Scand. J. Immutiol. 9, 303-314, 1979
Isolation and Properties of Detergent-Solubilized HLA Antigens Obtained from Platelets L. TRAGARDH, L. KLARESKOG, B. CURMAN. L. RASK & p. A. PETERSON Department of Cell Research, Biomedical Centre. University of Uppsala. Uppsala. Sweden
Trilgardh, L., Kliircskog. L,. Curnian, B.. Rask, L, & Peterson. P,A. Isolalion and Properties of Detergent-Solubiiized HLA Aniigens Obtained from Plalelets. Scand. J. linnuinul. 9, 303-314, 1979, Deoxycholate-solubilized HLA antigens have been isolated from plalelets and comprised a mixture of 43,000- and 39,000-daUon polypcptide chains iissocialed with [ij-microglobiilin. Limited proteolysis experiments suggested that the .ly.OOO-dallon chain is a fragment of the intact 43,000-dalton chain. Further proteolysis of the 39.0(K)-daltoii iViigment yields a 33,000dalton component. The 39,00U-dalton molecule is more acidic than both the 43,000- and the 33,000-dalton chains. Differences in the amino acid compositions of the 43,000- and 39,000dalton species demonstralc ihai the peptidefs) released on generation ofthe 39,000-dalton comconiponent are charged. The proieolytic split mosl probably occurs in the COOH-terminal end. which, owing to its content of charged amino acids, most probably is not integrated into the hydrocarbon mairix ofthe membrane. The 39,000- and 43,000-d all on components bind detergent in micellar form and can be incorporated into liposonies. The 33,000-dalton fragment has lost the ability to bind detergent micelles and is not incorporated into liposomes Lena Tnigdnlh. Departnwni of Cell Research., The Wath-nber^ Laboratory, Box 562. S-751 22 Uppsala. Sweden.
The major hisiocompatibiliiy complex (MHC) contains several distinct loci that control the expression of cell surface antigens. The classical transplantation antigens are derived from three loci. A, B and C. A fourth locus, D, also controls the expression of cell surface molecules (see ref. 16). The present article only deals wiih the A, B and C antigens which for brevity are called HLA antigens in this communication unless A. B and C loci products have to be distinguished. The HLA antigens are composed of two types of subunits. The smaller one, figmicroglobulin (fiam). is common to all antigens, whereas the larger subunit is unique [II. 18, 23], However, the physicochemical and immunological properties of the A, B and C antigen heavy chains are very similar [25, 27].
HLA antigens, there are two reasons for trying to isolate HLA antigens from other sources. First, since HLA antigens are present on most if not all nucleated cells and display an extensive, serologically defined genetic polymorphism, it seetns important to ascertain that the structure of HLA antigens obtained from individual lymphoblastoid cell lines is representative for the structure of HLA antigens present on other types of cells. Second, the costs involved in growing lymphoblastoid cell lines in quantities sufBcient for biochemical work render alternative sources for the isolation of the HLA antigens attractive. This communication describes the isolation of detergent-solubilized HLA antigens obtained from platelets. Some properties ofthe isolated HLA antigens have been examined.
The isolation of chemical quantities of detergent-solubilized HLA antigens has been accomplished [4, 24, 26. 28, 29, 31], The starting material in most cases has been cells from in vitro grown lymphoblastoid cell lines. Although this is an excellent source for the isolation of
MATERIALS AND
METHODS
Cc//.i, Outdated platelets were obtained by plateIelpheresi5. Lymphocytes were obtained by
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leucocylopheresis from patients with chronic lymphatic leukaemia. Surgically reninveii spleens were obtained from patients sufTering from oesophageal cancer, abdominal Irauma or haemolytic anaemia, Antisera. The antisera against HLA aniigen subunits have been described [25], Alloantisera against HLA-A and HLA-B anligens were kindly donated by Dr B, Lindblom. Tesis on large numher ofcells demonstrated ihat they were monospecific in cytolysis assays. Special rnatcriah. Deoxycholutc was purchased from Merck AG, Darmstadt, Cctyltrimethylammonium bromide (CTAB) was the product oT Sigma Chemical Co., St Louis, Mo, Tween-80 was obtained from Kebo AB, Stockholm, Sephadex G-200 and G-50. Sepharose 4B, Sepharose 6B and DEAE-Sephadex A-50 (Pharmacia Fine Chemicals AB, Uppsala) were prepared in accordance with the instructions supplied, Guanidine hydrochloride was obtained from Sigma and treated with charcoal prior to use. Phenylniethylsulfonyl fluoride (PMSF) was the product of Sigma Chemical Co. Lens culinaris haemagglutinin was isolated as described [12], Preparation of itieinhrane fraetions. Crude platelet membranes were isolated by cenlrifugation at 20.000 g for 60 min from repeatedly freeze-thawed platelet concentrates. The preparation of crude memhrane fractions from spleen cells and leukaemia cells followed the procedure oullined elsewhere [2.1]. Concentrations of proteins. Concentration of proleins during the isolation procedure was accomplished by ultrafillration [2]. The recovery of lhe HLA antigens after ulirafiltralion always exceeded 90"o. Inuniowlofficat techniques. 1 mmunodilfusion in gel was performed in 1% agarose gels containing 0.01 M Tris-HCI buirer, pH 8.0, O.I M NaCl. and 0,01",, Twecn-80. Immunoelectrophoresis was also carried oiii in the Tween-80-conlaining buffer, HLA antigens were monitored during the isolation procedure by a Pam radioimmunoassay [9]. The occurrence of HLA antigens in highly purified preparations was analysed by indirecl immunoprccipiiation [19], Affinity chromatography and immunosorhent purification. Sepharose 4B. aclivalec! by ihe cyanogen bromide method [1], was used to couple Lens eiilimtris hacmagglutinin and IgG isolated from rabbit antisera against %m. [5], The isolation of ihe IgG anlibody fraction was accomplished by chromaiography on a column of p^m-coupled Sepharose 4B [I], Desorplion of material bound to the leciin column was achieved by 10% a-methylmannoside (Sigma Chemical Co,. Si Louis, Mo.) in the ekienl. Protein bound to the immunosorbent column was eluted as described in the text. Electrophoresis ami isocketrie foctning. Polyacrylamide gel eleclrophoresis in sodium dodecyl sulphate (SDS) [17], charge shift eleclrophoresis in agarose gels [!3] and two-dimensional isoeieciric focusing and SDS polyacrylamide gel electrophoresis [20] was performed as described. In lhe isoelectrie focusing step 6",, polyacrylamide gels ill the pH interval .1,5-10 (Ampholine, LKB Produkter AB, Siockholm) were used. Radioactive labelling. Highly purilied HLA antigens were labelled wilh ^-''1 [14]. HLA anligens were also labelled in the carbohydrate portion afier neuraminidase treatment [10].
Determinations of diffusion coefficient, sedimenration constants, molecular weights, anilfrietional ratios. These measurements and the calculations were carried out as described [15], Amino uciil analyses mid NH^-terminal amino acid determinations. Separate amino acid analyses were performed on the 3.1,000-. 39.000- and 4.1.000-dalton HLA aniigen heavy chains. Mixtures coniaining the polypeptide chains were reduced and alkylated, traeelabelled with fluorescamin. and subjected to SDS polyacrylamide gel electrophoresis. The components, detected under ultraviolet lighi. were extracted from the gel, and excess SDS was removed by gel chromatography on columns (30 - 1 cm) of Sephadex G-50 equilibrated wilh 0,02 M (NH4)HCO,. After repeated lyophilization lhe samples were hydrolysed in 6 N HCI at 110 C for 24 or 72 h. Amino acid analyses were carried out on a Beckman 121B automatic amino acid analyser. Tryptophan was esiimalcii speclrophotometrically by the procedure of Hdelhoeh [8], Mixtures containing 33.000-, 39,000- and 43.000dalion heavy chain of the HLA antigens were labelled with dansyl chloride [34] and then separated by SDS polyaerylamide gel eleclrophoresis. The separated polypeptide chains were extracted from the gel, hydrolysed in 6 N HCi at 100 C for 20 h, ant! the liberated dansyl-amino acids were ideniineti by two-dimensional chromatography [35]. Other methods. Proiein [7] and phosphorus [6] determmaiions and analytical gel chromaiography [15] were carried out as described, Liposomes were prepared according to methods used earlier [6]. Lens enlinaris alRnily chromatography was used for detergent exchange.
RESULTS Isolation of detergent-solubilized HLA antigens Tests were made to optimize the solubilization of the HLA antigens. Less than 25% of the fotai protein could be solubilized (Fig. I). At the two lowest protein concentrations examined the ratios of the amount of Pam solubilized to tiie total protein solubilized were constant. At higher total protein concentrations relatively less ^io'''! was solubilized. The data for the alloantigenic HLA-antigen subunit were identical with those for p^m (not shown). Accordingly, for the purification ofthe HLA antigens crude membrane fractions containing 10 mg/ml of total protein were treated with 10 mM sodium deoxycholate. The isolation procedure for the HLA antigens (Table I) has been found to be reproducible and gives highly purified material in reasonable yield from platelets, spleen cells, and chronic lymphatic leukaemia cells. The solubilized platelet protein was subjected to Lens culinaris
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FIG. 6. Charge shift electrophoresis of'-''l-labelled HLA antigens conlaining 39.000- and 4.1.000-dalton heavy chains. The figure is a coniposiie representation of three elecirophoretic experiments performed in Tween-80 ( ). in Tween-80 and deoxycholate (- ) and in Tween-80 and CTAB ( ). The arrows denote the origin (O) and the migration position of fi^m (fla-M) in all three experiments.
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FIG. 7. Gel chromaiography on a column (62 x 1 cm) of Sepharose 6B equilibrated with 0.02 M TrisHCI buffer, pH 8.0, containing 0.15 M NaCI of a mixture of ^^M-labelled. detergent-solubilized HLA antigens and '"•I-labelled. papain-solubilized HLA antigens after iiposome formation in the presence of this mixture.
HLA anligens lo become integrated into liposomes. '-'*l-labelled HLA antigens, composed of 43,000- and :i9,000-dalton heavy chains, were mixed with '^M-labelled papain-solubilized HLA antigens, whose heavy chains displayed a molecular weight of 33.000. Liposomes were made in the presence of this mixture. The liposomes formed were subjected to eel chromatography on a column (62 x 1 cm) of Sepha-
rose 4B equilibrated with 0.02 M Tris-HCI buffer. pH 8.0, containing 0.15 M NaCI. It can be seen in Fig. 7 that the majority of the '^^i radioactivity occurred at a K^^ of about 0.4 and followed the distribution of the lipid phosphorus, whereas all of the ^'"I radioactivity emer ed close to the total volume of tbe column. Material from the two radioactive peaks was separately subjected to SDS polyacrylamide gel
Deter^'ent-Solubilized HLA Antigens electrophoresis. The '-•'^l-labelled material comprised polypeplide chains with the apparent molecular weights 12,000, 39,000 and 43.000. and the '^'1-labelled material was composed of components with molecular weights of 12,000 and 33,000. Thus, the 43,000- and 39.000dalton components were integrated into the liposomes, whereas the 33,000-dalton component was not. This is in keeping with the notion thai the 43,000- and 39,000-dalton chains have a hydrophobic portion of the polypeptide chain which is rennoved on proteolytic generation of the 33,000-dalton fragment. DISCUSSION The isolation ofthe HLA antigens was accomplished by modifications of previously described procedures [4, 28]. The adopted procedure gave a good yield of the HLA antigens. It is, however. obvious that platelets express much less of the HLA antigens than do lymphoblastoid cell lines (cf. Ref. 29). Therefore relatively large amounts of platelets have to be processed to obtain the HLA antigens in chemical amounts. This does not seem to be a major drawback since blood centres usually outdate considerable amounts of platelets. The highly purified HLA anligens were composed of three types of polypeptide chains. The relative amount of the 39,000-dalton species varied from preparation to preparation. Evidence was obtained that this material represents a proteolytic fragment of the 43,000-datton chain. It seems likely that the proteolysis occurs during the membrane preparation stage or before since the relative proportions of the 39,000-dalton chain to the 43,000-dalton chain remain relatively constant once the purification procedure has been initiated. Limited proteolysis of the highly purified 43,000-dalton chain gives rise initially to the 39,000-dalton species and subsequently to a fragment with a molecular weight of 33.000. Similar results have been obtained also for HLA antigens derived from lymphoblastoid cell lines [30]. The isoelectrie focusing profiles and the amino acid compositions suggested that the 43,000-dalton chain loses a basic peptide encompassing some tliirty-fivc amino acid residues on generation of the 39,000-dalton fragment. The acidic peptide segment of about forty-odd residues released on further digestion of the
39,000-dalton chain also seems to influence the isoelectrie point since the 33,000-dalton fragment has a more basic pl than the 39,000dalton molecule. Since the NHo-terminal amino acid ofthe intact HLA antigen heavy chain and of the proteolytically derived fragments is gtycine and the isolated proteolytic fragments carry all of the carbohydrate, which is located in the NH^-terminal portion of the heavy chain [21, 24], it seems Mkely that proteolysis removes peptides from the COOH-terminai end of the molecule [30]. The intact HLA antigen heavy chain and the 39,000-dalton fragment, but not the 33,000polypeptide. exhibit the type of hydrophobic amino acid sequence that is required for the integration inlo the hydrocarbon matrix of the membrane. Itcan therefore be concluded that the platelet HLA antigen heavy chain has a COOHterminal peptide, about thirty-five residues long, which is basic in character and contains charged amino acid residues. Preceding that peptide region is a segment that is hydrophobic enough to bind detergent micelles. Similar conclusions have been reached by Springer & Strominger [30] for HLA antigens derived from lymphoblastoid cell lines, although their data suggest that the charged COOH-terminal peptide is somewhat larger than reported here. Walsh & Crumpton [33] have elegantly shown that this portion of the molecule resides on the cytoplasmic side of the membrane. The amino acid composition of the HLA antigen heavy chain is remarkably similar to the amino acid compositions of heavy chains of defined alloantigenic specificities (cf. Refs. 30, 32). Likewise, the physicochemical properties of the isolated HLA antigens are in general agreement with those reported for HLA antigens derived from other sources [27, 28]. However, in contrast to Snary ct al. [28] we have not been able to distinguish HLA-A from HLA-B antigens by sedimentation velocity analyses.
ACKNOWLEDGMENTS The expert technical assistance of Mr Jtirgen Ericson is gratefully acknowledged. We are grateful to Dr Jan Siifwenberg for kindly performing the plateletphereses and the leucocytophoreses. This work was supported by grants from the Swedish Cancer Society.
314
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REFERENCES 1 Axcn. R.. Poratli. J. & Ernback. S. Chemical i;nupling of pcpiJdes and proieins lo polysaccharides by means of cyanogen haliiics. Nalure. 214, 1302. 1967. 2 BcrggariJ. I. Proieins. giycoproleins and mucopolysaccharides in normal humiiii urine. I. Fractionaiion of non-dialysable materials by ullrafiltration and by zone electropboresis. Ark. Kt-mi. 18, 291. 1961. y Berggartl. I. & Bcarn. A.G. Isolation and properties of a low molecular weight [i^-glohulin occurring in human biological fluids. J. biol. Chem. 243. 4095. 1%8. 4 Britigcn. J., Snary. D.. Crumplon. M.J.. Barnstable, C . Goodfellow P. & Bodmer. W.F. Purilicaiion of HLA-A and -B aniigens. Naitirc. 261, 200. 1976. 5 Ciiairecasas. P. Protein purification by afTmity chromatography. J. hiol. Chem. 245, .''059. 1970. 6 Curman, B.. Ostberg, L. & Peterson. P.A. Incorporation of murine MHC aniigens inlo liposomes and their elTect in the secondary mixed lymphocyte reaction. I^atiire. Ill, 545. 1978. 7 Dulley. J.R. & Greve, P.A. A simple technique for eliminating interference by detergents in the Lowry method of protein determination. Analyt. Biochem. 64, L16. 1975. 8 tdelhoch. H. Spectroscopic determination ol tryptophan and tyrosine in proteins. Biochfmixiry. 6, 1948. 1967. 9 Evrin, P.I-.. Peterson. P.A.. Wide. L. & Berggard. I. Radioimmunoassay of P^-microglobulin in human biological fluids. Scanil. J. clin. Lah. Invest. 28. 4.19. 1971. 10 Gahmberg. C.G.. Hiiyry, P. & Andersson, L.C. Characterization of surface glyeoproteins of mouse lymphoid cells. J. Cell Biol. 68, 642. 1976. 11 Grey, H.M.. Kubo. R.T.. Colon, S.M.. Poulik. M.D., Cresweli, P., Springer. T.. Turner. M. & Strominger. J.L. The small subunit of HL-A antigens isp^-microglohulin. J. exp. Meil. t38, 1608. 1971. !2 Haynian, M.J. & Crumpton. M.J. Isolation of glyeoproteins from pig lymphocyte plasma membrane using Lens eiilinaris phytohemagglutinin. Biochem. hiophys. Res. Cotnmtin. 47. 923. 1972. 1.1 Heleniiis, A. & Simons, K. Charge shift electrophoresis: Simple method for distinguishing between amphiphilic and hydrophilie proteins in detergent solution. Proc. nat. Acad. Set. iUSA). 74, 529, 1977. 14 Himier, W.M. & Greenwood. F.C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Naitire, 194. 495. 1962. 15 Karlsson, F.A.. Peterson. P.A. & Berggard. I. A structural feature of human immunoglobulin light chains. J. hiol- Chem. 241. 1065. 1972. 16 KaU. D. & Bcnaceralf. B. (cd.). The Role of Proihicts of the Htstocompatibility Gene Complex in Immune Responses. Academic Press. New York. 1976. 17 Laemmli, V.R. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 111. 680, 1970. 18 Nakamoro, K., Tanlgaki. N. & Pressman. D. Multiple common properties of human fl^-microglobuiin and the common portion fragment derived from HL-A antigen molecules. Proc. nat. Acad. Sei. lUSAllO,2S6i. 197.1. 19 Ostberg. L., Segc. K.. Rask. L. & Peterson. P.A.
Isolation of radiolabelled H-2 antigens. Folia hiol.. Praha. 11, ill. 1976. 20 O'Farrell. P.H. High resolution two-dimensional electrophoresis. J. hiol. Chem. 250, 4007. 1975. 21 Parham. P.. Alpert. B.N.. Orr. H.T. & Strominger, J.L. Carbohydrate moiety of HLA antigens. J. hiol. Clicm.152, 7555. 1977. 22 Peterson. P.A.. Anundi. H.. Curman. B., Klareskog. L.. Kvist. S.. Ostberg. L., Rask. L.. Sandberg, L. & Sege. K. Structural studies of f^.^-microglobulinassociated and other MHC-antigens. Colii Spring Harhor Symp. qwint. Biol. 41, 331. 1977. 23 Peterson. P.A., Rask, L. & Lindhiom. J.B. Highly purified papain-solubili/ed HL-A antigens contain p,j-niicroglobulin. Proc. nat. Acad. Sci. {USA). 71. 35. 1974. 24 Peterson. P.A.. Rask. L.. Sege. K.. Klareskog. L.. Anundi. H. & Ostberg. L. F'volutionary relationship between immunoglobulins and transplantation antigens. Proe. nat. Aead. Sci.iUSA). 11, 1612, 1975. 25 Rask, L., Lindblom. J.B. & Peterson. P.A. Structural and immunological similarities between HLA aniigens from three loci. Eur. J. Immunol. 6,93. 1976. 26 Robb, R.J., Mann. D.L. & Strominger. J.L. Rapid purification of detergent-solubilized HLA antigen by afiinity chromatography employing anti-^jmicroglobulin serum. J. hiol. Chem. 251, 5427. 1976. 27 Snary. D.. Barnstable. C.J., Bodmer. W.F. & Crumpton. M.J. Molecular structure of human hislocompatibility antigens: the HLA-C series. Eur. J. Immioiol. 8, 580. 1977. 28 Snary. D.. Goodfellow. P.. Haymann, M.J.. Bodmer, W.F. & Crumpton, M.J. Subcellular separation and molecular nature of human histocompatibility antigens (HL-A). Nature. 247, 457. 1974. 29 Springer. T.A., Mann, D.L.. De Franco, A.L. & Strominger, J.L. Detergent solubilization, purification and separation of specificities of HLA antigens from a cultured human lymphoblastoid line. RPMI 4265. J. hiol. Chem. 252. 4682, 1977. .10 Springer. T.A. & Strominger. J.L. Delergentsoluble HLA antigens contain a hydrophilie region at the COOH-terminus and a penultimate hydrophobic region. Proc. nat. Acad. Sci. {USA). 73, 2481. 1976. 31 Springer. T.A.. Strominger. J.L. & Mann, D.L. Partial purification of detergent-soluble HL-A antigen and its cleavage by papain. Proe. nat. Aead. Sci. {USA).1\, 1539, 1974. 32 Terhorst. C , Parham. P.. Mann. D.L. & Strominger. J.L. Structure of HLA antigens; amino-acid and carbohydrate compositions and NH^-terminal sequences of four antigen preparations. Proc. nat. Acad. Sci. [USA). 73, 9It). 1976. .'3 Walsh. F.S. & Crumpton. M.J. Orientation of cellsurface antigens in the lipid bilayer of lymphocyte plasma memhrane. Nature. 269, 307. 1977. .14 Weiner, A.M., Platt. T. & Weber, K. Aminoterminal sequence analysis of proteins purified on a nanomole scale by gel elecirophoresis. J. hiol. Chem. 247. 3242, 1972. .15 Woods. K.R. & Wang. K.-T. Separation of dansylamino acids by polyamide layer chromatography, Biochim. biophys. Acta. 133. 369. 1967. Received II September 1978 Received in revised form 27 November 1978
Isolation and Properties of Detergent-Solubilized HLA Antigens Obtained from Platelets L. TRAGARDH, L. KLARESKOG, B. CURMAN. L. RASK & p. A. PETERSON Department of Cell Research, Biomedical Centre. University of Uppsala. Uppsala. Sweden
Trilgardh, L., Kliircskog. L,. Curnian, B.. Rask, L, & Peterson. P,A. Isolalion and Properties of Detergent-Solubiiized HLA Aniigens Obtained from Plalelets. Scand. J. linnuinul. 9, 303-314, 1979, Deoxycholate-solubilized HLA antigens have been isolated from plalelets and comprised a mixture of 43,000- and 39,000-daUon polypcptide chains iissocialed with [ij-microglobiilin. Limited proteolysis experiments suggested that the .ly.OOO-dallon chain is a fragment of the intact 43,000-dalton chain. Further proteolysis of the 39.0(K)-daltoii iViigment yields a 33,000dalton component. The 39,00U-dalton molecule is more acidic than both the 43,000- and the 33,000-dalton chains. Differences in the amino acid compositions of the 43,000- and 39,000dalton species demonstralc ihai the peptidefs) released on generation ofthe 39,000-dalton comconiponent are charged. The proieolytic split mosl probably occurs in the COOH-terminal end. which, owing to its content of charged amino acids, most probably is not integrated into the hydrocarbon mairix ofthe membrane. The 39,000- and 43,000-d all on components bind detergent in micellar form and can be incorporated into liposonies. The 33,000-dalton fragment has lost the ability to bind detergent micelles and is not incorporated into liposomes Lena Tnigdnlh. Departnwni of Cell Research., The Wath-nber^ Laboratory, Box 562. S-751 22 Uppsala. Sweden.
The major hisiocompatibiliiy complex (MHC) contains several distinct loci that control the expression of cell surface antigens. The classical transplantation antigens are derived from three loci. A, B and C. A fourth locus, D, also controls the expression of cell surface molecules (see ref. 16). The present article only deals wiih the A, B and C antigens which for brevity are called HLA antigens in this communication unless A. B and C loci products have to be distinguished. The HLA antigens are composed of two types of subunits. The smaller one, figmicroglobulin (fiam). is common to all antigens, whereas the larger subunit is unique [II. 18, 23], However, the physicochemical and immunological properties of the A, B and C antigen heavy chains are very similar [25, 27].
HLA antigens, there are two reasons for trying to isolate HLA antigens from other sources. First, since HLA antigens are present on most if not all nucleated cells and display an extensive, serologically defined genetic polymorphism, it seetns important to ascertain that the structure of HLA antigens obtained from individual lymphoblastoid cell lines is representative for the structure of HLA antigens present on other types of cells. Second, the costs involved in growing lymphoblastoid cell lines in quantities sufBcient for biochemical work render alternative sources for the isolation of the HLA antigens attractive. This communication describes the isolation of detergent-solubilized HLA antigens obtained from platelets. Some properties ofthe isolated HLA antigens have been examined.
The isolation of chemical quantities of detergent-solubilized HLA antigens has been accomplished [4, 24, 26. 28, 29, 31], The starting material in most cases has been cells from in vitro grown lymphoblastoid cell lines. Although this is an excellent source for the isolation of
MATERIALS AND
METHODS
Cc//.i, Outdated platelets were obtained by plateIelpheresi5. Lymphocytes were obtained by
0300-9475/79/0400-0303 §02.00 © 1979 Blackwell Scientific Publications
303
304
L. Tragardhetal
leucocylopheresis from patients with chronic lymphatic leukaemia. Surgically reninveii spleens were obtained from patients sufTering from oesophageal cancer, abdominal Irauma or haemolytic anaemia, Antisera. The antisera against HLA aniigen subunits have been described [25], Alloantisera against HLA-A and HLA-B anligens were kindly donated by Dr B, Lindblom. Tesis on large numher ofcells demonstrated ihat they were monospecific in cytolysis assays. Special rnatcriah. Deoxycholutc was purchased from Merck AG, Darmstadt, Cctyltrimethylammonium bromide (CTAB) was the product oT Sigma Chemical Co., St Louis, Mo, Tween-80 was obtained from Kebo AB, Stockholm, Sephadex G-200 and G-50. Sepharose 4B, Sepharose 6B and DEAE-Sephadex A-50 (Pharmacia Fine Chemicals AB, Uppsala) were prepared in accordance with the instructions supplied, Guanidine hydrochloride was obtained from Sigma and treated with charcoal prior to use. Phenylniethylsulfonyl fluoride (PMSF) was the product of Sigma Chemical Co. Lens culinaris haemagglutinin was isolated as described [12], Preparation of itieinhrane fraetions. Crude platelet membranes were isolated by cenlrifugation at 20.000 g for 60 min from repeatedly freeze-thawed platelet concentrates. The preparation of crude memhrane fractions from spleen cells and leukaemia cells followed the procedure oullined elsewhere [2.1]. Concentrations of proteins. Concentration of proleins during the isolation procedure was accomplished by ultrafillration [2]. The recovery of lhe HLA antigens after ulirafiltralion always exceeded 90"o. Inuniowlofficat techniques. 1 mmunodilfusion in gel was performed in 1% agarose gels containing 0.01 M Tris-HCI buirer, pH 8.0, O.I M NaCl. and 0,01",, Twecn-80. Immunoelectrophoresis was also carried oiii in the Tween-80-conlaining buffer, HLA antigens were monitored during the isolation procedure by a Pam radioimmunoassay [9]. The occurrence of HLA antigens in highly purified preparations was analysed by indirecl immunoprccipiiation [19], Affinity chromatography and immunosorhent purification. Sepharose 4B. aclivalec! by ihe cyanogen bromide method [1], was used to couple Lens eiilimtris hacmagglutinin and IgG isolated from rabbit antisera against %m. [5], The isolation of ihe IgG anlibody fraction was accomplished by chromaiography on a column of p^m-coupled Sepharose 4B [I], Desorplion of material bound to the leciin column was achieved by 10% a-methylmannoside (Sigma Chemical Co,. Si Louis, Mo.) in the ekienl. Protein bound to the immunosorbent column was eluted as described in the text. Electrophoresis ami isocketrie foctning. Polyacrylamide gel eleclrophoresis in sodium dodecyl sulphate (SDS) [17], charge shift eleclrophoresis in agarose gels [!3] and two-dimensional isoeieciric focusing and SDS polyacrylamide gel electrophoresis [20] was performed as described. In lhe isoelectrie focusing step 6",, polyacrylamide gels ill the pH interval .1,5-10 (Ampholine, LKB Produkter AB, Siockholm) were used. Radioactive labelling. Highly purilied HLA antigens were labelled wilh ^-''1 [14]. HLA anligens were also labelled in the carbohydrate portion afier neuraminidase treatment [10].
Determinations of diffusion coefficient, sedimenration constants, molecular weights, anilfrietional ratios. These measurements and the calculations were carried out as described [15], Amino uciil analyses mid NH^-terminal amino acid determinations. Separate amino acid analyses were performed on the 3.1,000-. 39.000- and 4.1.000-dalton HLA aniigen heavy chains. Mixtures coniaining the polypeptide chains were reduced and alkylated, traeelabelled with fluorescamin. and subjected to SDS polyacrylamide gel electrophoresis. The components, detected under ultraviolet lighi. were extracted from the gel, and excess SDS was removed by gel chromatography on columns (30 - 1 cm) of Sephadex G-50 equilibrated wilh 0,02 M (NH4)HCO,. After repeated lyophilization lhe samples were hydrolysed in 6 N HCI at 110 C for 24 or 72 h. Amino acid analyses were carried out on a Beckman 121B automatic amino acid analyser. Tryptophan was esiimalcii speclrophotometrically by the procedure of Hdelhoeh [8], Mixtures containing 33.000-, 39,000- and 43.000dalion heavy chain of the HLA antigens were labelled with dansyl chloride [34] and then separated by SDS polyaerylamide gel eleclrophoresis. The separated polypeptide chains were extracted from the gel, hydrolysed in 6 N HCi at 100 C for 20 h, ant! the liberated dansyl-amino acids were ideniineti by two-dimensional chromatography [35]. Other methods. Proiein [7] and phosphorus [6] determmaiions and analytical gel chromaiography [15] were carried out as described, Liposomes were prepared according to methods used earlier [6]. Lens enlinaris alRnily chromatography was used for detergent exchange.
RESULTS Isolation of detergent-solubilized HLA antigens Tests were made to optimize the solubilization of the HLA antigens. Less than 25% of the fotai protein could be solubilized (Fig. I). At the two lowest protein concentrations examined the ratios of the amount of Pam solubilized to tiie total protein solubilized were constant. At higher total protein concentrations relatively less ^io'''! was solubilized. The data for the alloantigenic HLA-antigen subunit were identical with those for p^m (not shown). Accordingly, for the purification ofthe HLA antigens crude membrane fractions containing 10 mg/ml of total protein were treated with 10 mM sodium deoxycholate. The isolation procedure for the HLA antigens (Table I) has been found to be reproducible and gives highly purified material in reasonable yield from platelets, spleen cells, and chronic lymphatic leukaemia cells. The solubilized platelet protein was subjected to Lens culinaris
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FIG. 7. Gel chromaiography on a column (62 x 1 cm) of Sepharose 6B equilibrated with 0.02 M TrisHCI buffer, pH 8.0, containing 0.15 M NaCI of a mixture of ^^M-labelled. detergent-solubilized HLA antigens and '"•I-labelled. papain-solubilized HLA antigens after iiposome formation in the presence of this mixture.
HLA anligens lo become integrated into liposomes. '-'*l-labelled HLA antigens, composed of 43,000- and :i9,000-dalton heavy chains, were mixed with '^M-labelled papain-solubilized HLA antigens, whose heavy chains displayed a molecular weight of 33.000. Liposomes were made in the presence of this mixture. The liposomes formed were subjected to eel chromatography on a column (62 x 1 cm) of Sepha-
rose 4B equilibrated with 0.02 M Tris-HCI buffer. pH 8.0, containing 0.15 M NaCI. It can be seen in Fig. 7 that the majority of the '^^i radioactivity occurred at a K^^ of about 0.4 and followed the distribution of the lipid phosphorus, whereas all of the ^'"I radioactivity emer ed close to the total volume of tbe column. Material from the two radioactive peaks was separately subjected to SDS polyacrylamide gel
Deter^'ent-Solubilized HLA Antigens electrophoresis. The '-•'^l-labelled material comprised polypeplide chains with the apparent molecular weights 12,000, 39,000 and 43.000. and the '^'1-labelled material was composed of components with molecular weights of 12,000 and 33,000. Thus, the 43,000- and 39.000dalton components were integrated into the liposomes, whereas the 33,000-dalton component was not. This is in keeping with the notion thai the 43,000- and 39,000-dalton chains have a hydrophobic portion of the polypeptide chain which is rennoved on proteolytic generation of the 33,000-dalton fragment. DISCUSSION The isolation ofthe HLA antigens was accomplished by modifications of previously described procedures [4, 28]. The adopted procedure gave a good yield of the HLA antigens. It is, however. obvious that platelets express much less of the HLA antigens than do lymphoblastoid cell lines (cf. Ref. 29). Therefore relatively large amounts of platelets have to be processed to obtain the HLA antigens in chemical amounts. This does not seem to be a major drawback since blood centres usually outdate considerable amounts of platelets. The highly purified HLA anligens were composed of three types of polypeptide chains. The relative amount of the 39,000-dalton species varied from preparation to preparation. Evidence was obtained that this material represents a proteolytic fragment of the 43,000-datton chain. It seems likely that the proteolysis occurs during the membrane preparation stage or before since the relative proportions of the 39,000-dalton chain to the 43,000-dalton chain remain relatively constant once the purification procedure has been initiated. Limited proteolysis of the highly purified 43,000-dalton chain gives rise initially to the 39,000-dalton species and subsequently to a fragment with a molecular weight of 33.000. Similar results have been obtained also for HLA antigens derived from lymphoblastoid cell lines [30]. The isoelectrie focusing profiles and the amino acid compositions suggested that the 43,000-dalton chain loses a basic peptide encompassing some tliirty-fivc amino acid residues on generation of the 39,000-dalton fragment. The acidic peptide segment of about forty-odd residues released on further digestion of the
39,000-dalton chain also seems to influence the isoelectrie point since the 33,000-dalton fragment has a more basic pl than the 39,000dalton molecule. Since the NHo-terminal amino acid ofthe intact HLA antigen heavy chain and of the proteolytically derived fragments is gtycine and the isolated proteolytic fragments carry all of the carbohydrate, which is located in the NH^-terminal portion of the heavy chain [21, 24], it seems Mkely that proteolysis removes peptides from the COOH-terminai end of the molecule [30]. The intact HLA antigen heavy chain and the 39,000-dalton fragment, but not the 33,000polypeptide. exhibit the type of hydrophobic amino acid sequence that is required for the integration inlo the hydrocarbon matrix of the membrane. Itcan therefore be concluded that the platelet HLA antigen heavy chain has a COOHterminal peptide, about thirty-five residues long, which is basic in character and contains charged amino acid residues. Preceding that peptide region is a segment that is hydrophobic enough to bind detergent micelles. Similar conclusions have been reached by Springer & Strominger [30] for HLA antigens derived from lymphoblastoid cell lines, although their data suggest that the charged COOH-terminal peptide is somewhat larger than reported here. Walsh & Crumpton [33] have elegantly shown that this portion of the molecule resides on the cytoplasmic side of the membrane. The amino acid composition of the HLA antigen heavy chain is remarkably similar to the amino acid compositions of heavy chains of defined alloantigenic specificities (cf. Refs. 30, 32). Likewise, the physicochemical properties of the isolated HLA antigens are in general agreement with those reported for HLA antigens derived from other sources [27, 28]. However, in contrast to Snary ct al. [28] we have not been able to distinguish HLA-A from HLA-B antigens by sedimentation velocity analyses.
ACKNOWLEDGMENTS The expert technical assistance of Mr Jtirgen Ericson is gratefully acknowledged. We are grateful to Dr Jan Siifwenberg for kindly performing the plateletphereses and the leucocytophoreses. This work was supported by grants from the Swedish Cancer Society.
314
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REFERENCES 1 Axcn. R.. Poratli. J. & Ernback. S. Chemical i;nupling of pcpiJdes and proieins lo polysaccharides by means of cyanogen haliiics. Nalure. 214, 1302. 1967. 2 BcrggariJ. I. Proieins. giycoproleins and mucopolysaccharides in normal humiiii urine. I. Fractionaiion of non-dialysable materials by ullrafiltration and by zone electropboresis. Ark. Kt-mi. 18, 291. 1961. y Berggartl. I. & Bcarn. A.G. Isolation and properties of a low molecular weight [i^-glohulin occurring in human biological fluids. J. biol. Chem. 243. 4095. 1%8. 4 Britigcn. J., Snary. D.. Crumplon. M.J.. Barnstable, C . Goodfellow P. & Bodmer. W.F. Purilicaiion of HLA-A and -B aniigens. Naitirc. 261, 200. 1976. 5 Ciiairecasas. P. Protein purification by afTmity chromatography. J. hiol. Chem. 245, .''059. 1970. 6 Curman, B.. Ostberg, L. & Peterson. P.A. Incorporation of murine MHC aniigens inlo liposomes and their elTect in the secondary mixed lymphocyte reaction. I^atiire. Ill, 545. 1978. 7 Dulley. J.R. & Greve, P.A. A simple technique for eliminating interference by detergents in the Lowry method of protein determination. Analyt. Biochem. 64, L16. 1975. 8 tdelhoch. H. Spectroscopic determination ol tryptophan and tyrosine in proteins. Biochfmixiry. 6, 1948. 1967. 9 Evrin, P.I-.. Peterson. P.A.. Wide. L. & Berggard. I. Radioimmunoassay of P^-microglobulin in human biological fluids. Scanil. J. clin. Lah. Invest. 28. 4.19. 1971. 10 Gahmberg. C.G.. Hiiyry, P. & Andersson, L.C. Characterization of surface glyeoproteins of mouse lymphoid cells. J. Cell Biol. 68, 642. 1976. 11 Grey, H.M.. Kubo. R.T.. Colon, S.M.. Poulik. M.D., Cresweli, P., Springer. T.. Turner. M. & Strominger. J.L. The small subunit of HL-A antigens isp^-microglohulin. J. exp. Meil. t38, 1608. 1971. !2 Haynian, M.J. & Crumpton. M.J. Isolation of glyeoproteins from pig lymphocyte plasma membrane using Lens eiilinaris phytohemagglutinin. Biochem. hiophys. Res. Cotnmtin. 47. 923. 1972. 1.1 Heleniiis, A. & Simons, K. Charge shift electrophoresis: Simple method for distinguishing between amphiphilic and hydrophilie proteins in detergent solution. Proc. nat. Acad. Set. iUSA). 74, 529, 1977. 14 Himier, W.M. & Greenwood. F.C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Naitire, 194. 495. 1962. 15 Karlsson, F.A.. Peterson. P.A. & Berggard. I. A structural feature of human immunoglobulin light chains. J. hiol- Chem. 241. 1065. 1972. 16 KaU. D. & Bcnaceralf. B. (cd.). The Role of Proihicts of the Htstocompatibility Gene Complex in Immune Responses. Academic Press. New York. 1976. 17 Laemmli, V.R. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 111. 680, 1970. 18 Nakamoro, K., Tanlgaki. N. & Pressman. D. Multiple common properties of human fl^-microglobuiin and the common portion fragment derived from HL-A antigen molecules. Proc. nat. Acad. Sei. lUSAllO,2S6i. 197.1. 19 Ostberg. L., Segc. K.. Rask. L. & Peterson. P.A.
Isolation of radiolabelled H-2 antigens. Folia hiol.. Praha. 11, ill. 1976. 20 O'Farrell. P.H. High resolution two-dimensional electrophoresis. J. hiol. Chem. 250, 4007. 1975. 21 Parham. P.. Alpert. B.N.. Orr. H.T. & Strominger, J.L. Carbohydrate moiety of HLA antigens. J. hiol. Clicm.152, 7555. 1977. 22 Peterson. P.A.. Anundi. H.. Curman. B., Klareskog. L.. Kvist. S.. Ostberg. L., Rask. L.. Sandberg, L. & Sege. K. Structural studies of f^.^-microglobulinassociated and other MHC-antigens. Colii Spring Harhor Symp. qwint. Biol. 41, 331. 1977. 23 Peterson. P.A., Rask, L. & Lindhiom. J.B. Highly purified papain-solubili/ed HL-A antigens contain p,j-niicroglobulin. Proc. nat. Acad. Sci. {USA). 71. 35. 1974. 24 Peterson. P.A.. Rask. L.. Sege. K.. Klareskog. L.. Anundi. H. & Ostberg. L. F'volutionary relationship between immunoglobulins and transplantation antigens. Proe. nat. Aead. Sci.iUSA). 11, 1612, 1975. 25 Rask, L., Lindblom. J.B. & Peterson. P.A. Structural and immunological similarities between HLA aniigens from three loci. Eur. J. Immunol. 6,93. 1976. 26 Robb, R.J., Mann. D.L. & Strominger. J.L. Rapid purification of detergent-solubilized HLA antigen by afiinity chromatography employing anti-^jmicroglobulin serum. J. hiol. Chem. 251, 5427. 1976. 27 Snary. D.. Barnstable. C.J., Bodmer. W.F. & Crumpton. M.J. Molecular structure of human hislocompatibility antigens: the HLA-C series. Eur. J. Immioiol. 8, 580. 1977. 28 Snary. D.. Goodfellow. P.. Haymann, M.J.. Bodmer, W.F. & Crumpton, M.J. Subcellular separation and molecular nature of human histocompatibility antigens (HL-A). Nature. 247, 457. 1974. 29 Springer. T.A., Mann, D.L.. De Franco, A.L. & Strominger, J.L. Detergent solubilization, purification and separation of specificities of HLA antigens from a cultured human lymphoblastoid line. RPMI 4265. J. hiol. Chem. 252. 4682, 1977. .10 Springer. T.A. & Strominger. J.L. Delergentsoluble HLA antigens contain a hydrophilie region at the COOH-terminus and a penultimate hydrophobic region. Proc. nat. Acad. Sci. {USA). 73, 2481. 1976. 31 Springer. T.A.. Strominger. J.L. & Mann, D.L. Partial purification of detergent-soluble HL-A antigen and its cleavage by papain. Proe. nat. Aead. Sci. {USA).1\, 1539, 1974. 32 Terhorst. C , Parham. P.. Mann. D.L. & Strominger. J.L. Structure of HLA antigens; amino-acid and carbohydrate compositions and NH^-terminal sequences of four antigen preparations. Proc. nat. Acad. Sci. [USA). 73, 9It). 1976. .'3 Walsh. F.S. & Crumpton. M.J. Orientation of cellsurface antigens in the lipid bilayer of lymphocyte plasma memhrane. Nature. 269, 307. 1977. .14 Weiner, A.M., Platt. T. & Weber, K. Aminoterminal sequence analysis of proteins purified on a nanomole scale by gel elecirophoresis. J. hiol. Chem. 247. 3242, 1972. .15 Woods. K.R. & Wang. K.-T. Separation of dansylamino acids by polyamide layer chromatography, Biochim. biophys. Acta. 133. 369. 1967. Received II September 1978 Received in revised form 27 November 1978
