1 ERYTHROCYTE COPPER CHAP ERONE FOR Cu, Zn SUPEROXIDE DISMUTASE (CCS) AS A COPPER STATUS INDICATOR IN THE BOVINE By JOEVA JADE HEPBURN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008
2 2008 Joeva Jade Hepburn
3 To my wonderful husband. Without his endless love and support, none of this would be possible.
4 ACKNOWLEDGMENTS I would first like to than k my mother Through her own strength, ambition and independence, she taught me to never back down fr om a challenge and to always dance. I would also like to express my deep love and gratitude to my husband who never wavered in his support and was always there for me in both the good a nd bad times. I would like to thank my committee members (Dr. Gail Kauwell and Dr. John Arthingt on) for making this ex perience possible. I thank my lab members (Sukru Gulec, Ning Ning Zhao, Hye Young Nam and Supak Jenkitkasemwong) for their assistance, encour agement, and good times. Finally, the most significant acknowledgement goes to my advisor Dr Mitchell Knutson. I w ill never be able to convey my full gratitude to him for being such a wonderful mentor and a constant source of inspiration. I can only strive to live by his princi ples: that you must push yourself to the limit in order to embrace your full potential.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4LIST OF TABLES................................................................................................................. ..........6LIST OF FIGURES.........................................................................................................................7ABSTRACT.....................................................................................................................................8INTRODUCTION...........................................................................................................................9 CHAP TER 1 LITERATURE REVIEW.......................................................................................................12Introduction to Copper............................................................................................................12Intestinal Copper Absorptive Pathway...................................................................................12Copper Intake..........................................................................................................................16Secondary Copper Deficiency in the Ruminant Digestive System........................................ 16Effects of Copper Deficiency in Cattle...................................................................................18Current Copper Status Indicators............................................................................................21Copper Chaperone for Cu, Zn Superoxide Dismutase........................................................... 22Copper Chaperone for Superoxide Dismutase as a Potential Copper Status Indicator.......... 28Specific Aims..........................................................................................................................302 MATERIALS AND METHODS........................................................................................... 36Study Designs for Specific Aims I, II and III......................................................................... 36Erythrocyte Processing......................................................................................................... ..36Liver Tissue Processing........................................................................................................ ..37Western Blot Analysis of Bl ood and Liver Cell Lysates........................................................ 37Statistical Analysis........................................................................................................... .......383 RESULTS...............................................................................................................................39Detection of CCS in Bovine Erythrocytes..............................................................................39Bovine CCS Expression Increa ses in Copper Deficiency...................................................... 39Bovine CCS Levels do not Change with Inflammation......................................................... 404 DISCUSSION.........................................................................................................................47LIST OF REFERENCES...............................................................................................................51BIOGRAPHICAL SKETCH.........................................................................................................58
6 LIST OF TABLES Table page 2-1 Proteins involved in copper m etabolism and/or transport................................................. 312-2 NAHMS classification of copper adequacy in the bovine.................................................322-3 NRC forage analysis of minerals that can lead to copper antagonistic effects.................. 322-4 Bovine blood parameters in the classification of anemia.................................................. 324-1 Liver and plasma copper values at harvest (Day 490)....................................................... 434-2 Liver concentra tions at baseline......................................................................................... 44
7 LIST OF FIGURES Figure page 2-1 Copper transport........................................................................................................... ......332-2 Bovine anatomy of the polygastric system........................................................................ 342-3 Thiomolybdates sequestering copper................................................................................. 352-4 Proposed mechanism for CCS-media ted copper insertion into SOD1.............................. 354-1 Alignment of human and bovine CCS............................................................................... 414-2 Anti-human CCS antibody cross reacts with bovine liver CCS........................................ 424-3 Anti-human CCS detects bovine erythr ocyte CCS in a dose-dependent manner.............. 424-4 Copper deficiency increases CCS e xpression in bovine erythrocytes............................... 434-5 CCS expression increases in bovine liver in copper deficiency........................................ 444-6 Ceruloplasmin activity increases with vaccination............................................................ 454-7 Bovine liver and erythrocyte CCS expression does not change in inflammation.............. 46
8 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ERYTHROCYTE COPPER CHAP ERONE FOR Cu, Zn SUPEROXIDE DISMUTASE (CCS) AS A COPPER STATUS INDICATOR IN THE BOVINE By Joeva Jade Hepburn August 2008 Chair: Mitchell Knutson Cochair: John Arthington Major: Food Science and Human Nutrition After phosphorous, copper deficiency is the se cond most widespread mineral deficiency found in grazing cattle. Lack of adequate copper in beef cattle can lead to health complications such as anemia, impaired immune function, a nd failure to thrive re sulting in significant economic losses. Given the severity of these pot ential complications, the need for a sensitive, convenient copper status indicator is warranted. Recent studies have shown that the levels of copper chaperone for Cu, Zn Superoxide Dismut ase (CCS) in erythrocytes are reflective of copper status in mice and rats. The objective of this study was to evaluate erythrocyte CCS as a possible status indicator in the bovine, and to test the respon se of CCS under conditions of inflammation, as this is a limitati on with current copper status i ndicators. We demonstrate that bovine erythrocyte and liver CCS protein levels significantly increase (P < 0.05) in copperdeficient cattle. Furthermore, CCS protein levels were did not change in a vaccine-induced inflammatory response implying that CCS does no t exhibit acute-phase pr operties. Despite the significant increases in CCS protein concentration to a copper deficiency, the response was not as robust as those observed previ ously with rodents. CCS may not be sensitive in detecting a moderate copper deficiency in cattle.
9 CHAPTER 1 INTRODUCTION After phosphorus, copper deficiency is considered the second m ost common mineral deficiency in beef cattle (1). The National An imal Health Monitoring and Surveillance program (NAHMS) reports that 43% of beef cattle are cl assified as copper deficient based on serum copper values. This deficiency pe rsists despite the fact that ove r 64% of beef cattle operations provide regular copper supplementation well above the National Research Council (NRC) recommendations of 10 ppm (mg/kg) per day on a dry matter basis to maintain copper adequacy (1). Copper deficiency in these animals arises from a secondary condition rather than a lack of sufficient dietary copper. Secondary copper deficiency results from hi gh levels of copper antagonists in the soil, feed, or water supply. Copper antagonists such as sulfur, iron, molybdenum, and sulfurcontaining compounds such as thiomolybdenum, ch elate copper in the stomach and rumen and render the molecule unavailable for absorption (2 ). In the NAHMS Beef 1997 report, forage samples across the US were analyzed for copper c ontent as well as levels of copper antagonistic minerals. In Florida, forage is mainly compri sed of Bahiagrass and Bermudagrass, and according to the analysis of Bermudagrass, 64.3% of the forage samples were not only marginally deficient in copper, but 87% of the samples also containe d marginal to high leve ls of copper antagonists (3). A lack of adequate copper in beef cattle can lead to health complications, even when the animal is only marginally deficient. Complications include anemia due to l ack of availability of copper in the ferroxidase cerul oplasmin (72), loss of hair pi gmentation due to the reduced copper-dependent conversion of Ltyrosine to melanin, impaired immune function and failure to thrive (2, 5).
10 Given the severity and prevalence of a c opper deficiency and it adverse effects, a sensitive copper status indicator is needed. Curren tly, the most accurate form of testing is a liver biopsy (1). The liver is the prim ary storage site of copper and a hepatic copper concentration of 75-90 ppm (dry matter basis) or below classifies the animal as copper deficient. Although liver samples provide the most accurate indicator of copper status in the system, the procedure is highly invasive to the animal and requires trained personnel. As a result, serum or plasma copper is often used due to the conve nience of sampling (1). The main limitation of serum and plasma copper as status indicators is that more than 80% of plasma copper is bound to ceruloplasmin an acute-phase protein that incr eases during periods of inflammati on and stress (80). In addition, serum copper concentrations vary by the age, ge nder, and species of the animal (2). These fluctuations limit the validity of the test. Another potential status indicator that has been explored is the copper-co ntaining enzyme diamine oxidase (DAO), which catalyzes the deam ination of histidine, diamines and diamine derivatives. The activity of the enzyme significantly decreases in copper deficiency, and has been shown to be reflective of copper status in the bovine (6). The main limitation with DAO is that it is highly active in the kidney and intes tines, and its activity has been shown to increase independent of copper status in animals with renal or intestinal disease. The protein Cu, Zn superoxide dismutase (SOD1) has also been investigated as a possible copper status indicator. Severa l experiments have shown that S OD1 protein levels significantly decrease in the erythrocytes of copper-deficient rats and mice due to the missing catalytic copper cofactor (7). Measurement of SOD1 protein concentrations c ould serve as a potential copper status indicator, though it may not provide an accurate reflec tion of copper status as the nonfunctional apo form of the protein is still de tectible. A more accurate measurement therefore
11 would be to examine SOD1 activity as it has been shown to be more reflective of copper status (8). Previously, studies have shown that the copper chaperone for Cu, Zn Superoxide Dismutase (CCS) protein levels significantly increases in the liver and erythrocytes of copperdeficient mice and rats (7, 9). The CCS protein coul d be a viable marker of copper deficiency in the bovine, but its expression, acutephase characteristics, as well as response to copper status has not yet been assessed in the bovine system. CCS could serve as a sensitive copper-status indicator in the bovine.
12 CHAPTER 2 LITERATURE REVIEW Introduction to Copper Copper is a trace m ineral that is essential to li fe. Copper is a d-block transition element that can assume two distinct valence states : the reduced monovalent cuprous ion (Cu+) and the oxidized divalent cupric ion (Cu2+). Copper has the ability to sw itch between valance states and this redox chemistry is the reason why it is a crucial component of many enzymes in the body. Table 2-1 lists various proteins that are associated with copper transport and/or metabolism. Though copper is an important mineral to sustai n life, it can also be detrimental to the body. Copper has the ability to generate hydroxy l free radicals through the Fenton reaction: Cu+ + H2O2Cu2+ + OH + OHresulting in irreversible oxidative damage to tissues and cells (14). Due to the potential negative effects of copper, it s extracellular as well as intracellular transport and utilization are tightly regulated in the body (15). Intestinal Copper Absorptive Pathway In m ost organic food matter, copper is prim arily bound to sulfur-containing amino acids and dissociates from these complexes in the early stages of digestion. Copper is absorbed in the duodenum, though the exact mechanism is currently be ing investigated. It has been proposed that copper absorption is mediated eith er exclusively or in combinati on by either copper transporter 1 (CTR1), divalent metal transporter 1 (DMT1) or by an unknown saturable process. The human copper transporter 1 (hCTR1) was discovered in 1997 through functional complementation analysis with the yeast Ctr1 gene, and was late r characterized to mediat e the monovalent copper (Cu+) uptake in an ATP-independent manner with high specificity and affinity (16, 17). To investigate whether CTR1 mediat es the apical absorption of c opper, a CTR1 intestinal knockout mouse was generated (17). Ablati on of intestinal CTR1 resulted in reduced copper transport
13 across the intestinal ep ithelial tract and copper-dependent enzyme deficiencies. Contrary to expectations, copper absorption with in the intestinal epithelial cell (IEC) was not inhibited, and the IEC exhibited massive copper accumulation that was eight to ten times that of the control mice. This suggested that copper still crossed the apical membrane and accumulated in a nonbioavailable pool. Transport to th e basolateral membrane for excre tion into portal circulation did not occur (17). Though it is unclear as to why c opper loads in a biologically unavailable pool in the IEC, these data show that CTR1 is essent ial for copper absorption into portal circulation. Furthermore, CTR1 is essential for life since the CTR1 null mutation is embryonically lethal (18). CTR1 can only mediate the transport of the monovalent cuprous ion (Cu+); however the majority of dietary copper exis ts in the divalent form (Cu2+). Reduction of Cu2+ at the apical surface of enterocytes therefore needs to occur before transport by CTR1. The six transmembrane endothelial antigen of the prosta te (STEAP) proteins are known to reduce copper and could potentially fulfill this role, particularly the STEAP2 and STEAP3 isoforms (19, 20). The divalent metal transporter 1 (DMT1) is the other potential candidate for intestinal copper absorption. DMT1 is known to transport iron across the apical membrane, but it can also transport other divalent cations such as Mn2+, Cu2+, Co2+, Ni2+, and Pb2+ (21, 65). A recent study demonstrated that when Caco-2 cells were treated with the antisense oligonucleotide to DMT1 in order to reduce DMT1 protein expression, iron as well as copper transport were significantly reduced. Furthermore, in cellular uptake studies, Fe and copper could inhibit the others uptake suggesting that both metals share a common transporter. This inhibition however was only observed in the presence of ascorbate sugge sting that DMT1 transports monovalent (Cu+), the predominant form in acidic conditions (82). DM T1 could possibly play a role in non-specific intestinal copper absorption especi ally if copper levels are high.
14 Once in the enterocyte, copper is exported into portal circulation by the Menkes protein ATP7A. The ATP7A copper exporte r is localized to the transgolgi network under basal copper concentrations and imports copper to the Golgi apparatus for inco rporation into metalloproteins. As copper accumulates, the protein translocates to the basolateral membrane of the enterocyte where it actively exports copper into portal circulation using the energy from ATP hydrolysis (22). Defects in the ATP7A transporter results in Menkes disease, a recessive, X-linked inherited disorder that results in chroni c copper loading in the enterocy te due to the dysfunctional copper exporter (23). This leads to a systemic copper deficiency because the copper cannot be released from the enterocyte for utilization in the peri phery. Complications from the disease include abnormal hair structure, commonly termed ki nky hair, mental retard ation, connect ive tissue defects, and severe anemia, which are all symp toms attributed to the dysfunction of copperdependent enzymes (24, 25). Once in portal circulation, copper travels to the liver bound to transcuprein (10), albumin (26, 27) or other soluble peptides and amino acids. The liver is the main storage site for copper and is an important regulator in copper homeostasis. In copper excess, the biliary secretory pathway becomes activated and much of the copper becomes incorporated into bile for secretion back into the small intestine (15) Bile is the major excretory pa thway of copper and while it is true that bile is actively reabsorbed, much of the copper is lost. Under conditions of copper adequacy, copper is transported to the Golgi apparatus in the liver through the Wilson protein ATP7B for incorporation in the multicoppe r ferroxidase ceruloplasmin. ATP7B is a transmembrane copper transporter that is complimen tary in structure and function to ATP7A, but is predominantly expressed in th e liver (62). Missense mutations of the metal-binding domains of the ATP7B copper exporter protein results in Wilsons disease, an autosomal recessively
15 inherited disorder of copper metabolism (28). The disease results in severe copper loading in the brain, liver and kidney and leads to hepatoenticular degeneration as a result of the impaired ability to export copper to the secretory pathway for biliary excretion or for incorporation into ceruloplasmin. Chronic copper loading increa ses DNA damage, enzyme inactivation, and lipid peroxidation (29). Once copper is incorporated into ceruloplasmin, th e protein is released into the circulation through the secretory pathway of the Golgi netw ork. Ceruloplasmin accounts for over 80% of the copper found in circulation and though it plays an e ssential role in iron metabolism, its role in copper metabolism has yet to be elucidated. Ce ruloplasmin has long been proposed to function as a copper transporter since it binds the ma jority of serum copper (15). This hypothesis however, has recently been challenged in light of the Cp knockout mouse that exhibits normal copper metabolism in the periphery indicating th at ceruloplasmin is not needed for copper transport (63). Further research is needed to id entify the protein responsi ble for copper delivery to the peripher al tissues. Peripheral tissues import copper through CTR1 (64) and copper becomes bound to soluble intracellular receptors known as metallochaper ones. The metallochaperones cytochrome C oxidase assembly protein (COX17), human ATX1 homologue (HAH1) and copper chaperone for Cu,Zn Superoxide Dismutase (CCS) function to deliv er copper to specific target proteins in the cell (15). COX17 delivers copper to the enzyme cy tochrome C oxidase located in the terminal region of the electron transport chain in the m itochondrion (30). The tran sport of copper to the secretory pathways of the cell is mediated by HAH1 (31). CCS transports copper to the SOD1 protein, which functions to protect the cell from free radica l damage (Figure 2-1) (32).
16 Copper Intake In the US, the recomm ended daily allowance for copper is 0.9 mg/day for adult humans, with the average adult consuming approximately 0.6-1.6 mg of dietary copper per day (33). Copper is present in a multitude of plant and animal sources. It is abundantly rich in shellfish and organ meats such as liver, and in seeds, grains, and nuts. Copper deficiency in humans is not a comm on occurrence, unless a genetic disorder is present. In grazing cattle however, this is a significant issue. The National Research Council recommends a dietary copper in take of 10 ppm (mg/kg) on a dr y matter basis to maintain adequate copper status in beef cattle, which is normally attain ed through supplementation (1). The United States Department of Agriculture (USDA) states in their 1997 NAHMS report that 64% of the beef cattle operations provided regular copper supplementation in the form of free choice minerals in which the ca ttle consumes mineral mixtures ad libitum (1). Nonetheless, many grazing cattle remain copper-deficient. (34). Approximately 43% of th e beef cattle in the US are classified, by serum copper values as having moderate (0.25 ppm-0.65 ppm) to severe (less than 0.25 ppm) copper defi ciency (Table 2-2) (1). Copper deficiency usually persists despite ad equate supplementation because a deficiency can result as a primary or a secondary problem. Pr imary copper deficiency occurs when there is inadequate copper in the diet, which is not us ually a problem in the US where as mentioned previously, copper supplementation is common. Most cattle suffer from copper deficiency as a result of a secondary antagonistic condition. Secondary Copper Deficiency in the Ruminant Digestive System Intestinal copper absorption is sim ilar in both the monogastric and polygastric digestive systems in that primary absorption takes place in the proximal region of the small intestine. The transit route, however, as well as the bioavailability of coppe r, is markedly different. The major
17 unique characteristic of the polyga stric digestive system is the four-compartment stomach known as the ruminant. It is composed of the rumen, reticulum, omasum, and the abomasum (Figure 2) (35). Ruminant digestion is orchestrated through the elegant symbiotic re lationship that exists between the host organism and bacteria l microbes that populate the rumen. Microbial organisms ferment the cellulose matte r from the ingested feed and as a result, they create volatile fatty acids such as acetate, proprionate, and butyrate as a waste by-product. The host organism absorbs and metabolizes these volatile fatty acids into energy. This symbiotic relationship between the microbes and the host or ganism also promotes copper antagonistic activity. The rumen maintains a reducing environm ent necessary to maintain microbial growth. Dietary sulfur consumed from the feed, soil, fertil izer, or water is thereby reduced to the sulfide form under these conditions. Free sulfide can either chelate copper directly forming the insoluble copper sulfide (CuS), or it can combine with di etary molybdenum to create substances known as thiomolybdates (36). Thiomolybdates can have up to four degrees of sulfur binding and exhibit extreme copper antagonistic effects (Figure 3). The higher the degree of sulfur binding, the greater the antagonistic activity (36). Thiomolybdates can i nhibit copper availa bility in two ways: by binding the copper in the gastrointestinal system or by interactin g with copper once in the systemic circulation. In one study, invest igators showed that absorbed thiomolybdates induced increased biliary copper losses from liver stores, re duced copper transport capacity, and dissociated copper from metalloenzy mes (37). Spears et al. (37) demonstrated that thiomolybdate formation is a function of the amount of dietary sulfur consumed. When dietary sulfur levels were low, molybdenum did not in hibit copper availabil ity; however, as th e concentration of sulfur increased, thiomolybdates formed more read ily and decreased copper availability. Iron has also been shown to antagonize copper absorp tion although the exact mechanism is currently
18 unknown. It has been suggested that Fe2+ can combine with sulfur (S2-) to form ferrous sulfide (FeS) complexes in the rumen. These complexes la ter solublize in the ab omasum, and dissociates allowing the free sulfide to chelat e copper (CuS) (66). Multiple st udies have confirmed that high levels of dietary iron reduce plas ma and liver copper to levels i ndicative of deficiency (67). The National Research Council (NRC) ha s established critical levels of copper-antagonistic metals in forage that would lead to a coppe r deficiency (Table 2-3) (3). In Florida, beef cattle forage consists main ly of Bahiagrass and Bermudagrass. According to the NAHMS 1997 Beef Study analysis of Bermudagrass, 64.3% of the forage samples were marginally deficient in copper and 87% of the samples contained marginal to high levels of copper antagonists (3). Effects of Copper Deficiency in Cattle The m ost notable effect of a copper deficiency is anemia. In 1928, the first studies were conducted that shed light on the relationshi p between copper and ir on in rats leading investigators to propose a possible role for copper in hemoglobin formation (38, 39). This hypothesis was confirmed in classical studies using swine that demonstrated that diets deficient in copper but adequate in all other nutrients produced the same anemia as was observed with an iron deficiency, but such that supplemental iron was unable to correct. Copper-deficient animals exhibited a reduction in mean corpuscular volume (MCV), as well as marked microcytosis and hypochromia due to the reduction in hemoglobin fo rmation (40). The pigs became lethargic and if not rescued with copper supplementation, would die from tissue anoxia within 2 months (40). Similar observations have been reported in coppe r-deficient cattle (74). Studies have shown that in severe and prolonged copper deficiency, cattle develop anemia, though it is not without some degree of variability. Calves have greater hemoglobin content and red ce ll counts than adult cattle, and sexually mature bulls are reported to ha ve higher levels of eryt hrocytes than females
19 of the same age (76). Despite the age and gender differences, anemia is clearly observed in copper-deficient cattle. In a recent study, coppe r deficiency was induced by feeding calves a copper-deficient diet containi ng 1.3 mg/kg copper, which was further supplemented with 4 mg/kg molybdenum and 3g/kg sulfur. The onset of copper deficiency occurred at approximately 15 3.2 weeks, and by the end of the study (30 w eeks), copper concentrations in the liver (16 g/g) and plasma (0.1-0.2 mg/L) dropped well be low the NRC established levels for copper deficiency. The mean hemoglobin value at this time point was 6.7 g/dL, indicative of anemia (72). Table 2-4 lists the ranges of hematological measurements fo r the bovine. The anemia that results from a copper deficiency is due to the reduction in both ceruloplasmin and hephaestin activity. Both proteins utilize th e redox potential of copper to catalyze the oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+), which is necessary for the incorporation of iron into the plasma transport protein, transferrin (41). Without the function of these two copper-dependent proteins, iron cannot be efficiently loaded onto transferrin, which trans ports iron to the bone marrow for hemoglobin synthesis. Inadequate iron deliv ery to the erythron results in anemia. In addition to the lethargy caused by the anemia a copper deficiency can be detected when animals begin to lose hair pigmentation. This is the visual clinical sign of an overt copper deficiency in cattle and first a ppears around the eyes typically gi ving the animal a spectacled appearance (75). Melanin, the protein attributed to pigmentation is formed by the hydroxylation of tyrosine to L-3,4 dihydroxyphe nylalanine (DOPA) followed by th e subsequent oxidation to dopaquinone by the copper-dependent enzyme tyrosi nase. Tyrosinase activity is credited to the redox potential of two copper cof actors and without sufficient c opper due to a deficiency, the enzyme loses its catalytic activity and the synthe sis of melanin does not occur (42). The classic graying of the hair results due to lack of proper pigmentation.
20 While anemia and pigmentation abnormalities present a significant problem, especially in the beef industry where animal performance and appearance are critical for economic value, the detrimental effects of copper deficiency arise primarily from immune complications. Immune dysfunction in animals has been linked to a deficiency in copper in both in vivo and in vitro systems. It is well established that copper defici ency leads to neutropeni a reducing the amount of neutrophils available to fight in fection thereby placing the organi sm at increased susceptibility for infection (43). Copper-deficient rats and mice, when exposed to an adjuvant challenge, have been shown to have significantly fewer an tibody-producing cells and lower antibody titers compared to controls (44, 45). Furthermore, splenic mononuclear cells (MNCs) extracted from copper-deficient rats exhibited reduced IL-2 activity when e xposed to the phytohemagglutinin (PHA) adjuvant at a level of 4050% that of controls. Although th ese effects have been observed with various cell lines and rode nt models, the effect of a co pper deficiency on the immune system in the bovine is less-well characterized. Studies of the in vivo bovine response to copper deficiency are inconsistent. A recent study examined the effects of a secondary copper deficiency induced by either ir on or molybdenum on the immune f unction of calves. Calves were injected with porcine erythroc ytes (PRBC) at two time points throughout the study to induce an inflammatory response. Blood samples were rout inely taken to assess humoral immune function by measuring the plasma concentrations of PRBC antibody. In vitro lymphocyte blastogenic response was also assessed. Alt hough antibody titers were lower a nd blastogenic responses were higher in the molybdenum-induced copper-deficient group compared to both the iron-induced copper deficient and control calves, the results were largely inconsistent and varied between trials (69). In a similar st udy, bovine immune function was evaluated by administering live herpes virus to control and molybdenum-i nduced copper-deficient calves. Lymphocyte
21 proliferation responses in the copper-deficient group varied depending on the mitogen stimulus. The findings of the study did dem onstrate significant changes in the acute-phase response, which could play a role in altered im mune function (68). Alternatively, the increased susceptibility to infection could result from reduced antioxidant activity in lym phocytes. Indeed a recent study reported that activities of c opper-dependent proteins cyto chrome C oxidase and Cu,Zn Superoxide Dismutase are signif icantly lower in peripheral blood lymphocytes, neutrophils and macrophages relative to controls (81). The combination of severe anemia and a tendenc y for increased susceptibility to infection contributes to an animals overa ll failure to thrive. Indeed, co pper-deficient calv es have been shown to grow 30% slower than copper-adequa te animals despite no differences in food consumption (72). This results in severe economi c losses especially in the beef industry where not only primary, but secondary copper defi ciency poses a significant problem. Current Copper Status Indicators Given the severe com plications seen with a copper deficiency, the need for an accurate copper status indicator is warrant ed, especially in th e cattle population wher e copper deficiency is a problem. Currently, liver biopsies represent the most accurate form of testing because the liver is the primary storage site of copper (1). However, the procedure is highly invasive to the animal, time consuming, and requires trained personnel. Instead, serum copper is often used due to the convenience of sampling (1 ). In bovines with adequate li ver copper stores, plasma copper concentrations will remain within normal ranges. The liver will exhaust its copper stores in order to maintain the level of plasma copper in th e blood. Accordingly, when liver stores become depleted below 40 ppm, plasma copper levels fall (71). Low plasma copper levels suggest copper deficiency, but by the time they are detected, the animal could already be suffering from the adverse effects (75). A study compared serum and plasma copper levels since the two indicators
22 are interconvertibly used to assess copper st atus. Serum copper levels were found to be consistently lower (approximately 67%) to that of plasma values suggesting that a significant amount of copper is lost to the clotting proce ss (79). Furthermore, approximately 34.5% of the animals were diagnosed according to their serum va lues as being marginally deficient in copper, which was not the case when compared to thei r plasma copper concentrations (77). Plasma copper provides a more accurate reflection of th e copper status of the system. Both indicators however are limited in that both plasma and seru m copper concentrations fluctuate with age and gender of the animal, thus limiting the validity of the test (72, 73). Additionally, more than 80% of copper in serum and plasma is bound to the fe rroxidase ceruloplasmin, an acute-phase protein that increases under periods of inflammation a nd stress (80). Inflammation increases plasma copper concentrations and coul d mask a copper deficiency. The copper chaperone for Cu, Zn Superoxide Dismutase (CCS) has been shown to be highly responsive to the copper status in mice a nd rats (7). In copper de ficiency, the expression of the intracellular metallochaperone CCS increase s in liver and erythrocytes (7, 9). CCS has not yet been characterized in the bovi ne and a response to inflammatory changes has also not been assessed. The copper chaperone for Cu, Zn supe roxide dismutase could serve as a status indicator for copper in the bovine. Copper Chaperone for Cu, Zn Superoxide Dismutase The copper chaperone f or Cu, Zn superoxi de dismutase was first discovered in S. cerevisiae in 1995 through molecular cloning techniques (46). The initial name was LYS7 as it was proposed to be involved in the conversion of homocitrate homoaconitate in the lysine metabolism pathway. The LYS7 gene predicte d a 249 amino acid protein that shared no significant homology to other known proteins. A LYS7 mutant was created in yeast to test the
23 function of the protein and it was discovered that the LYS7 strain exhibited pleiotrophic phenotypes that were not exclusive to a defect in lysine metabo lism. This suggested that the function of LYS7 was not limited to lysine biosynthesis (46). Two years later, in 1997, a hypothesis was propo sed that LYS7 was functionally linked to a protein known as Cu, Zn Superoxide Dismutase (SOD1) (32). SOD1 is a cytosolic protein that is abundantly and ubiquitously ex pressed (47). It exists as a homodimer in the body and each monomer requires both a single zinc atom for structural stability, as well as the redox potential of a copper atom for catalytic function. SOD1 catal yzes the disproportionation of the superoxide ion to form the more stable products mol ecular oxygen and hydrogen peroxide (47). This reaction is crucial for cellular antioxidant defense to prevent oxidative damage to proteins, lipids, and nucleic acids (48). In the absence of the co pper cofactor, SOD1 lacks functional activity. It was proposed that LYS7 served as a metallochaper one to deliver copper to this target protein. The SOD1 and LYS7 null mutants were both auxotrophic to methionine and lysine when grown in aerobic conditions and lacked SOD1 activity when exposed to the oxidant nitro blue tetrazolium (NBT). In addition, immunofluorescence indicates that both proteins colocalize in the cytosol (32). Multiple human expressed sequence tag (EST) homologues to LYS7 were discovered, and through sequencing, a 274 amino acid human ort hologue protein that shared 28% homology to LYS7 was identified. The human orthologue, when transformed into the mutant LYS7 and SOD1 yeast strains, failed to restore SOD1 activity in SOD1 mutants, but was able to fully restore the activity in LYS7 mutants. This demonstrated that the human protein was indeed an orthologue to LYS7. Thus a name more appropri ate to its function was given: copper chaperone for Cu, Zn superoxide dismutase (CCS) (32). Wa s CCS specific to only SOD1, or could another
24 well characterized chaperone performing the same function? Yeast mutants were transformed with another known metallochaperone, ATOX1, to evaluate the restorat ion of SOD1 activity. ATOX1 was unable to restore SOD1 function in both the LYS7 and SOD1 mutants, indicating that CCS was a specific and essential chaperone for SOD1 (32). The discovery of CCS as metallochaperone to SOD1 led to the hypothesis that CCS could play a role in the fatal neurodegenerative dise ase familial amyotrophic lateral sclerosis (FALS). Amyotrophic lateral sclerosis (ALS ) is characterized by the sele ctive loss of motor neurons located in the spinal cord, brain stem and motor cortex (49). FALS represents 10% of ALS cases, and approximately 25% of these result from positi onal mutations in SOD1 that confer a toxic gain-of-function activity (48, 50). Mutations in SOD1 result in loose folding properties and when CCS loads copper to SOD1, the copper mol ecule is no longer buried within the protein. Exposure of the copper atom to the cellular enviro nment promotes the inte raction of copper with hydrogen peroxide and peroxynitrit ecatalyzing the formation of free radicals (50). CCS is ubiquitously expressed and colocalizes with S OD1 in multiple regions in the human central nervous system, particularly in motor neurons and astrocytes. (51). The hypothesis was proposed that if the source of copper could be blockedCCS to the FALS-SOD1 mutantthen the neurodegenerative process could be abrogated. In order for this to be a viable hypothesis, it had to be proven that SOD1 as well as the FALS-SOD1 could bind copper in vivo (50). This led to a series of investigations to determine the m echanism of copper incorporation into the target protein SOD1. The sequence of human CCS was analyzed and compared to the target protein SOD1 (52). Sequence alignmen t revealed a 47% homology between CCS and SOD1 from amino acids 86-234 identified as dom ain II. The first 85 amino acids(domain I), contained an MXCXXC metal bindi ng motif (where X represents any amino acid) that was also
25 common and essential in the copper ATPases and the ATOX1/HAH1 metallochaperones. The final domain, domain III, demonstrated no known ho mology to other proteins, but did contain a putative peroxisomal localization sequence (AHL) at the carboxyl terminus (52). SOD1 exists as a homodimer in vivo but there was increasing evidence th at suggested that a heterodimer complex could potentially exist between CCS a nd SOD1 to coordinate the copper transfer process due to the homologous sequences found in doma in II. To test this hypothesis, a series of column binding assays were performed using comp lete or abrogated CCS contructs (domain I or domain II/III) that were linked to glutathione S-transferase (GST) or to the amino acid histidine. The linked CCS molecules were i mmobilized on target-specific agarose beads and immersed in a solution containing COS-1 cell lysates that were transiently transfected with the SOD1 gene. Western analysis revealed that the SOD1 protein bound to the fu ll-length CCS protein as well as to the CCS contructs that contained domains II/I II. SOD1 did not bind to CCS contructs that only contained domain I, suggesting that the MXCXXC motif was not necessary for SOD1 binding. These results indicated that coppe r incorporation of SOD1 from CCS involved dir ect protein-toprotein interaction of a heterodimer complex. The model of the heterodimer complex was fu rther supported by crystal structure analyses. Lamb et al. (53) solved the initial crystal stru cture for apo-CCS and revealed that the protein actually presented as a dimer in vivo A detailed investigation of domain II revealed that the domain was similar to SOD1 but lacked the catalytic elements of copper binding (48). The dimer interface amino acid resi dues of the second domain of CCS shared a 64% homology with the residues that are located on the dimer interf ace of SOD1 and are highly conserved both structurally and sequentially. Furthermore, the mean change in accessible surface area ( ASA) in
26 CCS was similar to the ASA of SOD1, which gave strong ev idence that a heterodimer complex can be formed without steric interference between the invo lved residues (48, 53). The function of the individual protein domains on CCS was assessed by expressing physiological levels of truncated forms of th e yCCS protein (yCCS-dI/II, yCCS-dII/III and yCCS-dIII) into LYS7 strains and monitoring SOD1 activ ity by growing yeast under aerobic conditions (54). As stated previously, yeast lacking SOD1 activit y are auxotrophic to methionine and lysine in aerobic conditions. Contrary to e xpectations, the yCCS-dI/ II construct did restore SOD1 activity whereas the yCCS-dII/III construct di d, though slightly weaker than the wild type protein. This was a pivotal point in the CCS studi es because prior to this knowledge the third domain was regarded as a minor player in the copper delivery process to SOD1. The primary focus was placed on domain I because it contained the MXCXXC copper-binding motif, and on domain II due to the structural similarities to th e target protein SOD1. Th e surprising activity that was observed with the isolated yCCS dII/III constructs led to the hypothesis that the third domain may contain a functional copper-binding site. Sequence analysis of the third domain revealed a putative CXC copper-binding motif that was perfectly conserved in the CCS family between mammals, fungi and insects. Treatment of a peptide representing this domain (Alanine216 Lysine249) with Cu(I) tested the functionali ty of the CXC motif. Domain III possessed the ability to bind coppe r, but was it essential for CCS function? A simple experiment proved that mutations of the key cysteine residue s in the third domain resulted in a complete abrogation of SOD1 activity. Domain III was ab solutely critical for CCS function. Taken together a model was proposed with domains I and III acting in tandem to deliver copper to the target protein SOD1 since they both contained copper-binding motifs (54).
27 Additional crystal structures studies proved that a heterodi mer complex existed between CCS and SOD1 that was stabilized through f our strong main-chain hydrogen bonds and that domain III was the factor that delivered copper to the active site of SOD1. The CXC motif of domain III was located adjacent to the active site of the SOD1 complex. This was in contrast to domain I, which was located ~35Ao from the SOD1 active site where it would not be in a position to deliver copper to the active site (55). Compiling all the results from multiple stud ies led to an intricate copper delivery mechanism model between CCS and SOD1 (Fi gure 4). The model was based on experiments tested in yeast, but has been extrapolated to e ukaryotic systems. The mo del is a 4-step process that involves first the heterodimeric bi nding between CCS and SOD1, and through the interaction of distinct cysteine amino acid residues of domain I and III, the copper molecule is delivered to the catalytic site of SOD1 post-translationally through the oxidation and reduction of disulfide bonds (32, 56). Remarkable progress had been made with CCS but several questions remained. It has been observed that SOD1 has the ability to acquire copper and self-activate in vitro independent of CCS, however under in vivo conditions, this selfactivation is not seen. Why is CCS required for in vivo but not in vitro activation? In an eleg ant study, apo-SOD1 was treat ed with either Cu(I)CCS, Cu(I)-GSH, or CuSO4 (57). All three donors had the abi lity to activate the apo-SOD1 protein, but when the potent copp er chelator bathocuproine di sulfonate (BCS) was added to solution reducing copper av ailability to only 10-17 M of free copper, only the Cu(I)-CCS donor retained the ability to activate the apo-SOD1 protein. The results supported the hypothesis that in vivo the cellular milieu has an over chelation of copper such that the concentration of
28 intracellular copper is virtually undetectable givi ng rise for the necessity for metallochaperones to deliver copper to target proteins. The compounding evidence shed no doubt that CCS was essential for the activation of SOD1 in yeast, but was it essential in mammals? A CCS-/mouse was created using homologous recombination techniques to inact ivate the CCS gene (58). The CCS-/had dramatic effects on SOD1 activity. SOD1 gel activity assays demonstr ated a pronounced reduction of SOD1 activity that was observed in all tissues. Metabolic labeling with 64Cu proved that the reduction in activity was attributed to the missing catalytic component. However, despite the reduction of activity, there was still retention of 10-20% of wild type SOD1 activity with the exception of liver, which was able to retain 30% activity. Nevertheless, the reducti on was severe enough to conclude that CCS was essential for in vivo copper incorporation for e fficient activation of the SOD1 enzyme. A FALS-linked SOD1-mutant mouse model was la ter created that lacked the CCS gene to test the original hypothesis that if the sour ce of copper could be blocked, the symptoms associated with FALS would be reso lved (49). Metabolic labeling with 64Cu in the FALS-SOD1 mutants demonstrated that copper was not being incorporated into SOD1. However, contrary to what was expected, mutant FALS-SOD1 retained the ability to induce motor neuron cell death independent of CCS (49). The cure for FALS did not lie in CCS inhibition in an attempt to abrogate copper delivery. Copper Chaperone for Superoxide Dismutase as a Potential Copper Status Indicator Shortly after the discovery that inhibition of CCS did not prevent disease progression in FALS, a novel function for the protein was prop osed. There had been a growing interest in finding a reliable copper status indicator that would reflect a ccurate m easurements of copper concentration since the current copper status indicators were inadequate (8). The focus was
29 placed on CCS as a potential copper status indicator given that it was essential to activate SOD1. In a pilot study, rats were fed diets with varying copper concentrations. CCS protein expression in erythrocyte and liver samples of rats fed adequate amounts of copper was relatively low. However, in rats fed a copper deficient diet, th e CCS protein levels were markedly up-regulated in a dose-responsive manner related to the severity of copper restriction (8). The increase in the abundance of CCS was likely a regulatory mechan ism to improve the efficiency of copper scavenging to allow the SOD1 antioxidant capacity to be maintained (9). Copper-deficient rats exhibited a 9-fold increase in CCS protein leve ls in liver compared to the control group. No changes, however, were observed in mRNA levels between groups suggesting that the increase in protein levels was from a decrease in degrad ation (9). CCS is regulated by a posttranslational mechanism involving copper. Subs equent studies showed that copper regulates CCS expression by promoting its degradation. The proposed mechanism is that holo-CCS, which would be abundant in a copper-adequate environment, adopts a conformation that is not as stable as the apo-CCS form, which is more predominantly found in a copper-deficient state. The instability of the holo-CCS reduces the lifespan of the protein and it is targeted for degradation by the 26S proteosome (9). Species-specific differences have been obser ved in the copper-dep endent regulation of CCS. In a study performed by Bertinat o et al. (9), CCS protein levels were examined in two liver cell lines maintained in a coppe r-deficient environment: one from rat and the other from human. The CCS protein levels from the rat cell line ga ve a more robust 6-fold increase in protein concentration compared to that of the human liver cell line, where a more modest 3-fold increase was observed. The species-specific variations in CCS response to copper status should be taken into consideration.
30 Although CCS protein levels have been shown to increase in liver samples in a copper deficiency, for a status indicator to be applicable to bovines, a more convenient sampling source is necessary. Two studies were performed to examine CCS erythrocyte expression in mice and rats. Both studies concluded that CCS expression in erythrocytes is res ponsive to changes in copper status (7, 8). CCS protein levels were significantly higher in the copper-deficient groups compared to controls and expression in erythroc ytes was similar to that which was observed in previous studies examining liver cells. The curr ent project proposes to investigate the use of erythrocyte CCS as an indicator of copper status in the bovine. Specific Aims Aim 1: Establish a m ethod to measure bovine erythr ocyte CCS levels: To use CCS as a potential status indicator for copper in the bovine, a convenient sampling source is desirable. Red blood cells are readily available and erythrocyte CCS pr otein has been shown to be reflective of copper status in rats and mice (7). Aim 2: Determine if bovine erythrocyte CCS levels are responsive to copper status: To determine if bovine CCS protein exhibit similar increases in concentra tion in experimentally induced copper-deficient cattle. Aim 3: Determine if CCS levels are modulated by inflammation: To determine if CCS protein levels are altered under conditions of inflammation as it would lim it the validity and use of CCS as a copper status indicator.
31 Table 2-1. Proteins involved in copper metabolism and/or transport Protein Function Transcuprein -high affinity Cu2+ carrier th at transports copper to the liver (10) Albumin -Serum protein that acts as a Cu2+ carrier for transport to the liver (11) Ceruloplasmin -Serum ferroxida se that oxidizes ferrous iron to ferric iron the form of iron bound by transferrin. Hephaestin -Membrane protein that utili zes the redox potential of copper to oxidize ferrous iron to the ferric form for binding to transferring Cytochrome C Oxidase -Terminal enzyme in the electron tran sport chain that utilizes copper for electron transfer needed for ATP synthesis. Cu, Zn Superoxide Dismutase (SOD1) -Cytosolic protein that functions in antioxidant defense. SOD1 utilizes copper as a cofactor to catalyze the di smutation of the superoxide ion into hydrogen peroxide and molecular oxygen. Lysyl Oxidase -Copper-dependent amine oxidase that catalyzes the post-translational modifications that are critical for th e biogenesis of conn ective tissue (12). Tyrosinase -Contains two copper cofactors th at catalyze the first enzymatic step in melanin pathwayconverting tyro sine to dihydroxyphenylalanine (DOPA), then subsequently to dopaquinone (13).
32 Table 2-2. NAHMS classifi cation of copper adequacy in the bovine (3) Adequate Marginal Deficiency Severe Deficiency Serum Copper (ppm) >0.65 0.25-0.65 <0.25 Liver Copper (ppm on DM basis) >90 75-90 <75 Dietary copper (mg/kg) >10 4.0-9.9 <4.0 Table 2-3. NRC forage analysis of minerals th at can lead to copper an tagonistic effects (3). Copper Antagonists Trace Element Minimal Marginal High MTC* Molybdenum (ppm) <1 1-3 >3 5+ Sulfur (%DM) <0.1 0.15-0.20 >0.20-0.30 0.40 Iron (ppm) 50-200 >200-400 >400 1000 *Maximum Tolerable Concentration Table 2-4. Bovine blood parameters in the classification of anemia (76) Normal Mild-Moderate Severe Hemoglobin (g/100ml) 8-12 7.9-4.1 < 4.0 Erythrocyte count (106/mm3) 5.0-8.0 4.9-2.6 < 2.5 Hematocrit (%) 30-40 29-26 <25
33 Figure 2-1. Copper transport. 1) Copper absorp tion through the duodenal enterocyte is mediated either exclusively by CTR1 or in combin ation with DMT1. 2) Copper import into secretory pathways or export into porta l circulation occurs through the Menkes protein ATP7A. 3) In portal circulation, copper is transported to the liver bound to albumin, transcuprein or other small pep tides. 4) Once in the liver, copper is transported into the Golgi apparatus by the Wilson Protein ATP7B. In the Golgi, copper is either incorporated into ceruloplasmin in copper adequacy, or is excreted in the bile in conditions of copper excess. 5) Ceruloplasmin accounts for over 80% of the copper present in the plasma. Copper is imported into peripheral cells through CTR1 and is transported to target prot eins by the intracellular metallochaperones CCS, HAH1 and COX17.
34 Figure 2-2. Bovine anatom y of the polygastric system Food enters the rumen via the esophagus which is the primary site fo r microbial fermentation. Once adequate fermentation has taken place, food then passes to the retic uluma temporary holding site before entering the omasum where fluid absorption takes place. Food then continues to the abomasum which is considered the true stomach. The digestive enzymes in the abomasum break apart the food molecules be fore entering the small intestines for absorption.
35 Figure 2-3. Thiomolybdat es sequestering copper. Molybdenum can combine with sulfur to form substances known as thiomolybdates. Thiomo lybdates can have up to four degrees of sulfur binding. The higher the degree of sulfur binding, th e higher the copperantagonistic effect. Figure 2-4. Proposed mechanism for CCS-m ediated copper insertion into SOD1. 1) Domain I and III acquire copper from the cellular m ilieu which induces conformational changes that cause the CCS dimer to dissociate. 2) The CCS monomer then combines with a SOD1 monomer to form a heterodimeric complex through domain II of CCS. The third domain delivers copper to the active site of the SOD1 molecule through oxidation and reduction of disulfide bonds th ereby activating the SOD1 protein. 3) The complex then dissociates and CCS scavenger activity is recycled. The activated SOD1 monomer combines with another of its kind to form the active SOD1 dimer.
36 CHAPTER 3 MATERIALS AND METHODS Study Designs for Specific Aims I, II and III For specific aim s I and II, bovine blood and liver samples used for this study were obtained from Dr. Jerry Spears (Department of Animal Sc ience, North Carolina State University). Whole blood and liver samples were extracted at harves t (day 490) from weanling heifers and steers that were randomized to one of three diet tr eatments for 27 weeks: Copper deficient (Cu; n=8), copper adequate (Cu+ ; n = 6), and copper deficient + suppl emented with high dietary manganese (Cu-+Mn ; n = 7). The composition of the diets me t or exceeded the NRC requirements in all nutrients with the exception of copper, which was omitted from the Cuand Cu-+Mn diet treatments. Copper deficiency was further induced by the administ ration of 2 mg/kg of molybdenuma potent copper antagoni st to the diet in the Cuand Cu-+Mn treated groups. Full diet protocol and experimental design are referenced as follows (S. Hansen, Department of Animal Science, North Carolina State University ). For specific aim III, blood and liver samples from 11 heifers were collected at day 0 to esta blish a baseline measur ement of CCS and liver copper before the subcutaneous administration of 2 mL of the Mannheimia haemolytica vaccine. After vaccine administration, daily blood samples (5 mL from jugular vein) were collected in heparinized tubes and placed immediately on ice fo r storage until analysis. Liver samples were obtained via liver biopsy on day 2 and 4 of the study and were frozen on dry ice and stored 80oC. Erythrocyte Processing The erythrocyte processing protoc ol was m odified from Prohaska et al (7). This protocol was applied to whole blood samples for all thr ee sources of blood tested: rat, human and bovine.
37 The rat and human samples were used for the pr eliminary detection and optimization studies of CCS. Once an optimized method was establis hed, the bovine samples were analyzed. Fresh whole blood samples were collected in heparinized tubes and stored at 4oC. One mL of blood was centrifuged at 1000 x g for 5 min at 4oC to pellet the red blood cells. The resultant supernatant was aspirated to remove th e plasma and buffy coat; the estimated final red cell volume was recorded. Erythrocytes were re-suspended by gently mixing in 1 mL of Alsevers solution (Sigma-A ldrich). Cells were centrifuged a nd washed as above a total of three times. After the final washing, a final red cell volume of ~200 L remained. Cells were lysed by adding 200 L of lysis buffer [10mM Tris pH 7.2 and 20 L of 7X complete mini protease inhibitor cocktail (Roche Diagnostics)]. Cell lysates were centrifuged at 13,000 x g for 10 min at 4oC to remove residual red cell debris The supernatant was stored at -80oC until analysis. Liver Tissue Processing Liver sam ples (0.5-0.85 g) were homogenized in 1 mL of lysis buffer by using a Polytron at setting 3 for 20 s. Liver homogenates were stored at oC until analysis. Western Blot Analysis of Bl ood and Liver Cell Lysates Proteins were quantified by using the RC DC pr otein assay (Bio-Rad). Proteins (80 g) were mixed with Laemmli buffer and heated to 95oC for 5 min before loading onto a 12% SDSPAGE gel. Proteins were el ectrophoretically size -fractionated and transferred to a 0.45 m nitrocellulose membrane (Optitran; Schleicher & Schuell). Equivalent protein loading and transfer were verified by Ponceau staining. N onspecific protein binding to the membrane was blocked by incubating the blot fo r 1 h in blocking buffer [5% nonfat dried milk suspended in Tris-buffered saline, pH 7.4, and 0.01% Tween-20 (TBST)]. Membranes were incubated with rabbit anti-human CCS (Santa Cruz Biotechnology) for 1 h. After primary antibody incubation, the membranes were washed several times with TBST before incubating with the secondary
38 antibody (horseradish peroxidase-linked donkey anti-rabbit IgG antibody; Amersham Biosciences UK Limited) for 40 min. Membranes were washed with TBST and next with TBS before the final incubation with the chemilumine scence substrate (SuperSignal WestPico; Pierce) for 5 min. Blots were exposed to x-ray film for imaging. Antibodies were subsequently stripped by incubating blot at low pH (25 mM glycine, pH 2.8, 1% SDS) for 5 min. Blots were re-probed with rabbit anti-actin or tubulin antibodies (Sigma-Aldrich) as an indicator of protein loading. Immunoreactive band intensities were quantifie d by densitometric analysis (Gene Tools; SynGene). Statistical Analysis All values are express ed as means SEM. Statistical analyses were performed using Prism 4.03 (GraphPad) software. Data from St udy Aim II were analyzed by unpaired t-test, whereas data from Study Aim III were analy zed by paired t-test. Differences with P <0.05 were considered significant.
39 CHAPTER 4 RESULTS Detection of CCS in Bovine Erythrocytes Measurem ent of bovine CCS by Western blot analysis requires an antibody that cross reacts with the bovine protein. We first test ed the immunoreactivity of a commercial antibody (Santa Cruz), raised against full-length human CCS protein. The probability seemed high that this polyclonal antibody would recognize bovine CCS given that bovine and human CCS are 88% orthologous (Figure 4-1). Western analysis of bovine liver lysates revealed a predominant immunoreactive band migrating with an apparent molecular mass of 30-35 kDa (Figure 4-2), which is similar to the caclul ated molecular mass of bovine CCS. This indicated that the antibody did cross-react with th e bovine species. Subsequent western analysis of bovine erythrocytes revealed two bands migrating betw een 30-35 kDa. Signal intensities increased as protein concentration increased (Figure 4-3). Bovine liver and human erythrocte lysates were used as positive controls for bovi ne erythroctye analysis and rev ealed bands of similar apparent molecular masses. Bovine CCS Expression Increases in Copper Deficiency The experim ental diets reduced the copper c ontent in the liver and plasma by 97% and 86%, respectively, compared to initial values. These animals were then classified as being copper deficient according the th e NRC guidelines (Table 5-1). Er ythrocyte and liver samples were obtained from copper-normal and copper-deficient animals and were evaluated for CCS content through western blot analysis. The Cuand Cu-Mn groups were combined into one copperdeficient group since they did not differ with respect to copper deficien cy. Immunoblot analysis revealed that CCS levels were higher in the c opper-deficient groups for both erythrocte (Figure 4-4A) and liver (Figure 4-5A) samp les. To verify that the increa se in CCS protein levels were
40 not due to differences in lane loading, th e membranes were stripped and reprobed with antibodies against structural prot eins. An anti-actin antibody was used for lane loading control in erythrocyte samples, whereas an anti-tubulin antibody was used for liver. Neither actin nor tubulin proteins exhibited changes in coppe r-deficient samples compared to controls. Quantification by densitometric anal ysis of erythrocyte and liver CCS protein content revealed a statistically significant 63% (F igure 4-4B) and 44% (Figure 45B) increase in the copperdeficient animals compared to their respective controls ( P < 0.05). Bovine CCS Levels do not Change with Inflammation Ani mals were classified as copper normal base d on liver copper concen trations prior to the vaccine challenge (Table 5-2). Administration of the Mannheimia haemolytica vaccination at baseline (Day 0) induced a positive inflammato ry response as indicated by a 75% increase in ceruloplasmin activity on day 4 (Figure 4-6). This increase is similar to the 70-97% increase in ceruloplasmin activity in turpen tine-induced inflammation in bovi ne (80). CCS protein levels were measured in liver and erythrocyte samp les taken at baseline and on the final day of treatment (Day 4). Figure 4-7A demonstrates that erythrocyte CCS protei n levels do not change from baseline to day 4 under inflammatory cond itions. Similar results are observed in the liver (Figure 4-7C). Densitometry and st atistical analysis confirmed that CCS protein levels exhibited no significant change in erythrocytes or liver (Figure 4-7B and D, respectively). Similar lane loading was verified through actin and tubulin Western analyses. Taken together the results demonstrate that bovine erythroc yte and liver CCS levels increas e in copper deficiency and do not change in a 4-day period after an vaccine-induced inflammatory stimulus.
41 Figure 4-1. Alignment of human and bovine CCS. Sequence alignment of bovine and human CCS. The human CCS sequence is positioned on the top line (hCCS) while the bovine protein sequence is found below (bCCS). Amino acid marker s are listed to the right and represent hCCS. Shaded regions represent identical amino acids and the boxed amino acids represent the conserved motifs MXCXXC and CXC that are required for copper bindin g. The lines correspond with the protein domains of hCCS and the domain legend is shown below. Sequence BLAST analysis reveals an 88% homology between human and bovi ne CCS protein sequence. The commercial anti-CCS antibody from Santa Cruz was synthesized against full length human CCS (1-274).
42 Figure 4-2. Anti-human CCS antibody cross reacts with bovine liver CCS. Proteins from bovine liver lysates were electr ophoretically size fractiona ted on 12% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was probed with rabbit anti-human CCS primary antibody and then s ubsequently probed with the anti-rabbit HRP-linked secondary antibody. Chemilumine scence revealed a predominant CCSimmunoreactive band around 30-35 kDa. Figure 4-3. Anti-human CCS det ects bovine erythrocyte CCS in a dose-dependent manner. The anti-human CCS antibody detects bovine CCS and is sensitive given the level of protein loaded. Beef liver lysates (BLL) a nd human blood (HB) were used as positive controls
43 Table 4-1. Liver and plasma copper values at ha rvest (day 490). Liver and plasma samples were collected from animals in each experimental group and analyzed for copper content using flame atomic absorption spectroscopy. Da ta represent ppm for liver and plasma copper levels SEM and were generously obtained from S. Hansen and Dr. Jerry Spears (Department of Animal Science, North Carolina University). Figure 4-4. Copper deficiency increases CCS expression in bovine erythrocytes. Weanling calves were fed a copper-adequate (Cu+) (n = 6) or a copper-deficient diet (Cu-) (n = 15) for 27 weeks after weaning. A) Protei ns from erythrocyt e homogenates were electrophoretically separated by usin g a 12% SDS-PAGE, transferred to nitrocellulose, and analyzed for CCS by Western blotting. Positions and masses of molecular weight markers (in kDa) are show to the right. To visualize lane loading, the blot was stripped and re-probed for actin. Representative samples from each group are shown. B) Band intensities fo r erythrocyte CCS were quantified by densitometry for all 21 samples. Data represent the mean SEM. Asterisk denotes statistical significance (P<0.05)
44 Figure 4-5. CCS expression increas es in bovine liver in copper deficiency. Weanling calves were fed a copper-adequate (Cu+ ) (n = 6) or a copper-deficie nt diet (Cu-) (n = 15) for 27 weeks after the weaning period. A) Pr oteins from liver homogenates were electrophoretically separated by usin g a 12% SDS-PAGE, transferred to nitrocellulose, and analyzed for CCS by Western blotting. Positions and masses of molecular weight markers (in kDa) are show to the right. To visualize lane loading, the blot was stripped and re-probed for tubulin. B) Band intensities for liver CCS were quantified by densitometry for all 21 samples. Data represent the mean SEM. Asterisk denotes statisti cal significance (P<0.05) Table 4-2. Liver concentrations at baseline. Liver samples were collected from each animal at baseline prior to vaccine administration and analyzed for copper content using the inductively coupled plasma mass spectroscopy (ICP-MS).
45 Figure 4-6. Ceruloplasmin activit y increases with vaccination. A Mannheima haemolytica (One Shot) vaccination was administered to 11 yearling heifers at baseline (Day 0). Plasma was collected daily through to day 4 and analyzed for ceruloplasmin activity using the ELISA method. Results represent the least square means (n = 10) in mg/dl SEM. Data based on PROC MIXED analysis Asterisks denote significance p < 0.01. Data obtained from Dr. John Arthington (Range Cattle Research and Education Center, University of Florida).
46 Figure 4-7. Bovine liver and er ythrocyte CCS expression does not change in inflammation. A Mannheima haemolytica (One Shot) vaccination was administered to 11 yearling heifers at baseline (Day 0). Liver extracts were collected at baseline, day 2, and day 4 of treatment and erythrocyte ex tracts were collected for a pe riod of 5 days (baseline to day 4). CCS protein expression was assesse d. A) CCS expression was measured in erythrocyte lysates at base line and day 4 by Western blot analysis (top panel). Representative blots from each group are shown. The membrane was subsequently stripped and re-probed with anti-Actin as a lane loading control (bottom panel). B) Relative intensities of erythrocyte CCS leve ls at baseline and Day 4 were quantified by densitometry. Data are representative of the mean SEM. C) Western analysis of baseline liver CCS protein expression was compared to day 4 CCS levels (top panel). The membrane was then stripped and re-p robed with anti-tubulin antibody to verify equal lane loading (bottom panel) D) Gr aphical representation of densitometric analysis of liver CCS levels at baseline and Day 4. Data represent the mean SEM.
47 CHAPTER 5 DISCUSSION The increase in bovine erythrocyte and liv er CCS levels under conditions of copper deficiency is sim ilar to findings of previous st udies of copper-deficient rats and mice (7, 8). However, the 0.63and 0.44-fold increase in bovine erythrocyte and liver CCS, respectively, was much less than the 7.2and 9.6-fold increase in CC S protein levels in eryt hrocytes and liver in copper-deficient rats (8). Given the severity of the copper-deficient state of these animals based on NRC reference standards, the nominal increase in CCS levels suggests that bovine erythrocyte CCS would not be a sensitive copper status indica tor in detecting a moderate copper deficiency. In the beef cattle industry, it would be important to detect a copper deficiency as early as possible so that intervention measures could be initiated before problems occur that would lead to an economic loss. There are several potential explanations as to why the bovine CCS response to copper deficiency is not as robust as with rodents. The first would be the sequence differences. The bovine CCS protein sequence is shorter (216 aa) compared to its orthologues in humans and rodents (274 aa) and does not possess the CXC motif in domain III that is critical for CCS function. Schmidt et al.(54) reported that when minor amino acid mutations were introduced in the CXC motif of the third domain, CCS functi on was completely abrogated. The fact that bovine CCS can function without this domain suggests that the bovine species employs an alternative mechanism for CCS regulation and stab ility and may provide an explanation as to why the bovine CCS protein is not as responsiv e to copper deficiency. The question, however, still remains as to how SOD1 can be activat ed without the CXC meta l binding motif in CCS. Recently, an independent CCS mediated-SOD1 ac tivation mechanism has been proposed based on the evidence demonstrated in CCS -/mice that retained a reduced, bu t significant form of
48 SOD1 activity (58). The enzyme glutathione peroxidase has been proposed to exhibit compensatory activity to deliver copper to SOD1 when CCS is lacking. This alternative pathway of copper incorporation into SOD1 could play a more prominent role in the bovine and warrants further investigation (59). Another potential explanation for the reduced bovine CCS response to a copper deficiency could be the cell type examin ed. Erythrocyte CCS was selected to represent a convenient sampling source. However, mature erythrocytes are quite unique in that they do not possess a nucleus or organelles and ar e consequently incapable of de novo protein synthesis. Therefore, after the orthochromatic erythroblast stage of erythropoiesis when e nucleation occurs, CCS protein levels are presumably fixed (60). T hough the nature of the erythrocyte is quite exceptional in that these cells lack the mechanic s for protein synthesis, the erythrocyte CCS can still be evaluated as a potentia l copper status indicator because the regulatory mechanism and stability of CCS is mainly post transcriptional. As previously reported, the increase in CCS protein levels observed in a coppe r deficiency occurs through a d ecrease in degradation rather than an increase in protein synthesis. Bertina do et al. (9) proposed that a copper-loaded CCS adopts a conformation that is less stable and incr eases the rate of degradation. Therefore, in a copper-deficient state, CCS would be mainly in the apo-form increasing the stability of the protein to promote maximal copper scavenger potent ial. This implies that the inability of de novo protein synthesis of the erythr ocyte cell system should not be a primary factor as to why a reduced response was observed with bovine er ythrocyte CCS to a copper deficiency. Furthermore, the erythrocyte cell system was also used to evaluate CCS in rodents and potent responses to a copper deficiency were observed (9). In contrast, bovine liver cells do possess a nucleus to support de novo synthesi s of CCS; yet liver CCS exhibi ted similar minor increases in
49 protein levels. Taken together, the nominal increa ses in CCS protein levels in response to a copper deficiency is most likely attributed to the protein sequence di fferences as discussed previously. In erythrocytes, CCS presented as a double ba nd at the 30-35 kDa marker, whereas in liver this doublet was not present suggesting that bovine erythrocyte CCS is slightly different from the liver form of the protein. It is possible that the erythrocyte form of the bovine CCS protein exhibits post-translational modifications such as glycosylation, phosphoryl ation or acetylation. Also, it is possible that the lower band could re present a degradation prod uct or splice variant. The findings of this study demonstrate that CCS protein levels likely do not change under conditions of inflammation. The inflammatory m odel selected was base d on a pilot study that tested the inflammatory response in the bovine sy stem of two commonly used vaccinations in the beef industry: the One Shot (Mannheimia haemolytica ) and UltraBac 7 (multiple Clostridium strains) (Dr. John Arthington, Un iversity of Florida, unpublished results). Ceruloplasmin, an acute phase protein, was measured for a peri od of 21 days to determine the degree of inflammation. The use of acute phase serum prot eins as markers for non-specific inflammation are well supported in the liter ature (12). Animals that were vaccinated with the One Shot vaccine had greater ceruloplasmi n concentrations compared to saline-injected controls and UltraBac7-treated animals. In addition, the ac ute-phase response generated by this vaccine gave earlier peak respon ses (Day 3 and Day 4 of treatment) compared to the UltraBac 7 Based on these results, the One Shot vaccine was selected to induce an inflammatory response and a 5-day study design was deemed sufficient to observe the maximal acute-phase response in this study. Although this time frame has been prov en to produce a potent acute phase response, and inflammation was confirmed by the significant increase in ceruloplasmin activity, consistent
50 with the pilot study, it is not known whether 5 da ys is sufficient to ev aluate the effect of inflammation on CCS. Inflammation is a major limitation with serum and plasma copper as a status indicator because the majority of seru m copper is bound to ceruloplasmin, which increases during inflammation. The increase in ceruloplasmi n, as well as other acute phase proteins under inflammatory conditions is quite rapid and usually occurs within 24 h of infection. Therefore a five-day model should be sufficient to capture the acute inflammatory state of the system. Furthermore, another study evaluated the effects of inflammation on erythrocyte copper using ICP-MS and found that though serum copper increased as expected, erythrocyte copper concentrations did not change (61) It is plausible to assume that since there were no changes in erythrocyte copper, that the c opper containing enzymes in the er ythrocyte such as CCS would also not exhibit any changes. Li ver CCS would be more susceptible to changes in protein levels since both transcriptional as well as the post-tr anscriptional modifications of CCS are possible, yet no changes in protein levels were observed. Bovine erythrocyte CCS levels increases in copper deficiency and could be used as a copper status indicator as it is represents a convenient sampling source. Moreover CCS levels do not vary with inflammationa limitation with many current indicators of copper status. The level of response to copper deficiency, however, is not as robust as that seen in rodents, indicating that erythrocyte CCS ma y not readily detect a moderate copper deficiency in cattle.
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58 BIOGRAPHICAL SKETCH Joeva Hepburn was born in Kingston, Jam aica in 1983. Joeva completed her B.S. in food science and human nutrition at the University of Florida in 2005 w ith a specialization in dietetics and a minor in French. Currently, Joeva is comp leting her master's degree in food science and human nutrition at the University of Florida th rough a combined program in which she will also fulfill the Dietetic Internship requirements (MSDI). Joeva has always ha d a profound interest in nutrition especially from the res earch perspective. In 2006, Joeva worked with Dr. Gail Kauwell in the food science and human nutrition depart ment studying folate metabolism using the microbiological assay. Currently, Joeva is work ing with Dr. John Arthington and Dr. Mitchell Knutson evaluating a copper chaper one protein and its applications to the bovine system. After graduation, Joeva plans to pursue her PhD degree in biochemistry and molecular biology through the College of Medicine at the University of Florida.