
Results and Discussion
cytl9 mRNA Knockdown by siRNA
Three different siRNAs were designed (Table 1). The efficiency of all three
siRNAs as well as a pool of the three siRNAs was determined (Figure 1). The data
demonstrates that the knockdown was successful with the pool of siRNAs, however
further optimization is still required. It appears that the first three siRNAs had no effect
on the cytl9 mRNA levels. The pool of all three siRNAs appears to knockdown cytl9
mRNA levels by about 26%, relative to control. The second attempt at cytl9 mRNA
knockdown demonstrated that 16,000 cells per well in a 96-well plate resulted in a
greater knockdown of cytl9 mRNA of about 40% compared to 45,000 cells used in the
first attempt (Figure 2). It appears that cytl9 mRNA levels might have been slightly
reduced in the negative control. In addition, the siRNA transfection did not result in a
decrease of viability relative to control (Figure 3). The data demonstrates that the
optimal cell density and the transfection agent are important to optimize. Taken together
the results suggest that the concentration of the siRNA requires further optimization or
perhaps new siRNAs can be designed and tested.
Antibody Specificity and Purification
Crude antiserum was shown to be able to detect the antigen up to about 25 ng
(Figure 4). The antibody was not able to detect cytl9 in any of the human liver cytosol
preparations tested. In addition, the antibody appears not to be very specific (Figure 5).
Once purified, the antibody was characterized and found to be able to detect the antigen
up to about 40 ng (Figure 6). The specificity of the antibody was increased dramatically
and is able to detect cytl9 in all tested human liver samples and in HepG2 cytosol tested
(Figure 7). In order to determine if the antibody is in fact recognizing cytl9 in these
cytosol preparations, the protein must be immunoprecipitated and sequenced.
Further studies are needed in order to better understand the role of arsenic
methylation in acute human arsenic exposures. However, two very important steps in
answering this question have been addressed, knockdown of cytl9 and the ability to
measure cytl9 protein levels through antibody specificity. We have to determine the
correlation between mRNA levels and protein levels. It may be possible to knockdown
the mRNA levels without affecting the protein levels if the protein has a long half-life.
The opposite may be true as well, we may not see much affect in the mRNA levels but
the siRNA may be able to suppress protein expression thereby reducing methylation.
Once we prove the protein levels are down, we can determine the effects of As exposure
as well as determine if cytl9 is the only arsenic methyltransferase in humans.
Table A-1. Sequence of double stranded siRNA
siRNA 1: 5'-CAU UGA GAA GUU GGC AGA GUU-3'
3'-UU GUA ACU CUU CAA CCG UCU C-5'
siRNA 2: 5'-UGU GAC UUU UUU CCA UGG CUU-3'
3'-UU ACA CUC AAA AAA GGU ACC G-5'
siRNA3: 5'-GUU GGC AGA GGC UGG AAU CUU-3'
3'-UU CAA CCG UCU CCG ACC UUA G-5'
cytl9 mRNA Knockdown
Figure A-1. Determination of the most efficient siRNA in the knockdown of cytl9
mRNA levels assayed by qPCR.
cytl9 mRNA Knockdown
Figure A-2. The siRNA pool knockdown of cytl9 mRNA relative to control.
Figure A-3. Determination of cell viability after siRNA knockdown by the XTT assay.
25ng 50ng 100ng 200ng 300ng
Figure A-4. Western blot of the specificity of the crude antisera to the antigen, purified
1 2 3 4 5
Figure A-5. Western blot of the specificity of the crude antisera to cytl9 in human liver
cytosolic preparations. 1) HL-93-F6; 2) HL-93-F7; 3) HL-94-F4; 4) HL-97-
21; 5) purified cytl9.
I I I I I I
40ng 60ng 125ng 250ng 500ng
Figure A-6. Western blot of the specificity of the purified antibody to the antigen,
purified cytl9 protein.
1 2 3 4 5 6
Figure A-7. Western blot of the specificity of the crude antisera to cytl9 in human liver
cytosolic preparations. 1) HL-97-21; 2) HL-H-F6; 3) HL-93-F7; 4) HL-94-
F4; 5) HL-714 6) HeG2 cytosol 7) purified cytl9.
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Alex J. McNally is the youngest son of Eduaurdo and Ligia McNally, who
immigrated from Managua, Nicaragua. He was born on April 6, 1980, in Miami, FL. He
was raised in Hialeah, FL, where he attended North Miami Senior High and graduated
with honors on May 1998. He then attended the University of Florida where he dualed
major in animal science and microbiology and cell science. He further enhanced his
undergraduate experience working as a student lab assistant at the University of Florida
microbiology and cell science building. He worked with cloning arogenate
dehydrogenase from Arabidopsis thaliana under the guidance of Dr. Carol Bonner and
Dr. Nemat Keyhani. After earning his Bachelor of Science, on August 2003, Alex
continued his graduate studies at the University of Florida, pursuing a Master of Science
specializing in toxicology in May of 2004. He emphasized his graduate research on the
characterization of cytl9, an arsenic methyltransferase, under the guidance of Dr. David
S. Barber. He now plans to pursue a career in a research laboratory where he can
enhance his knowledge and practical experience.