Differences between a- and /3-Lipovitellin from Hen Egg Yolk

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1 Agric. Biol Chem., 45 (3), , Differences between a- and /3-Lipovitellin from Hen Egg Yolk Jun-ichi Kurisaki, Kunio Yamauchi, Hiroyuki Isshiki and Shinobu Ogiwara Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received September 1 1, 1980 Differences between a- and /?-lipovitellin were examined, especially in regard to the polypeptide and carbohydrate composition of apolipoprotein. Both lipoproteins were composed of at least to 140,000 daltons. eight polypeptides with similar molecular weights ranging from 35,000 Polypeptides with 110,000 daltons were commonmajor constituents. The close similarity similar amino acid of component polypeptides in the two lipoproteins was also assumed from compositions and the identical immunological properties of the two lipoproteins. However,somenotable differences were found in the composition of the polypeptides. a-lipovitellin contained much more polypeptide with 85,000 daltons than /?- lipovitellin. Both apolipovitellins were found to be glycoprotein containing mannose, galactose, glucosamine and sialic acid. The sialic acid in a-lipovitellin exceeded that in ^-lipovitellin by six times, though only slight differences were found in the content of neutral and amino sugars. The relatively acidic nature of a-lipovitellin comparedwith ^-lipovitellin is attributed not only to the relative predominance in protein phosphorus but also to the predominance in the sialic acid. Lipovitellin is one of the two lipoproteins contained in hen's egg yolk and comprises about one sixth of the yolk solid. It is spherical in shape with a molecular weight of 4xlO5, composed of about 80% protein and 20% lipid and has a great affinity for phosvitin forming the granule.j) Alpha- and /Mipovitellin are discriminated in electrophoresis and separated on hydroxyapatite2} or ion-exchange chromatography.3'4) They differ in phosphorus content in the protein moiety2~4) and in dissociation-association behavior depending on ph.5) The properties of lipovitellin have been thus extensively studied mainly by Cook and co-workers.1"3'^ However, the properties of the protein moiety, which must contribute significantly to the characteristics of the parent lipoprotein, are still not clear mainly because of the high insolubility of the apolipoproteins free of lipids. In our present study, detailed comparison was madeon the protein moieties of a- and /^-lipovitellin in polypeptides and carbohydrate composition and we found a close similarity in the size and number of the component polypeptides and a marked difference in sialic acid content. MATERIALS AND METHODS Preparation oflipovitellin. Fresh infertile eggs from White Leghorn were used within one day after laying. Egg yolk was separated and the granule fraction was obtained as described previously.6) The preparation of lipovitellin-phosvitin complex and the purification of a- and /?-lipovitellin were performed, essentially based on the method of Wallace45 as follows: the granule fraction was dissolved in a buffer containing 0.06 m 2-amino-2-methyl-l-propanol (Monol) and 0.01 m citric acid and centrifuged at 106,000 x g for 17 hr. The floating pellicle was discarded and the subnatant solution of lipovitellin-phosvitin complex was obtained. The solution was exhaustively dialyzed against the buffer (0.06 m Monol and 0.01 m citric acid), centrifuged to remove some precipitates and applied to a TEAEcellulose (>200mesh, Bio Rad) column (3x60cm) equilibrated with the buffer. Usually 600 to 700 mg oflipoprotein was charged on the column. The protein was eluted at 3 to 5 C with a concave gradient of ph produced by a three-chambered gradient apparatus, as described by Wallace,45 containing a total volume of 3 liters and monitored at 280nm with a ISCO UA-5 absorbance monitor. The lipoproteins fractionated were exhaustively dialyzed against deionized water and

2 700 J. Kurisaki, K. Yamauchi, H. Isshiki and S. Ogiwara precipitated. The precipitates were collected by centrifugation at 15,000Xg for 15min, dissolved in the starting buffer, then dialyzed against the buffer and rechromatographed. The purified a- and /3-lipovitellin fractions were exhaustively dialyzed against deionized water as above. The resultant precipitates were dissolved in either the buffer or 0.3 m NaCl and dialyzed against the respective solutions. Agarose gel electrophoresis. In agarose gel electrophoresis, the buffer containing 0.06 m Monol and 0.01 mcitric acid was employedfor the superior resolution of a- and /Mipovitellin. Electrophoresis was performed at 7 V/cm for 1 hr. The agarose content was 0.8% in the gel. After electrophoresis, the gel was fixed with picric acid and stained with Amino black 10B for protein and oil red O for lipids. Immunological experiments. Antibodies against a- and jft-lipovitellin were prepared, essentially based on the methods of Harboe and Ingild7) as follows : 2 mg/ml of the lipoprotein in 0.3 m NaCl solution was mixed and emulsified with an equal volume of Freund's incomplete adjuvant (Difco) in syringes joined together by a double-habbed needle. On days 0, 14, 28 and 42, 1 ml of the emulsion was cutaneously injected into the backs of rabbits and blood was obtained on the fiftieth day. And then, every sixth week, blood was drawn from the rabbits, having received the emulsion containing antigen a week before each bleeding. After bleeding, serum was obtained in a usual way and stored at -80 C until use. Immunoelectrophoresis8) and double immunodiffusion8) were carried out in 0.8 %agarose gel containing 0.06m Monol and 0.01 m citric acid. After the formation of immunoprecipitine lines, the plates were exhaustively washed with the buffer and subsequently with saline and stained with Coomassie brilliant blue R 250 (CB). SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis was performed according to Weber and Osborn.9) Lipoprotein in 0.3 m NaCl solution was mixed with an equal volume of 0.2m phosphate buffer containing 2% SDS and 1 % mercaptoethanol, heated for 10 min at 60 C and subjected to electrophoresis in 7.5 %acrylamide gel. Protein was stained with CBand the destained gel was scanned at 546 nm with a ISCO UA-5 absorbance monitor equipped with a ISCO model 659 gel scanner. Carbohydrate was stained with periodic acid Schiff reagent. Molecular weights of apolipoproteins were estimated by using molecular weight markers : bovine serum albumin containing dimeric form, hen's egg ovalbumin and bovine /Mactoglobulin. Chemical analyses. Protein was estimated by the method of Lowry et al.m Lipid was determined by weighing after extraction with chloroform-methanol (2: 1 v/v) and desiccation at 60 C in vacuo. Apolipoproteins from a- and ^-lipovitellin, obtained by the removal of lipids with chloroform-methanol (2: 1 v/v), were used in protein phosphorus, amino acid and carbohydrate analyses. The phosphorus of protein and lipids was determined by the method of Rouser et al.n) For the expression of phospholipid content, the phosphorus values were multiplied by the factor of 25. Triacylglycerol was determined according to Fletcher.12) Choresterol was estimated by substracting phospholipid and triacylglycerol from total lipids. Amino acid analysis was performed on a Hitachi 835 amino acid analyzer after hydrolysis of protein with 6 n HC1 at 110 C for 20 hr in a sealed evacuated tube.13) Neutral sugar was determined by gas-liquid chromatography after methanolysis and trimethylsilylation.14) Amino sugar was determined by the amino acid analyzer after 3 hr to 12 hr ofhydrolysis of protein and the maximum value was adopted. Sialic acid was determined by the method of Warren15) after hydrolysis with 0.1 n H2SO4 at 80 Cfor 1 hr. RESULTS Preparation and composition of a- and ftlipo vitellin Figure 1 shows the representative chromatogram of lipovitellin-phosvitin complex on TEAE-cellulose. The first peak corresponded to ^-lipovitellin and the second to ' Fraction number Fig. 1. Separation ofa- and /3-Lipovitellin on TEAE- Cellulose. About 650 mgof lipovitellin-phosvitin complex was applied to the column (3 x 60 cm) equilibrated with the buffer containing 0.06 mmonol and 0.01 mcitric acid. Elution was performed with a concave gradient of ph according to Wallace4) at a flow rate of 150 ml/hr and fractions of 20ml were collected. Two portions indicated by the bars were pooled and rechromatographed., absorbance at 280 nm;,ph.

3 Differences between a- and ^-Lipovitellin from Hen Egg Yolk 701 Table I. Chemical Composition of a- and /#-Lipovi telli n Fig. 2. Agarose Gel Electrophoresis oflipovitellins. Samples were dissolved in the buffer containing 0.06 m Monol and 0.01 m citric acid and applied to the gel (0.8% agarose in the buffer). Electrophoresis was performed at a constant voltage of 7 V/cmfor 1 hr. Only the protein staining is given. 1, lipovitellinphosvitin complex prior to the application to the ion-exchange chromatography; 2, a-lipovitellin ; 3, /?- lipovitellin. a-lipovitellin. The purity of either lipoprotein after rechromatography was confirmed as shown in Fig. 2. The chemical composition of purified a- and /Mipovitellin is shown in Tables I and II. No significant difference in the protein content and lipid composition was found between a- and ^-lipovitellin. The amino acid composition of protein moiety of both lipoproteins did not differ by more than 1 %. The protein phosphorus content of a-lipovitellin was about twice as high as that of ^-lipovitellin. Identity of a- with ft-lipovitellin in immunological criteria Lipovitellin-phosvitin complex was electrophoresed and reacted with either anti a- or /Mipovitellin serum. As shown in Fig. 3A, either antiserum formed a single precipitine line with lipovitellins. In immunodiflusion, only a single precipitine line was given, when anti a-lipovitellin serum reacted with lipovitellin-phosvitin complex, purified a-lipovitellin or /3-lipovitellin (Fig. 3B, left). Moreover, the respective precipitine lines fused with one another and no spur was detected in the gel. * N.D., not determined. Also anti /Mipovitellin serum could not discriminate between two lipoproteins (Fig. 3B, right). Polypeptide composition and molecular weights of apolipoproteins Figure 4 clearly shows the similarity in polypeptide composition. Both lipoproteins consisted of at least eight polypeptide chains and the size distributions of apolipoproteins from both lipoproteins were also similar to each other. The bands were designated numerically, as in Fig.4, in order of size, such as a-1 which represented the polypeptide in a-lipovitellin with the highest molecular weight. Someminor differences, however, were noted: bands 3 (a-3 and /?-3) with molecular weights

4 702 J. Kurisaki, K. Yamauchi, H. Isshiki and S. Ogiwara of 110,000 were common major components but, in addition, a-5 in a-lipovitellin was also major. In a-lipovitellin, a single band (a-5) was detected in the region of the doublet bands of p~5 and /?-6, while only fi-1 in /3-lipovitellin instead of the doublet of a-6 and a-7 in a-lipovitellin. Also, all the protein bands found in electrophoretic patterns were stained with periodic acid Schiff reagent (figure not shown). Fig. 3. Immunoelectrophoretic and Double Immunodiffusion Analyses of Lipovitellins. A, immunoelectrophoietic patterns; B, double immunodiffusion patterns. Electrophoresis was performed as described in Fig.2. Also in immunodiffusion, the gel contained 0.8 % agarose in the buffer (0.06 m Monol and 0.01 m citric acid). 1, lipovitellinphosvitin complex; 2, a-lipovitellin; 3, /Mipovitellin; a, anti a-lipovitellin serum; b, anti /Mipovitellin serum. Carbohydrate content of apolipoprotein The results of carbohydrate analyses are summarized in TableIII. Neutral sugar of apolipoproteins consisted of only mannoseand galactose. No other peak of neutral sugar was detected on the gas-liquid chromatogram. Mannose was major in both lipoproteins and the slight predominance of a-lipovitellin in neutral sugar content was due to the lesser content of galactose in /?-lipovitellin. Only glucosamine was found in apolipoproteins as amino sugar. The difference in amino sugar QL daltons (xio"4) ll daltons (xi(f4) Fig. 4. SDS-Polyacrylamide Gel Electrophoretic Patterns and Approximate Molecular Weights of Apolipoproteins. Lipoproteins in 0.3 m NaCl solution were mixed with an equal volume of 0.2 m phosphate buffer containing 2% SDS and 1 % mercaptoethanol, heated for 10 min at 60 C and subjected to electrophoresis in 7.5% acrylamide gel according to Weber and Osborn.9) Protein was stained with CBand the destained gels were scanned at 546 nm. Molecular weights of apolipoproteins were estimated by using bovine serum albumin, /?-lactoglobulin and hen's egg ovalbumin as molecular weight markers. A, a-lipovitellin; B, ^-lipovitellin.

5 Differences between a- and ^-Lipovitellin from Hen Egg Yolk 703 Table III. Carbohydrate Composition of Apolipoproteins from a- and ^-Lipovitellin between two lipoproteins was not significant in amount. Sialic acid content of apolipoprotein from a-lipovitellin was six times higher than that of /?-lipovitellin. DISCUSSION No significant difference, except for the protein phosphorus content, has been found between a- and /Mipovitellin in chemical composition, such as the content and composition of lipid and amino acid composition.1} In our present study, the similarity of the two lipoproteins mentioned above was confirmed (Tables I and II) and further, the immunological discrimination of the two lipoproteins was shown to be impossible. In double immunodiffusion and immunoelectrophoresis, there was no spur between the precipitine lines formed by a- and /Mipovitellin with antiserum to either lipovitellin (Fig. 3). It clearly shows that the two lipoproteins were immunologically identical. The similarity of the two lipoproteins was thus evident but the polypeptide composition of these lipoproteins has not been compared in detail, especially in minor components. The protein of a- and ^-lipovitellin was not fully studied before Franzen et al.1q>17) because of the difficulty in dissociating the lipid-free protein in aqueous media. They dissolved the succinylated and -carboxymethylated apolipovitellin in 6 m guanidine hydrochloride and reported that the protein moiety of /?-lipovitellin consisted of two polypeptide chains with molecular weights of about 30,000 and 45,000.17) Bergink et ai,18) however, utilized SDS-polyacrylamide gel electrophoresis and reported that a-lipovitellin was composedof three polypeptides with molecular weights of 135,000, 105,000 and 40,000, while 0-lipovitellin cotained two polypeptide chains with molecular weights of 135,000 and 30,000. Christmann et al.19) also reported that 0-lipovitellin was composed of two polypeptide chains with molecular weights of 140,000 and 30,000. In our present study, the close similarity, which had not been previously pointed out, was noted in the number and the molecular weights of the polypeptide components (Fig. 4). All the polypeptides except for a-7 contained in a-lipovitellin corresponded in mobility, i.e. in molecular weight, to those in 0-lipovitellin. Such pairs of polypeptide components with similar molecular weights found in each lipoprotein as a-3 and 0-3 are supposed to be closely related each other, considering the similarity lipoproteins in the characteristics and in amino acid of the parent composition and the immunological identity; though, further experiments are needed. However, some notable differences were found in the polypeptide compositions: a-7 and 0-5 were minor components but characteristic of the respective lipoprotein; a-3 (110,000 daltons) and a-5 vitellin, (85,000 while daltons) only 0-3 were major in a-lipo- (110,000 daltons) in 0-lipovitellin. These three major components, a-3, a-5 and 0-3, probably correspond to the polypeptides of 135,000, 105,000 and 135,000 daltons, reported by Bergink et al.18) respectively, but the somewhat lower molecular weights estimated in our present study should be due to the higher concentration of acrylamide in the gel (7.5% instead of 5%), as pointed out in the case of SDS-electrophoresis of glycoproteins,20) because all the polypeptides contained carbohydrates. The carbohydrate content (probably hexose) of lipovitellin was previously reported to be 0.75% by Ito and Fujii.21) Our present study showsmuchmorecarbohydrate in the protein moiety of lipovitellin (TableIII). In both lipovitellins, mannose was predominant in neutral sugar and galactose was minor constituent. Glucosamine was the only amino sugar detected in lipovitellins as well as in very

6 704 J. Kurisaki, K. Yamauchi, H. Isshiki and S. Ogiwara low density lipoprotein in yolk.22) Between the two lipoproteins, only a slight difference was observed in the contents of neutral and amino sugar. It was noted, however, that sialic acid content in a-lipovitellin was more than six times over that in /Mipovitellin. Several characteristics were thus commonto both lipoproteins but a-lipovitellin was rich in sialic acid as well as in phosphorus3) compared with /3-lipovitellin. The relatively acidic nature of a-lipovitellin in comparison with /?- lipovitellin is due not only to the relative predominance of phosphate groups but also to that of sialic acid attached to the protein moiety of the lipoprotein. Recently, it was confirmed that lipovitellins as well as phosvitin are the proteolytic cleavage products of the precursor, vitellogenin, which is synthesized in the liver under the regulation ofestrogenic hormones.18'19'23) Therefore, such differences between two lipovitellins as shown in the present study might be attributed to either the differences in vitellogenin, occurring in the liver prior to the proteolytic cleavage, or the differences in the site of the cleavage. The precursor-product relationships between two distinct vitellogenins, more recently reported by Wang and Williams,24) and the two lipovitellins should be more extensively studied. Acknowledgments. We are indebted to Mr. N. Azumafor his technical advice in carbohydrate analyses. Weare also grateful to Dr. T. Morichi of the National Institute of Animal Industry, Japan, for the supply of fresh eggs. REFERENCES 1) W. H. Cook and W. G. Martin, "Structural and Functional Aspects of Lipoprotein in Living Systems," ed. by E.Tria and A.M. Scanu, Academic Press Inc., New York, 1969, p ) G. Bernardi and W. H. Cook, Biochim. Biophys. Acta, 44, 96 (1960). 3) M.W.Radomski and W.H.Cook, Can. J. Biochem., 42, 1203 (1964). 4) R. A. Wallace, Anal. Biochem., ll, 297 (1965). 5) R. W. Burley and W. H. Cook, Can. J. Biochem. Physiol., 40, 363 (1962). 6) K. Yamauchi, J. Kurisaki and K. Sasago, Biol. Chem., 40, 1581 (1976). Agric. 7) N. Harboe and A. Ingild, Suppl. 1, 161 (1973). Scand. J. Immunol, 2, 8) O. Ouchterlony and L. A. Nilsson, "Handbook of Experimental Immunology," Vol. 1 (3rd Ed.) ed. by D. M. Weir, Blackwell Scientific Publications, Oxford, 1978, p ) K.Weber and M. Osborn, /. Biol. Chem., 244, 4406 (1969). 10) O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, /. Biol. Chem., 193, 265 (1951). ll) G. Rouser, A.N. 1, 85 (1966). Siakotos and S. Fleischer, Lipids, 12) M. J. Fletcher, din. Chim. Ada, 22, 393 (1968). 13) S. Moore and W.H. Stein, "Methods in Enzymology," Vol. 6, ed. by S. P. Colowick and N. O. Kaplan, Academic Press Inc., New York, 1963, p ) J.R.Clamp, T.Bhatti and R.E.Chambers, "Glycoproteins," Part A, ed. by A. Gottschalk, Elsevier Publishing Company, Amsterdam, 1972, p ) 16) L. J. Warren, /. S.Franzen, Biol. C. Chem., 234, 1971 (1959). M.Bobik, R. N.Stuchell and L. D. Lee, Arch. Biochem. Biophys., 123, 127 (1968). 17) J.S. Franzen and L.D. Lee, Arch. Biochem. Biophys., 140, 295 (1970). 18) E. W. Bergink, R. A. Wallace, J. A. Van de Berg, E.S.Bos, M.Gruber and G.AB, Amer. ZooL, 14, 1177 (1974). 19) J.L.Christmann, M.J.Grayson and R.C.C. Huang, Biochemistry, 16, 3250 (1977). 20) J.P.Segrest and R.L.Jackson, "Methods in Enzymology," Vol.28, ed. by V. Ginsburg, Academic Press Inc., New York, 1972, p ) Y. Ito and T. Fujii, /. Biochem., 52, 221 (1962). 22) J.Augustyniak and W.G.Martin, Can. J. Biochem., 46, 983 (1968). 23) R. G. Deeley, K. P. Mullinix, W. Wetekam, H.M. Kronenberg, M. Meyers, J. D. Eldridge and R. F. Goldberger, /. Biol. Chem., 250, 9060 (1975). 24) S. Wang and D. L. Williams, Biochemistry, 19, 1557 (1980).

Antigenic Analysis of Isolated Polypeptides from Visna Virus

INFECTION AND IMMUNITY, June 1976, p. 1728-1732 Copyright 1976 American Society for Microbiology Vol. 13, No. 6 Printed in USA. Antigenic Analysis of Isolated Polypeptides from Visna Virus P. D. MEHTA,*