Cell, Vol. 18.979-991,
Assembly and Maturation Antigens In Vivo Michael S. Krangel, Harry T. Orr and Jack L. Strominger Harvard University The Biological Laboratories Cambridge, Massachusetts 02138
HLA-A and HLA-B antigens are integral membrane glycoproteins which consist of a glycosylated heavy chain embedded in the membrane in noncovalent association with /3z-microglobulln, a water-soluble polypeptide. The assembly and maturation of these antigens has been studied in vivo in the human B lymphoblastoid cell line T8-1 (HLA-Al, -A2, -88, -B27). Two antigenically distinct populations of HLA-A and -B heavy chains can be detected by antisera which recognize determinants sensitive to the conformation of the heavy chain. One heavy chain population is associated with &-microglobulin, whereas the other population is not. These populations can be further distinguished by their oligosaccharide structure and their localization within the cell. Pulse-chase experiments demonstrate a precursor-product relationship between these heavy chain populations and suggest the following pathway for the assembly and maturation of HLA-A and -B antigens. The completed heavy chains initially carry high mannose oligosaccharides and are largely or wholly unassociated with &-microglobulin. During the next lo-15 min, association with &-microglobulin occurs and the heavy chain conformation Is altered. Beginning at about 30 min after synthesis, the oligosaccharides are converted from the high mannose form to the complex form, and between 80 and 80 min after synthesis, the mature antigens appear at the cell surface. These observations are discussed in relation to in vivo and in vitro studies on the biosynthesis of a variety of secreted proteins and membrane proteins. Introduction HLA-A and -B antigens are integral membrane glycoproteins which are found on almost all human cells. These highly polymorphic antigens appear to play a crucial role in a variety of immune phenomena. Structurally, they consist of two polypeptides, a polymorphic heavy chain which is noncovaiently associated with a nonpolymorphic light chain, &-microglobulin (&m) (Cresswell et al., 1974). The heavy chain is a 41,000 dalton glycoprotein (as estimated from the amino acid sequence and the carbohydrate composition of the HLA-B7 heavy chain) (Strominger et al., 1979) which is embedded in the membrane via a hydrophobic stretch of amino acids near its carboxy terminus (Springer and Strominger, 1976). Whereas
of HLA-A and HLA-B
the bulk of the polypeptide resides on the external surface of the cell, it is thought that a small carboxy terminal hydrophilic region extends into the C~OplaS~ (Walsh and Crumpton, 1977). The extracellular region carries a single asparagine-linked oligosaccharide which is composed of residues of N-acetylglucosamine, mannose, galactose, fucose and sialic acid (Parham et al., 1977). /In-microglobulin, a nOnglYCOSYiated, water-soluble polypeptide of 11,600 daltons, is associated with the extracellular portion of the heavy chain. HLA-A and -B antigens provide an excellent system for studying the biosynthesis, assembly and maturation of a multimeric eucaryotic membrane glycoprotein. Cell lines are available which synthesize these antigens in quantities and at rates sufficient for study, and well characterized antisera are available for their analysis. In addition, the structure of these molecules is now understood in great detail. The biosynthesis of eucaryotic membrane proteins has been intensively studied both in vivo and in vitro in a number of model systems, including secretory proteins and, more recently, the viral membrane proteins of vesicular stomatitis, Sindbis, Semliki Forest and influenza viruses. The synthesis of these proteins in vivo occurs on membrane-bound polysomes (Palade, 1975; Morrison and Lodish, 1975; Wirth et al., 1977). Translation of mRNA in vitro results in the synthesis of an amino terminal “signal sequence” of approximately 20 residues, which precedes the sequence of the mature protein (Devillers-Thiery et al., 1975; Palmiter et al., 1977; Burstein and Schechter, 1978; Lingappa et al., 1978). This “signal sequence” has been postulated to direct the nascent chain to the endoplasmic reticulum and through the membrane in vivo (Blobel and Dobberstein, 1975). When an in vitro translation system is supplemented with microsomes, the nascent polypeptide is transferred across the microsomal membrane. As transfer occurs, the signal sequence is removed and the polypeptide is glycosylated (Rothman and Lodish, 1977; Lingappa, DevillersThiery and Blobel, 1977; Katz et al., 1977; Garoff, Simons and Dobberstein, 1978; Lingappa et al., 1978). Transfer of the polypeptide across the membrane is complete for secreted proteins, whereas integral membrane proteins become anchored in the membrane at some point during transfer. The initial glycosylation of asparagine-linked oligosaccharides in vivo occurs on the growing polypeptide (Kiely, McKnight and Schimke, 1976; Sefton, 1977; Bergman and Kuehl, 1978). Glycosylation results from the transfer of a large “high mannose” oligosaccharide from a lipid-linked intermediate to the appropriate asparagine residue on the polypeptide (Lennarz, 1975; Waechter and Lennarz, 1976; Robbins et al., 1977; Li, Tabas and Kornfeld, 1978; Tabas, Schlesinger and Kornfeld, 1978; Nakamura and Compans, 1979). This type of oligosaccharide generally consists
of an N,N’-diacetylchitobiose core followed by five or more mannose residues and a variable number of glucose residues. Many mature glycoproteins carry asparagine-linked oligosaccharides termed “complex.” These oligosaccharides contain outer residues of sialic acid, galactose, N-acetylglucosamine and sometimes fucose attached to a core of mannose residues linked to N,N’-diacetylchitobiose (Kornfeld and Kornfeld, 1976). During the maturation of these glycoproteins, outer glucose and mannose residues are first trimmed from the high mannose precursor and the terminal sugars characteristic of complex oligosaccharides are subsequently added (Robbins et al., 1977; Kornfeld, Li and Tabas, 1976; Tabas et al., 1976; Nakamura and Compans, 1979). These final glycosylation reactions occur as the polypeptide is in transit through the endoplasmic reticulum and the Golgi apparatus. The fully mature glycoprotein is then either secreted or expressed on the cell surface. The factors which govern the intracellular transport of membrane proteins from their site of synthesis to the cell surface or to various intracellular membranes are not well understood. It has recently been demonstrated that the mRNAs coding for HLA-A and -B heavy chains and for p2microglobulin can be translated in vitro to yield products with amino terminal extensions (“signal sequences”) of 20-24 residues. When microsomes are added during translation, these products are segregated across the microsomal membranes and processing occurs, which presumably consists of the removal of the N-terminal extensions and the glycosylation of the heavy chain (Ploegh, Cannon and Strominger, 1979). In this paper, the subsequent assembly and maturation of HLA-A and -B antigens has been analyzed in vivo in the human B lymphoblastoid cell line T5-1. This cell line is of particular interest due to the availability of mutants derived from it which are defective in the expression of HLA-A and -B antigens (Pious, Soderland and Gladstone, 1977). Results Antigenically Distinct Populations of HLA-A and -6 Heavy Chains Can Be Detected in T51 The human B lymphoblastoid cell line T5-1 expresses the HLA antigenic specificities Al, A2, 86 and 827 (Pious et al., 1977). The biosynthetic products of this cell line were analyzed by immunoprecipitation from detergent lysates of 35S-methionine-labeled cells. The antisera used were W6/32, a mouse monoclonal antibody which recognizes a determinant on all HLA-A, -B, and-C heavy chains only when they are associated with /3*m (Barnstable et al., 1976; Parham, Barnstable and Bodmer, 1979); anti-heavy chain serum (anti-H), a rabbit heteroserum raised against the highly purified, guanidine-denatured HLA-B7 heavy chain; anti-&m serum, a rabbit heteroserum raised against human
Prim; and human alloantisera which recognize each of the four allospecificities synthesized in T5-1. lmmunoprecipitates were analyzed by SDS-PAGE and fluorography and are presented in Figure 1. Lane 1 is a sample of total lysate. The major species, having an apparent molecular weight of 44,000, is probably actin (Barber and Crumpton, 1976; Barber and Delovitch, 1979). This polypeptide is a variable contaminant in both nonspecific (not shown) and specific immunoprecipitates, and actually serves as a good reference for comparing small mobility differences. As seen in lane 2, W6/32 primarily precipitates material having an apparent molecular weight of 43,000 which is often, but not always, resolved into a well defined doublet, and a single band having an apparent moleqular weight of 12,000. These species presumably represent four different HLA heavy chain allospecificities (HLA-Al, -A2, -88, -827) and Prim, respectively. A rabbit anti-&m serum apparently precipitates the same spectrum of products (lane 4). A representative alloantiserum precipitation is presented in lane 5; this HLA-AP-specific antiserum precipitates material which co-migrates with the lower band of the 43,000 dalton doublet precipitated by W6/32 or antiPrim, along with Brim. lmmunoprecipitations utilizing antisera recognizing the remaining allospecificities (data not shown) suggest that the HLA-B6 and -B27 heavy chains also co-migrate with the lower band of the 43,000 dalton doublet, whereas the HLA-Al heavy chain co-migrates with the upper band. In clear contrast to these antisera, anti-H serum precipitates material (which is sometimes resolved into a doublet) having an apparent molecular weight of 42,000, without any material of 12,000 daltons (lane 3). This material appears to co-migrate with some minor species present in the W6/32 immunoprecipitate. To further characterize the relationships between the products recognized by the various antisera, serial immunoprecipitations were conducted (lanes 6-13). It can be seen that W6/32 removes from solution all the heavy chains precipitable by the anti&m serum (lane 61, all the heavy chains precipitable by the alloantiserum (lane 91, but none of the heavy chains precipitable by anti-H serum (lane 7). Anti-H serum, on the other hand, does not remove heavy chains precipitable by any of the other three antisera (lanes 10, 12 and 13). This demonstrates that the species recognized as W6/32 and anti-H are completely nonoverlapping and that W6/32precipitable material, but not anti-H-precipitable material, is allo-reactive. In addition, since W6/32 removes almost all of the Prim from solution (lane 61, only a small fraction of the cellular fl?rn must be free. Among the material which is immunoprecipitated by the anti&m serum but not associated with HLA-A and -B heavy chains is a species migrating somewhat more rapidly than &m (lane 6). The identity of this species is unknown.
and -B Antigens
Cells were labeled overnight with %-methionine and detergent lysates were prepared. Aliquots were immunoprecipitated and analyzed by SDSPAGE and fluorography. For serial immunoprecipitations. an aliquot of lysate was incubated with the antiserum desired for preclearing. followed by the immunoadsorbent as usual. After pelleting, the supernatant was incubated with the immunoadsorbent and pelleted again to remove any remaining immune complexes. The resulting supernatant was divided into equal portions which were immunoprecipitated with the appropriate antisera. All final immunoprecipitations were from equivalent portions of the original lysate. Lane 1 is sample of the labeled polypeptides synthesized in T5-1. Lanes 2-5 are direct immunoprecipitates. Lanes 6-9 are the result of immunoprecipitations following an initial preclearing with W6/32. Lanes 1 O-l 3 are the result of immunoprecipitations following an initial preclearing with anti-H. The antisera were as follows: lanes 2.6 and 10. W6/32; lanes 3,7 and 11, anti-H; lanes 4.8 and 12, anti-,$m; lanes 5.9 and 13, anti-HLA-A2.
To determine whether both the 43,000 dalton species precipitated by W6/32 and the 42,000 dalton species precipitated by anti-H were indeed HLA heavy chains, amino terminal sequencing of radiolabeled immunoprecipitates was performed. All HLA-A and -B heavy chains examined so far contain methionine at position 5, phenylalanine at position 8 and serine at positions 2, 4, 11 and 13. Some, but not all, specificities contain an additional methionine at position 12 and an additional phenylalanine at position 9 (Strom-
inger et al., 1979). /lam, which should be sequenced along with the heavy chains present in W6/32 immunoprecipitates, should not add any complexity to the results, since in the sequenced region it contains no methionine, no phenylalanine and only a single serine residue at position 11. As seen in Figure 2, immunoprecipitates from cells radiolabeled in either methionine, phenylalanine or serine residues yielded the expected results, constituting unambiguous evidence for the identification of
300 200 100
600 z 0
400 500 200 ?
I IO I5
CYCLE of W6/32 NUMBER and Anti-H Immunopre-
Figure 2. Amino Acid Sequencing cipitates
lmmunoprecipitates from detergent lysates of “S-methionine-. 3Hphenylalanineor 3H-serine-labeled cells were eluted from the immunoadsorbent in 2% SDS and sequenced as described in Experimental Procedures. The radioactivity released in the chlorobutane extract at each cycle is presented. (A) W6/32-precipitable material from 35S-methionine-labeled cells: (6) anti-H-precipitable material from 35S-methionine-labeled cells: (C) W6/32-precipitable material from 3H-phenylalanine-labeled cells: (D) anti-H-precipitable material from 3H-phenylalanine-labeled cells; (E) W6/32-precipitable material from 3H-serine-labeled cells: (F) anti-H-precipitable material from ‘Hserine-labeled cells. The sequence in (F) could not be extended to 14 cycles due to technical problems.
HLA-A and -6 heavy chains in both W6/32 and antiH immunoprecipitates. Based upon a 94% repetitive yield, the amount of radioactivity in the 35S-methionine peaks at cycle 12 is consistent with the idea that in each case one fourth of the heavy chains or one of
the four allospecificities have a methionine at position 12. Similarly, it is probable that in each case two of the four allospecificities have a phenylalanine at position 9. These data suggest that the different HLA-A and -B heavy chains may be represented in similar proportions in each of these immunoprecipitates. The sequence data also rules out the possibility that the mobility difference between W6/32-reactive and antiH-reactive heavy chains is due to a difference in the polypeptides at their amino termini. Structural Comparisons These data suggest that W6/32 and anti-H recognize two distinct populations of HLA-A and -B heavy chains. One population is associated with /3*rn and carries alloantigenic determinants and the other population lacks both. Conformational differences between these populations were probed by examining their susceptibility to proteolysis. Native HLA-A and B antigens display only limited susceptibility to papain proteolysis, whereas separated heavy chains are completely degraded (Parham et at., 1977). When cell lysates were incubated with varying amounts of papain and then immunoprecipitated (Figure 31, anti-Hreactive material, but not W6/32-reactive material, was completely degraded. The unique antigenic activities of the two populations of heavy chains can be completely accounted for by conformational differences, as shown in Figure 4. A sample of lysate was first immunoprecipitated with W6/32 and the immunoprecipitate was then eluted using 6 M guanidine-HCI. Under these conditions, chain denaturation and dissociation should occur. When the eluate was subsequently dialyzed to remove guanidine, none of the resulting material could be immunoprecipitated by W6/32 (lane 4). However, the heavy chains were precipitated by anti-H (lane 5) and Pam was precipitated by an anti-&m serum (lane 6). Hence the conformational properties of HLA-A and -B heavy chains can be manipulated in vitro to convert them from one antigenic form to the other. This implies that conformational differences can account for the antigenic properties of the different heavy chain populations detected in cell lysates. Since guanidine treatment did not change the SDS-PAGE mobility of the heavychains, it is improbable that the mobility difference between the two populations detected in cell lysates is directly responsible for their distinct antigenie properties. The structural features resulting in the mobility difference between the W6/32-reactive and anti-H-reactive heavy chains generated in vivo were next explored. Carbohydrate differences were investigated by labeling the cells overnight in the presence of tunicamycin. This antibiotic prevents glycosylation by inhibiting the formation of the lipid-linked intermediates used in the synthesis of asparagine-linked oligosaccharides (Kuo and Lampen, 1974; Takatsuki, Kohno and Tamura, 1975). B lymphoblastoid cell lines
and -B Antigens
Aliquots of a detergent lysate of 35S-methionine-labeled cells were incubated with 0, 0.2, 1 or 5 Ag of activated papain in a total volume of 50 ~1, as described in Experimental Procedures. After terminating the reaction, aliquots were chilled, divided into equal portions.and immunoprecipitated with either W6/32 or anti-H. The results were analyzed by SDS-PAGE and fluorography.
cultured in the presence of tunicamycin have been shown to synthesize nonglycosylated forms of HLA-A and -6 antigens (H. L. Ploegh, H. T. Orr and J. L. Strominger, manuscript in preparation). As seen in Figure 5A, when cells were labeled in the presence of 2 pg/ml tunicamycin, more than 50% of the HLA heavy chains synthesized migrated with an apparent molecular weight of 39,000. Since W6/32-reactive and anti-H-reactive heavy chains were both converted to 39,000 dalton species, this suggested that both heavy chain populations were glycosylated in the absence of tunicamycin and that differences in oligosaccharide structure could account for their distinct mobilities. The basis for this structural difference was turther investigated. The enzyme endo+N-acetylglucosaminidase H (endo H) cleaves high mannose, asparagine-linked oligosaccharides between core N-acetylglucosamine residues. Complex oligosaccharides are resistant to its action (Tarentino and Maley, 1974; Trimble et al., 1978). When W6/32 and anti-H immunoprecipitates were treated with endo H, the anti-
lmmunoprecipitates from a detergent lysate of 3sS-methior$e-labeled cells were analyzed by SDS-PAGE and ffuorography. An aliquot of lysate was divided into equal portions which were immunoprecipitated with either W6/32 (lane I), anti-H (lane 2) or anti-&m (lane 3). A W6/32 immunoprecipitate from an identical allquot of lysate was eluted with 6 M guanidine-HCI, 50 mM Tris-HCI (pH 7.5). After the addition of NP40 to 1% and ovalbumin to 100 @g/ml. the eluate was dialyzed extensively against 10 mM Tris-HCI (pH 7.5) to remove the guanidine-HCI. The dialysate was then divided into equal portions and immunoprecipitated with either W6/32 (lane 4). anti-H (lane 5) or anti-&m (lane 6).
H-reactive heavy chain6 were converted to a mobility of 39,000 daltons, whereas only a small fraction of the W6/32-reactive heavy chains were similarly converted (Figure 58). Longer exposures of fluorographs reveal, however, that a small fraction of the anti-Hreactive heavy chains are indeed resistant to the action of endo H (data not shown). Hence whereas the majority of the anti-H reactive heavy chains appear to carry high mannose oligosaccharides, most of those reactive with W6/32 are probably carrying complex oligosaccharides. The heterogeneity in the oligosaccharide composition of the W6/32-reactive heavy chains accounts for some of the heterogeneity observed in W6/32 immunoprecipitates, particularly the faint bands often observed co-migrating with anti-Hreactive material at 42,000 daltons. Subcellular Localization An experiment designed
to test for the cell surface
Figure 5. Characterization 32- and Anti-H-Reactive
of the Oligosaccharide Heavy Chains
(A) Effect of tunicamycin. Cells were labeled overnight with 35Smethionine in the presence or absence of 2 pg/ml tunicamycin. lmmunoprecipitates from detergent lysates were analyzed by SDSPAGE and ftuorography. (Lane 1) a W6/32 immunoprecipitate from cells labeled in the absence of tunicamycin: (lane 2) an anti-H immunoprecipitate from cells labeled in the absence of tunicamycin; (lane 3) a W6/32 immunoprecipitate from cells labeled in the presence of tunicamycin; (lane 4) an anti-H immunoprecipitate from cells labeled in the presence of tunicamycin. (6) Susceptibility to endo H digestion. Aliquots of a detergent lysate of 35S-methionine-labeled cells were immunoprecipitated with W6/ 32 or anti-H. lmmunoprecipitates were eluted from the immunoadsorbent. divided into equal portions and either treated with endo H as described in Experimental Procedures or mock-incubated. Samples were subsequently precipitated on ice with 15% TCA. centrifuged at 8000 x g for 10 min and washed once with ice-cold acetone. Pellets were dissolved in SDS-PAGE sample buffer and were analyzed by SDS-PAGE and fluorography. (Lane 1) a W6/32 immunoprecipitate without endo H treatment; (lane 2) a W6/32 immunoprecipitate with endo H treatment; (lane 3) an anti-H immunoprecipitate without endo H treatment; (lane 4) an anti-H immunoprecipitate with endo H treatment.
localization of the different populations of heavy chains is presented in Figure 6. The technique used relies on the selective binding of antibodies to cell surface molecules when incubated with intact cells. To accomplish this, cells were incubated with antiserum and washed to remove unbound antibodies. They were then lysed in detergent in the presence of an excess of unlabeled cell extract, which served to
Figure 6. Subcellular Species
10’ cells were labeled overnight with 35S-methionine and divided into four aliquots in a 4:4:1:1 ratio. The larger aliquots were treated with W6/32 or anti-H as described in Experimental Procedures to detect antigens present on the cell surface. The smaller aliquots were lysed and immunoprecipitated as usual with either W6/32 or anti-H. The results were analyzed by SDS-PAGE and fluorography. (Lane 1) W6/ 32-reactive species in the whole cell lysate; (lane 2) anti-H-reactive species in the whole cell lysate; (lane 3) W6/32-reactive species on the cell surface; (lane 4) anti-H reactive species on the cell surface.
prevent any antibodies which might have dissociated from cell surface molecules during or after solubilization from rebinding to other labeled molecules. Immune complexes were then precipitated with the immunoadsorbent, and immunoprecipitates were analyzed as usual. W6/32-reactive material was detected on the cell surface (lane 31, whereas anti-H-reactive material, not visible at all in this figure (lane 4), could only sometimes be detected in small quantities on overexposed fluorographs (data not shown). Thus at most only 1 or 2% of the heavy chains present on the surface of T5-1 were precipitable by anti-H. In contrast, both populations were easily detected in detergent lysates of these cells (lanes 1 and 21, indicating
and -6 Antigens
that anti-l-l-reactive heavy chains are largely confined to locations inside the cell. It is not likely that the antiH reactive heavy chains which are detected on the cell surface are actually precipitated due to artifactual cell lysis during antibody incubation, since all of the anti-H-reactive heavy chains detected at the cell surface have an apparent molecular weight of 43,000. This suggests that they, unlike the vast majority of anti-H-reactive heavy chains in the cell, carry complex oligosaccharides. Since anti-H-precipitable heavy chains are highly susceptible to proteolysis, one explanation for the relatively small quantity detected at the cell surface would be that, although they do reach the surface in large quantities, they are rapidly removed by proteases present either in the medium or on the cell surface. This question was approached by incubating detergent lysates of labeled cells for up to 3 hr at 37% in the presence of medium in which T5-1 cells had been cultured for 3 days. Anti-H-reactive heavy chains proved quite stable to such treatment, although they were rapidly degraded by similar treatment with trypsin (data not shown).
I IO MINUTES
Figure 7. Anti-H-Reactive Heavy 32-Reactive Heavy Chains
I I I 15 20 25 AFTER PULSE Chains
Are the Precursors
30 of W6/
(A.B) 2 X 10’ cells were labeled for 3 min at 37°C in the presence of 500 pCi ?S-methionine in 4 ml of methionine-free medium. Half of this suspension was diluted into 10 ml of chase medium (A). and puromycin was added at 100 Fg/ml to the remaining half (6). Incubation at 37’C was continued and aliquots of 10’ cells were removed at the designated times and rapidly chilled on ice. These were washed once with ice-cold phosphate-buffered saline and detergent lysates were prepared. Incorporation of radioactivity into total protein was determined by TCA precipitation. 5% of each lysate was incubated on ice in the presence of 15% TCA, with 100 pg of bovine serum albumin added as a carrier. Precipitates were centrifuged at 6000 x g for 10 min and were washed once with ice-cold HCI. Pellets were dissolved in 0.1 M NaOH and the radioactivity was determined as described in Experimental Procedures. No increase in radioactivity was observed subsequent to the pulse. The remainder of each lysate was divided into equal portions and immunoprecipitated with either 1 Fl of W6/32 or 5 ~1 of anti-H. The results were analyzed by SDSPAGE and fluorography. (C) For quantitation, film was preexposed to an optical density of 0.15. Fluorographs were scanned using a densitometer and radioactivity was determined as the area under each peak. Cold chase and puromycin data were normalized with respect to the average amount of radioactivity incorporated into the TCA precipitates in each experiment. The results are plotted as follows: (0) heavy chains in W6/32 immunoprecipitates during a cold chase: 0 heavy chains in anti-H immunoprecipitates during a colcl chase: (A) sum of the heavy chains
Anti-H-Reactive Heavy Chains Are Precursors of W6/32-Reactive Heavy Chains Thus the heavy chains precipitated by anti-H were not associated with &m, and the majority both carried a high mannose oligosaccharide and were detected internally, but not at the cell surface. These observations suggested the possibility that anti-H-precipitable heavy chains might be immature forms of W6/32precipitable heavy chains. The biosynthetic relationship between these heavy chain populations was tested directly in pulse-chase experiments. Cells were pulsed for 3 min with 36Smethionine and then chased with medium containing an excess of unlabeled methionine. Aliquots were removed at various times of chase, lysates were prepared and portions were immunoprecipitated with either W6/32 or anti-H and analyzed by SDS-PAGE. Figure 7A demonstrates that the amount of anti-Himmunoprecipitable heavy chains decreased over the course of a 30 min chase period, whereas the amount of both heavy chains and Pnrn increased in W6/32 immunoprecipitates during this same time period. This is consistent with the idea that anti-H-reactive heavy chains are converted into W6/32-reactive heavy chains concomitant with their association with Pam. Also compatible with these data is the possibility that anti-H-reactive material is simply degraded intracellularly following synthesis, possibly as a result of its in W6/32 and anti-H immunoprecipitates during a cold chase; (0) heavy chains in W6/32 immunoprecipitates after puromycin addition: (0) heavy chains in anti-H immunoprecipitates afler puromycin addition: (a) sum of the heavy chains in W6/32 and anti-H immunoprecipitates afler puromycin addition. Crhe results for &m in W6/32 immunoprecipitates are not shown.)
failure to combine with j&m at an early time. Then the increase in W6/32precipitable heavy chains and Pam during the chase could be explained as the result of the completion of nascent heavy chains with which &m had already associated. This possibility was tested in the experiment presented in Figure 7B. It was reasoned that if the increase in W6/32-precipitable species were due to completion of nascent chains during the chase period, then the addition of puromycin after a 3 min pulse should prevent this increase, since puromycin will prevent the elongation of nascent chains. Hence cells were labeled in a fashion identical to that in the previous experiment, but were treated with 100 pg/ ml puromycin instead of chasing. This concentration of puromycin was effective in inhibiting the incorporation of %-methionine into TCA-precipitable material within 15 sec. It can be seen from Figure 78 that the kinetics are similar to those observed during a cold chase. A direct quantitative comparison of these results is presented in Figure 7C, and a virtual identity in the kinetics of increase and decrease of both antigenie species is apparent. This suggests strongly that the formation of W6/32-precipitable material during the chase period is due to conversion from either an as yet undetected antigenic species or from anti-Hprecipitable heavy chains and freeDem. The possibility that an undetected antigenic Species is the precursor of W6/32-reactive material is considered improbable, however, since the total amount of W6/32-reactive and anti-H-reactive heavy chains was constant in both experiments throughout the time period examined (see Figure 7C). The possibility that the loss of anti-Hprecipitable heavy chains with time is due to degradation is considered improbable, as a result of experiments using the cell line Daudi. This cell line, which lacks the ability to synthesize j&m (Goodfellow et al., 19751, produces anti-H-reactive heavy chains (Ploegh et al., 1979). Although these heavy chains are not detected at the cell surface, they have been found to be stable intracellularly for relatively long periods of time (Ploegh et al., 1979). This supports the idea that the disappearance of anti-H-reactive heavy chains in T5-1 is due to their association with /3*rn. lmmunoprecipitations from T5-1 with an anti-&m serum demonstrate that j&m is synthesized in excess of heavy chains and that the total amount of free and bound &m is constant throughout the chase period (data not shown). This indicates that the appearance of /&rn in W6/32 immunoprecipitates is due to its association with heavy chains from a pool of free /%.m. Maturation of W6/324teactive Complexes It can be seen from Figure 7 that the apparent molecular weight of newly formed W6/32-reactive heavy chains is not 43,000, as is observed in long-term labelings. Indeed, they appear to migrate slightly more rapidly than anti-H-reactive heavy chains. This is in
10’ cells were labeled for 5 min at 37°C with 250 &i 35S-methionine in 2 ml of methionine-free medium and were diluted into 10 ml of chase medium. Aliquots were removed at the appropriate times, detergent lysates were prepared and duplicate immunoprecipitations were performed with 1 pl of W6/32 or 5 yl of anti-H as indicated (5 x 1 O5 cell equivalents per immunoprecipitation). To control for SDSPAGE artifacts due to the variable amounts of Ig in immunoprecipitates. 4 pl of normal rabbit serum were added to the W6/32 incubations. One set of immunoprecipitates was subsequently eluted with SDS-PAGE sample buffer and analyzed by SDS-PAGE and fluorography. The other set was eluted and treated with endo H as described in Experimental Procedures. Samples were then precipitated on ice with 15% TCA, centrifuged at 8000 x g for 10 min and washed once with ice-cold acetone. Pellets were dissolved in SDS-PAGE sample buffer and analyzed by SDS-PAGE and fluorography.
part an SDS-PAGE artifact due to the larger amounts of immunoglobulin heavy chains present in anti-H immunoprecipitates. When this is carefully controlled for (see Figure 61, newly synthesized W6/32-reactive heavy chains co-migrate exactly with anti-H-reactive heavy chains, at an apparent molecular weight of 42,000. A band of apparent molecular weight 43,000 did appear in W6/32 immunoprecipitates after 20-30 min of chase (Figure 7). When cells were pulsed for 5 min and chased for longer periods of time, it became clear that the W6/32-precipitable heavy chain doublet of 42,000 daltons was slowly converted to a 43,000 dalton doublet (Figure 8). lmmunoprecipitations with alloantisera confirmed that heavy chain allospecificities present in both bands of the 42,000 dalton doublet were converted to a mobility of 43,000 daltons (data not shown). The nature of this mobility shift was investigated by treating portions of the immunoprecipitates with endo H. The conversion of the 42,000 dalton species to the 43,000 dalton species paralleled the conversion of the oligosaccharide from an endo H-sensitive form to an endo H-insensitive form (Figure 8). This probably represents the conversion of the oligosaccharide from the immature, high mannose form to the complex oligosaccharide characteristic of the mature glycoprotein. Neuraminidase, which had no observable effect on the 42,000 dalton W6/32precipitable heavy chains, converted the 43.000 dalton W6/32-precipitable heavy chains to a mobility of 42,000 daltons (Figure 9). The most likely interpreta-
Figure 9. Neuraminidase tive Material
and -6 Antigens
2 x 10’ cells were labeled for 5 min at 37°C with 125 fiCi %methionine in 0.5 ml of methionine-free medium and were diluted into 2 ml of chase medium. Aliquots were removed at the indicated times and detergent lysates were prepared. Each lysate was divided into equal portions which were either incubated with neuraminidase as described in Experimental Procedures or mock-incubated. These were subsequently immunoprecipitated with W6/32 and analyzed by SDS-PAGE and fluorography.
tion is that the observed mobility difference between heavy chains carrying high mannose and complex oligosaccharides is due solely to the presence of charged sialic acid residues on the mature, complex oligosaccharides. To determine at what time after their synthesis W6/ 32-reactive species reach the cell surface, cell surface binding assays were performed on pulse-labeled cells chased for varying lengths of time. W6/32-reactive species were first detected at the cell surface after 60 min of chase and were maximally detected by 80 min of chase (Figure 10). Discussion The experiments presented here describe the events in the assembly and maturation of HLA-A and -B antigens in the lymphoblastoid cell line T5-1. Completed HLA-A and -B heavy chain polypeptides are initially observed carrying high mannose oligosaccharides. These HLA-A and -B heavy chains are largely or wholly unassociated with /3*rn immediately after synthesis. During the following 15 min, chain association occurs and the conformation of the heavy chains is converted from one antigenic form to another. Beginning at approximately 30 min after synthesis, the high mannose oligosaccharides are converted to complex oligosaccharides characteristic of mature HLA-A and -B antigens. Between 60 and 80 min after synthesis, the mature antigens can be detected at the cell surface. The properties of the antisera and the nature of the determinants they recognize on HLA-A and -B heavy
10. Cell Surface
10’ cells were labeled for 10 min at 37°C with 500 j&i %-methionine in 2 ml of methionine-free medium and were diluted into 10 ml of chase medium. 10’ cells were removed and chilled at the appropriate times and were treated with 2 pl of W6/32 to detect cell surface antigens, as described in Experimental Procedures.
chains are intriguing. The mouse monoclonal antibody W6/32 has been shown to recognize a heavy chain determinant present on all HLA-A, -B and -C allospecificites examined (Barnstable et al., 1978; Parham et al., 1979). Both the W6/32 determinant and alloantigenie determinants appear to be highly sensitive to the conformation of the molecule, requiring for their expression the presence of&m in a complex with the heavy chain (for the one reported exception, see Nakamuro, Tanigaki and Pressman, 1975). The antiH serum, on the other hand, appears highly specific for antigenic determinants expressed by the separated heavy chain, but not by the native complex. An antiserum with a similar specificity has been previously described (Tanigaki et al., 1974). The heavy chain determinants recognized by anti-H may not be expressed by the complex due to physical shielding by Pnrn. Alternatively, they may only be expressed by a different conformer of the heavy chain. The lack of reactivity of the separated heavy chain with W6/32 and with alloantisera, as well as its susceptibility to proteolysis, might suggest the latter. Such a conclusion is also supported by the results of
circular dichroism measurements (Lancet, Parham and Strominger, 1979). In either case, one might expect that such an antiserum could influence the association-dissociation equilibrium between the subunits of HLA-A and -B antigens (Hyafil and Strominger, 1979). If the antiserum actually induced dissociation, or if substantial dissociation occurred in the absence of antiserum, one would expect to precipitate anti-Hreactive heavy chains from detergent lysates even after an initial preclear with anti-H to remove this material. The results of the serial immunoprecipitations (Figure 1, lane 11) make it clear that this does not occur under the conditions used in these studies. Incubation of aliquots of cell lysate at 37°C has, however, been found to lead to some dissociation of W6/32-reactive complexes into anti-H-reactive heavy chains and free &m. No additional effect of added anti-H serum on this dissociation has been observed. The complementary specificities of W6/32 and antiH have permitted the clarification of some details concerning the association of the subunits of HLA-A and -B antigens in vivo. Most or all of the chain assembly in T5-1 takes place post-translationally. After a 3 min pulse, only 25% of the heavy chains appear to have already associated with /&rn. Although it is possible that some &rn associates with nascent heavy chains, the presence of these complexes may well be explained by the association of Prim with completed heavy chains during the pulse. Whether or not &m can in fact associate with partially synthesized heavy chains is an intriguing question. There are few clues as to where Prim contacts the heavy chain in the native complex. Whereas interactions with the amino terminal region of the heavy chain might allow for some association with partially synthesized heavy chains, substantial interactions nearer the carboxy terminus might preclude this. In this regard, it is interesting to note that some covalent assembly of completed immunoglobulin light chains onto partially synthesized immunoglobulin heavy chains has been demonstrated to occur in MOPC 11 cells, and that this assembly occurs only after after the heavy chains have reached a well defined size (Bergman and Kuehl, 1979). It has not been possible to demonstrate the in vitro association of Pzrn with the isolated heavy chains produced by the Daudi cell line, or with isolated heavy chains after their synthesis and processing in vitro (Ploegh et al., 1979). Attempts to do so with the isolated heavy chains obtained after separation in vitro have produced conflicting results (Lancet et al., 1979; Parham et al., 1979). Our experiments demonstrate that &m can associate with completed heavy chains in vivo. The failure to consistently reproduce this in vitro may be due to conformational changes occurring in heavy chains soon after their synthesis which render them incapable of subsequent association. This might explain why some heavy chains appear not to
associate with &rn in vivo even after long periods of time (see Figures 7 and 8). On the other hand, particular conditions in the endoplasmic reticulum which have not yet been reproduced in vitro may be required for association. The observed kinetics of association in vivo may be merely a reflection of the on-rate for association under the conditions present in the endoplasmic reticulum. On the other hand, it is possible that the subunits are inserted into the endoplasmic reticulum in different locations, and the limiting factor in their association may be the migration of one to the point of insertion of the other. Similarly, an as yet undetected processing step might be rate-limiting. The mRNA for HLA-A and -B heavy chains has been translated in vitro to yield products with amino terminal extensions. When supplemented with microsomes, processing occurs, which probably consists of the removal of this extension and the glycosylation of the heavy chain (Ploegh et al., 1979). The data presented here suggest that the initial glycosylation reactions in vivo result in the assembly of a high mannose oligosaccharide, even though the carbohydrate composition of mature HLA-A and -B antigens is that of a typical complex oligosaccharide (Parham et al., 1977). At approximately 30 min after synthesis, a gradual conversion from the high mannose form to the complex form begins. Similar processing has been demonstrated for the asparagine-linked oligosaccharide of IgG, as well as for several viral membrane glycoproteins. In these systems, processing consists of the stepwise removal of glucose and mannose residues followed by the addition of terminal sugars characteristic of complex oligosaccharides (Robbins et al., 1977; Kornfeld et al., 1978; Tabas et al., 1978). The earliest stages of trimming which have been described in these systems have probably not been detected in this study, since early processing intermediates would carry oligosaccharides which were still substrates for endo H. The relationships among the various maturational steps leading to the expression of HLA-A and -B antigens at the cell surface are only partially understood. That glycosylation of the heavy chain is not at all required for its association with /3*rn or for its expression at the cell surface has been demonstrated directly using the antibiotic tunicamycin (H. L. Ploegh, H. T. Orr and J. L. Strominger, manuscript in preparation). The association of /&rn with the heavy chain does, however, seem to influence the rate at which the heavy chain oligosaccharide is processed. It is evident in Figure 8 that the oligosaccharides present on anti-H-reactive heavy chains have not been processed by 40 min after synthesis, even though approximately half of those present on W6/32-reactive heavy chains have. Processing of the oligosaccharides present on those heavy chains which for some reason have not associated with fi2rn does indeed occur, but
and -B Antigens
only slowly, not beginning until 1 hr after synthesis and continuing for at least 2 hr thereafter (our unpublished observations). This relatively slow rate of processing of anti-H-reactive heavy chains is also observed in Daudi (H. Ploegh, unpublished observations), suggesting that it may be a general phenomenon. It is possible that the anti-H-reactive heavy chains present in T5-1 and in Daudi move only slowly to the intracellular location where the processing enzymes are located. The possibility that efficient recognition by processing enzymes may be dependent upon the conformation of the heavy chain cannot, however, be ruled out. The association of j&m with the heavy chain appears also to be a requirement for the movement of the heavy chain to the cell surface. The anti-H-reactive heavy chains present in Daudi remain intracellular at all times, as judged by the observations that anti-H serum is not cytotoxic for Daudi and that it does not precipitate material from the surface of Daudi (Ploegh et al., 1979). By these same criteria, anti-H-reactive heavy chains are detectable on the surface of T5-1. The origin of the small amount of processed anti-Hreactive heavy chains on the surface of T5-1 is, however, not clear. They may be present as a result of the movement of anti-H-reactive heavy chains from locations inside the cell to the cell surface. Their presence is more easily accounted for, however, by the dissociation of W6/32-reactive complexes once they reach the cell surface. This notion is plausible, since, as mentioned above, the dissociation of such complexes can be detected at 37°C in cell lysates, and this would also be fully consistent with the evidence gathered from Daudi, since in Daudi no W6/32-reactive complexes are produced as a result of its inability to synthesize Blrn. Taken together, then, the evidence obtained both from T5-1 and from Daudi may indicate an important role for&m in the intracellular movement of the heavy chain and in its appearance at the cell surface. This effect may be the result of the distinct conformational states of the heavy chain defined by the antisera used in this study. The events in the assembly and maturation of HLAA and -B antigens in the human B lymphoblastoid cell line JY appear to occur in a manner qualitatively similar to that outlined for T5-1. Cell surface expression of HLA-A and -B antigens in T5-1 occurs approximately 60-80 min after the synthesis of their subunits, soon after oligosaccharide processing has taken place. Chain association, oligosaccharide processing and cell surface expression all seem to occur at a rate approximately twice as fast in JY as in T5-1, however, resulting in the appearance of these antigens at the cell surface within 30 min after synthesis (H. Ploegh, unpublished observations; H. L. Ploegh. H. T. Orr and J. L. Strominger, manuscript in preparation). Thus quantitative, but not qualitative, differ-
ences are likely to exist among various B lymphoblastoid cell lines. A similar variability is observed in the expression of other glycoproteins in a variety of cell types. Expression of the VSV G protein at the surface of CHO cells occurs approximately 40-50 min after synthesis (Knipe, Lodish and Baltimore, 1977). The surface appearance of H-2 antigens in mouse spleen cells (Vitetta, and Uhr, 1975) and of glycophorin in K562 erythroleukemic cells (Jokinen, Gahmberg and Anderson, 1979) occur within 30 min of synthesis. Although the time course of maturation of these glycoproteins is somewhat variable, the nature and the sequence of the maturational events which occur during the biosynthesis of such glycoproteins appear to be quite general. Experimental
Cdl8 The human 6 lymphoblastoid cell line T5-1 (HLA-Al , -A2, -68, -827) was routinely cultured in RPM1 1840 medium supplemented with 10% Fetal calf serum, 100 U/ml penicillin and 100 wgg/ml streptomycin (GIBCO). Cells were tested for mycoplasma (Bioassay Systems) and found to be negative. Radioactive Labeling Cells were labeled by incubation in methionine-free RPM1 1640 (GIBCO. Select-Amine Kit) supplemented with 10% dialyzed fetal calf serum in the presence of 35S-methionine (500 Ci/mmole; New England Nuclear). Cells were washed once with methionine-free medium, resuspended at 1 X 10s/ml in fresh medium and preincubated at 37’C for 45 min. ?S-methionine was added at 250 rCi/ml and incorporation was allowed to proceed for 8-l 0 hr at 37OC. Cells were labeled either with ‘f-f-serine (2 Ci/mmole: New England Nuclear) or with ‘H-phenylalanine (54 Ci/mmole; New England Nuclear) at 62 &i/ml in a similar manner, utilizing either serine- or phenylalaninefree RPM 1640 (GIBCO, Select-Amine Kit), respectively. When present, tunicamycin (a gifl from Eli Lilly Co.) was added to the preincubation medium at 2 pge/ml. Cells were pulse-labeled after washing twice with methionine-free medium, resuspending at 5 X 1 O6 cells per ml and preincubating for 1 hr at 37’C. ?S-methionine was added to 125-250 &i/ml and incorporation was allowed to proceed for 3-l 0 min. The suspension was then diluted into 5 vol of RPM 1640 medium containing 5 times the normal concentration of methionine, and incubation at 37’C was continued. Aliquots (5 X 10’ cells per immunoprecipitation) were removed at the appropriate times and rapidly chilled on ice. Alternatively, labeling was stopped by the addition of 100 pg/ml puromycin (Sigma). In either case, no increase in TCA-precipitable counts was observed subsequent to the pulse. Detergent Lysates Labeled cells were washed once with ice-cold phosphate-buffered saline (GIBCO) and were solubilized by incubating on ice for 45 min at 5 X lo6 cells per ml in 10 mM Tris-HCI (pH 7.5). 1% NP40 (Particle Data Laboratories), 1 mM DTT (Sigma) and 0.1 mM PMSF (Sigma). Lysates from continuous labelings were centrifuged for 1 hr at 100,000 x g; lysates from pulse labelings were centrifuged for 15 min at 12,000 X g. Supernatants were stored at -70°C if not used immediately. Antisera and lmmunoprecipitation The antisera used were: (1) W6/32. a mouse monoclonal antibody which recognizes a determinant present on all HLA-A and -B and -C heavy chains when they are associated with &m (Barnstable et al., 1978; Parham at al., 1979); (2) anti-heavy chain serum (anti-H). a rabbit heteroserum raised by A. Fuks against the papain-solubilized
HLA-B7 heavy chain prepared by gel filtration in 6 M guanidine-HCI; (3) anti-&-microglobulin serum (anti-&m). a rabbit heteroserum raised against human urinary µglobulin by Ft. Robb; (4) Stewart, an HLA-AP-specific alloantiserum; (5) Raymond, an HLA-Alspecific alloantiserum; (6) SFCC I1 27 (a gift from E. Yunis), an HLABB-specific alloantiserum; (7) SFCC X206 (a gift from E. Yunis), an HLA-B27-specific alloantiserum and (6) normal rabbit serum. lmmunoprecipitation was carried out essentially as described by Kessler (1975). using formalin-fixed. heat-killed Staphylococcus aureus (Cowan I Strain). Briefly, aliquots of detergent lysate were incubated for 0.5-l hr on ice with 5 ~1 normal rabbit serum, followed by the immunoadsorbent. Suspensions were centrifuged for 5 min at 10.000 x g. Supernatants were subsequently treated with l-5 ~1 of specific antibody for 1 hr on ice, followed again by the immunoadsorbent. Specific immunoprecipitates were washed 4 times with NET buffer [0.15 M NaCI, 5 mM EDTA, 50 mM Tris-HCI. 0.02% azide (pH 7.5)] supplemented with 0.5 M NaCI, 0.5% NP40, 0.1 mM PMSF and 1 mg/ml ovalbumin. and washed twice with NET supplemented with 0.5% NP40 and 0.1 mM PMSF. lmmunoprecipitates were then eluted by boiling for 3 min in SDS-PAGE sample buffer, 2% SDS or 0.15 M Tris-HCI (pH 7.6). 1% /3-mercaptoethanol and 1% SDS. Binding of Cell Surface HLA-A and -B Antigens Detection of cell surface HLA-A and -B antigens was performed essentially as described by Vitetta and Uhr (1975). 5 X 1 O5 continuously labeled or lo8 pulse-labeled cells were washed once with phosphate-buffered saline, resuspended in 250-500 pl phosphatebuffered saline and incubated for 0.5 hr on ice with the specific antiserum. Cells were washed twice with ice-cold phosphate-buffered saline to remove unbound antibodies and then lysed into 500 pl of a detergent lysate containing a 10 fold excess of a previously prepared unlabeled cell extract. These lysates were then centrifuged for 15 min at 12.000 x g and supernatants were treated with the immunoadsorbent as described above. Enzyme Incubations Papain (Worthington) was activated by diluting into lysis buffer containing 2 mM OTT and was incubated at 37’C for 1 hr with an equal volume of labeled cell extract. The reaction was terminated by the addition of 0.1 vol of 100 mM neutralized iodoacetic acid. Trypsin sensitivity was tested by incubating 30 ~1 of labeled cell lysate with 1 Fg TPCK-trypsin (Worthington) at 37’C in a total volume of 60~1. The reaction was terminated by the addition of PMSF (Sigma) to 0.1 mM. Neuraminidase (Vibrio cholerae, 500 U/ml; Behring Diagnostics) incubations were performed by diluting labeled cell lysates with an equal volume of 0.1 M sodium acetate (pH 5.5). 1.6% NaCI. 0.2% CaC12 and adding 10 cl of enzyme at 0, 0.5. 1 and 2 hr during a 4 hr incubation at 37’C. The reaction was stopped by chilling on ice. Endo-P-N-acetylglucosaminidase H (a gift from 0. Wirth and P. Robbins) incubations were performed after eluting immunoprecipitates by boiling in 0.15 M Tris-HCI (pH 7.6) 1% SDS, 1% 6mercaptoethanol for 3 min. These were diluted with 9 vol of 0.15 M sodium citrate (pH 5.5). Incubations were for 6-l 2 hr at 37°C with a 1: 100 ratio (v/v) of enzyme solution (30 Fg/ml) to incubation mixture. The reaction was stopped by TCA precipitation. Guanidine Densturatlon HLA-A and -B heavy chains by eluting immunoprecipitates mM Tris-HCI (pH 7.5). The HCI. 50 mM Tris-HCI fpH followed by dialysis against
were denatured and separated from fi2rn with 6 M guanidine-HCI (Heico), 50 eluate was diluted into 6 M guanidine7.5) 1% NP40. 100 ).rgs/ml ovalbumin. 10 mM Tris-HCI (pH 7.5).
SDS-PAGE SDS-PAGE was carried out as described by Laemmli (1970) on 715% linear gradient slab gels, using electrophoresis grade reagents (BioRAD). Radioactive bands were detected by fluorography (Banner and Laskey, 1974) on Kodak 58-5 X-ray film. For quantitation. Kodak XR-5 X-ray film was preexposed as described by Laskey_a?d Mills (1975). and fluorographs were scanned using an ORTEC 4310 densitometer.
Amlno Acid Sequence Analysis Amino acid sequencing of immunoprecipitates was performed on an updated Beckman Liquid Phase Sequenator 690, using the 0.1 M Quadrol program (Brauer, Margolies and Haber. 1975) in the presence of 3 mg Polybrene (Tarr et al., 1976). Samples were applied after elution from the immunoadsorbent in 2% SDS, with 50 nmole sperm whale myoglobin as a carrier. The repetitive yield had been previously determined to be 94%. Liquld Scintillation Counting Samples to be counted were dissolved nostics) and counted in a Beckman Counter.
in Liquiscint (National DiagLS 233 Liquid Scintillation
Acknowledgments We gratefully acknowledge the contributions to this work of Dr. Abe Fuks, for preparing the anti-heavy chain serum, and of Dr. Dyann Wirth. both for providing endo H and for helpful suggestions. This research was supported by a research grant from the NIH. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact. Received
June 6. 1979;
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