HLA-DRβ chain residue 86 controls DRαβ dimer stability

1346

E A. W.Verreck, A. Terjmijtelen and F. Koning

Frank A. W. Verreck., Annemarie Termijtelen and kits Koning

Department of Immunohaematology and Bloodbank, University Hospital Leiden, Leiden

Eur. J. Immunol. 1993.23: 1346-1350

HLA-DRP chain residue 86 controls DRaP dimer stability Major histocompatibility complex class I1 molecules exist in two forms, which can be distinguished on the basis of their stability in sodium dodecyl sulfate (SDS) as SDS-stable and SDS-unstable ab dimers. The ratio of stable vs. unstable ab dimers varies between murine H-2 alleles and isotypes, but the molecular basis for this observation is unknown. Here we show that for the human HLA-DRB1 and HLA-DRB3 gene products this ratio is controlled by the valine/glycine dimorphism at position 86. Haplotypes coding for DRP chains with a valine at position 86 express higher numbers of stable dimers compared to similar haplotypes expressing DRP chains with a glycine at that position. Reverse-phase highperformance liquid chromatography analysis of iodinated peptides, which were eluted from D R dimers with either a DRB1*1101 or a DRB1*1104 chain which differ only at position 86, indicated that these DR dimers contain (partially) distinct sets of peptides. The valine/glycine dimorphism is highly conserved, present in most HLA-DR alleles and influences peptide-binding. Analysis of the occurrence of theVal% and the GlyS6 gene products revealed that these are not equally present in the population. Depending on the DR specificity either the Valg6 of Gly% allelic variant is favored. Thus, the natural, highly conserved dimorphism at HLA-DR f3 chain position 86 influences peptide selection. The dimorphism is therefore likely to influence antigen presentation and forms the molecular basis for the observed differences in stability of Valg6-and GlyS6-containingDR dimers in the presence of SDS.

1 Introduction

unstable HLA-DR class I1 dimers is controlled by the nature of the amino acid at position 86 of the @ chain. This appears to reflect differences in peptide binding.

MHC class I1 molecules bind and present processed peptide antigens. At least two forms of class I1 dimers can be distinguished based on their stability in SDS: a stable conformation, resistant to dissociation in 2 % SDS, and an 2 Materials and methods unstable conformation which rapidly dissociates in 2 YO SDS [l-61. It has been shown that for the formation of 2.1 Cell lines SDS-stable dimers degradation of the class 11-associated invariant chain and the presence of peptide is required All HLA-homozygous EBV-transformed B cell lines were [3, 4,7,8]. Recent evidence, however, indicates that a established in our own laboratory or obtained via the substantial amount of both stable and unstable cell surface Histocompatibility Workshops. expressed class I1 dimers contain peptide antigens [6]. Studies by Germain and Hendrix [4] have demonstrated that in the mouse allele- and isotype-specific variations 2.2 Surface iodination exist in the ratios between the stable and unstable dimers. This raises the questions whether similar allelic variations Approximately lo7 cells were harvested, washed three can be observed for human HLA class I1 dimers, what the times with PBS and lactoperoxidase-catalyzed cell surface molecular basis for such variations is and whether they have iodination was carried out as described previously [9, 101. biological relevance. To investigate this we took advantage Subsequently, these cells were lysed in 1 ml lysis buffer of the availability of a large panel of well-defined human containing 0.5 YO NP40, 50 mM Tris-HC1, 150 mM NaCl, B cell lines that are homozygous for the HLA gene 0.1 mM PMSF, 10 mM iodoacetamide, 1 pg/ml leupeptin, products (homozygous typing cells), and of the fact that 1 pg/ml chymostatin, 1pg/ml antipain, 1 pg/ml pepstatin, sequence data on the expressed class I1 gene products are pH 8. After incubation for 30 min at 4°C the lysates were known. The results indicate that the ratio of stable to centrifuged at 13000 x g for 15 min at 4°C and supernatants were used for immunoprecipitations. [I 113481 Supported by the Dutch Kidney Foundation. Correspondence: Frank Verreck, Department of Immunohaematology and Bloodbank, BLDG 1, E3-Q, University Hospital Leiden, PO.Box 9600, NL-2300 RC Leiden, The Netherlands Key Words: HLA-DR I fi chain position 86 I Dimorphism I Sodium dodecyl sulfate stability I Peptide binding 0014-2980/93/0606-1346$10.00+ .2510

2.3 Immunoprecipitation and SDS-PAGE analysis

The lysates were precleared by adding 75 pl normal rabbit serum and 100 pl protein A-Sepharose beads (Pharmacia), followed by gentle shaking at room temperature for 2 h. After removal of the beads HLA-DR-specific immunoprecipitations were carried out by mixing 4 p1 ascites fluid of the DR-specific mAb B8.11.2 [ll-131 and 100 p1 lysate for 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1993

Eur. J. Immunol. 1993. 23: 1346-1350

1h at room temperature, followed by the addition of 10 p1 protein A-Sepharose beads (Pharmacia) and mixing for another hour at room temperature. To dissect the HLADRBl and HLA-DRB3 gene products first an immunoprecipitation with mAb 7.3.19.1 was performed. This procedure results in the complete depletion of all DRaf33 dimers from the lysate ([11, 121 and results not shown). The DRap3-depleted lysate was subsequently used for immunoprecipitation with the D R specific mAb B8.11.2 resulting in the recovery of D R a p l dimers. The beads were washed four times with 0.5 ml lysis buffer and finally once with 0.5 ml buffer containing 125 mM Tris, 150 mM NaC1, pH 8. Next they were resuspended in 100 pl sample buffer containing 2 % SDS, 125 mM Tris, 10 % glycerol, 0.1 % BFB,pH 6.8. The samples were left at room temperature for 30 min and split into two equal aliquots. One was boiled 3 min before analysis by SDS-PAGE on 12 YOgels. The gels were dryed and autoradiography carried out at -70 "C. To quantitate the precipitates counting of the excised gel spots corresponding t o the position of the af3dimers and free a and f3 chains was performed on a Packard Cobra Gamma Counter. 2.4 Reverse-phase W L C analysis of DR-associated peptides

HLA-DR dimer stability

1347

A

LANE

1

2

3

4

5

6

7

8

NON-BOILED/BOILED

NB

B

NB

B

NB

B

NB

B

9 1 0

NB

B

aB

a

B

front putativepeptide DM1*

0301

1101

1301

1301

1302

B COUNTS PER MINUTE

a6

1649

986

931

1590

1653

0

702

876

262

447

1047

B

643

946

234

433

1106

a + B

1345

1822

496

880

2153

0301

1101

1301

1301

1302

.................................................... LJJ u eL28

RATIO aB/(a + 6 )

HLA-DR molecules were isolated from cell surface iodinated B cell lines. DR-bound peptides were eluted with 0.1 % trifluoroacetic acid and separated from high molecular weight material by ultracentrifugation using a Centricon 10 filter (Amicon). The eluted peptides were subsequently separated by reverse-phase HPLC (using a Lichrosorb RP18 HPLC column from Chrompack and an acetonitrile gradient for elution). Radioactivity in the collected fractions of 1ml was determined using a gamma counter.

3 Results 3.1 Allele-specific stability of HLA-DR h e r s Cell surface iodination and DR-specific immunoprecipitations were carried out on a panel of HLA homozygous typing cells. Prior to SDS-PAGE analysis the immunoprecipitates were eluted in 2 % SDS, split into two equal portions of which one was'boiled. A representative experiment shows that in all non-boiled samples both ap dimers and free a and fl chains are present (Fig. l A , odd lanes) and that boiling results in the loss of the afl dimers and gain of free a and p chains (Fig. lA, even lanes). The results in Fig. 1show that differences in the ratio of stable to unstable dimers between the HLA-class I1 allelic products can be detected easily (Fig. l A , lanes 1, 3 , 5 7, and 9). To quantitate these ratios the gel slices corresponding to the ap dimers and free a and f3 chains were excised and counted in a gamma counter. From these counts the ratio of stable t o unstable dimers was calculated (Fig. lB), giving an arbitrary measurement for the stability of the cell surfaceexpressed DR molecules. These ratios demonstrate that, under the experimental conditions used, the DRB1*0301 and DRB1*1301 gene products give rise to higher numbers of stable af3 dimers (ratio > 1) than the DRB1*1101 and DRB1*1302 gene products investigated (ratio < 1).

DM1*

Figure 1. Allele-specific stability of HLA-DR molecules. (A) SDS-PAGEanalysisof DR-immunoprecipitatesobtained from five human HLA-homozygous EBV transformed B cell lines. Samples

were either not boiled (odd lanes) or boiled (even lanes) prior to analysis. Note that loss of the stable dimer results in a larger amount of such molecular weight material that runs ahead of the front. Two-dimensional SDS-PAGE analysis of these samples revealed that dissociation of the stable afi dimer results in the release of this low molecular weight material (results not shown, [6]) that may, therefore, represent HLA-DR-bound peptide. Since the mAb B8.11.2 reacts with all DR molecules the ratios reflect the sum of the stability of DRafil and DRaf33 dimers. However, expression of the the DRapl dimer is much higher as that of the DRafi3 dimer (results not shown) so that the ratios shown are largely determined by the stability of the DRafil dimers. (B) Quantification of the ratio of stable- to -unstable HLA-DRdimers. Gel slices corresponding to the position of the DRafi dimers and the free a and f3 chains in the odd lanes were excised and the counts per minute determined by gamma counting. From these counts the stable- to -unstableratio was calculated as follows: cpm ap/cpm (a + 0). In a second experiment the following ratios were found: DRB1*0301:1.46; DRB1*1101:0.55; DRB1*1301:2.03; DRB1*1301:1.86; DRB1*1302:0.96.

3.2 Molecular basis of HLA-DR h e r stability Scrutiny of the amino acid sequences in the putative peptide-binding region of the DRB1*1301 and DRB1*1302 p chains (Fig. 2) revealed that they differ by only one amino acid: the more stable DRB1*1301has a valine at position 86 (Valss) while the less stable DRB1*1302 has a glycine at that position (Glyss). Similarly,the relatively stable DRB1*0301 sequence has Val&, while the relatively unstable DRB1*1101 gene product has Gly&. To determine whether this valine/glycine dimorphism at position 86 indeed plays a role in defining the stability of

1348

E A. W. Verreck, A. Terjmijtelen and F. Koning

Eur. J. Immunol. 1993.23: 1346-1350

T cellzdefined specificit4es Haplotypo Amino acid mquanca

HLA-DR molecules we extented the panel of cell lines for further studies. Ten cell lines expressing DRaPl and DRaP3 dimers were selected. They were surface iodinated and their DRaPl and DRaP3 dimers were isolated. These immunoprecipitates were analyzed on SDS-PAGE and the ratios of stable to unstable dimers were calculated (Fig. 3).

For both the DRBl and the DRB3 locus products of the DR5 and DR6 haplotypes it was found that the presence of Valg6results in the expression of higher numbers of stable aP dimers than the presence of Gly%. Although other amino acid substitutions are found in the putative peptidebinding region of the DRB chains investigated (Fig. 2), the

POS.88 DRS

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0

0s

1

15

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identified (Fig. 3). This strongly indicates that the intrinsic character of these allelic gene products is controlled by the valine/glycine dimorphism and is not significantly influenced by potential differences between the endogeneously generated peptide pools present in the cells investigated. We also determined the stability of the DRaP-dimers in the DR1, DR3 and DR4 specificities (Fig. 4). A similar influence of the valine/glycine dimorphism on the formation of stable and unstable aP-dimers was observed in the panel of DR3- and DR4-positive cells investigated (Fig. 4A). In contrast to the DR3, DR4, DR5 and DR6 specificities where a ratio of > 1is indicative for the more stableVal% and a ratio of < 1represents the more unstable Gly&, it was found that the Glyg6-containingDR1 specificity (DRB1*0101, Dwl) forms dimers with a stable- to

ratio stablelunstable

Figure 3. Stability of DRBl and DRB3 gene products in DR5 and DR6 specificities. DRafil and DRap3 dimers were isolated by On SDS-PAGE. sequential immunoprecipitation and Ratios of stable- to -unstable DR dimers were calculated as in legend to Fig. 1. Cells expressing either both stable (TEM) and both unstable DRapl and afi3 dimers (SPO-010, SWEIG), stable DRafil and unstable 4 3 dimers (JP, JVM, HBS, HHK, APD), or unstable DRafil and stable DRafi3 dimers (KT11, KRA) were present in the panel of cell lines investigated.

> 4 d&onstr&&g that the v&e/glycine dimorphism also influences the stability of DR dimen in the DR1 specificity. Those results indicate, however, that other factors also influence the formation of stable class I1 dimers. The sequence differences between the DR1 and DR3/4‘5/6 P chains at positions 9 to l 3 and positions 26 to 32 variable region 1and 2, respectively,see Fig. 2) are likely to be important in this respect.

Eur. J. Immunol. 1993.23: 1346-1350

OR4

CSll Line

POS.86

(a) DR3

HLA-DR dimer stability

DRB1.0301

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DRB1.0302

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DRB1.0402

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DRB1-0104

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0

1

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2

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Cell Line

POS.88 DRBlW102

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DRB1.0102

V

DRB1*0101

G

DRB1'0101

G

0

5

D

1

2

3

4

5

ratio stablelunatablr

Figure4 Stability of ap dimers in HLA-DR1, HLA-DR3 and HLA-DR4 specificities. DR specific immunoprecipitations were carried out and analyzed by SDS-PAGE. Ratios of stable- to -unstable dimers were calculated as in legend to Fig. 1. (a) Ratios for the Val% positive DRB1*0301, DRB1*0402 and DRB1*0404 specificities expressed by cell lines HAR, JBlO and LS40, respectively and for the Gly&-positive DRB1*0302 and DRl31*0405 haplotypes expressed by the cell lines RSH and YOT, respectively. Since the mAb B8.11.2 reacts with all DR molecules the ratios reflect the stability of DRapl and DRap3 dimers in the case of DR3-positivecells and of DRafJland DRap4 dimers in the case of DRCpositive cells. However, the DRap3 dimers expressed by the DR3-positive cells are both Gly& positive whereas the DR4 associated DRafJ4dimer isVa186positive. The differences between the ratios in the DR3 and DR4 haplotypes, therefore, reflect the VaVGly dimorphism of the DRapl dimers. (b) Ratios for the Va186-containing specificity DRB1*0102 (expressed by LWAGS and MZ070782) and the GlyS6-containingspecificity DRB1*0101 (expressed by BVR and SA). Since DR1-positive cells express only a DRapl dimer the different ratios probably reflect the VaVGly dimorphism. However, in DR1 a second dimorphism is found on position 85 (see Fig. 2). The influence of this has not been determined.

3.3 Analysis of peptides eluted from DRBl*llOl and DRBl*1104 containing DR dimers To demonstrate that theVaYGly dimorphism influences the selection of peptides we isolated DR molecules from cell surface iodinated B cell lines expressing either DRB1*1101 or DRB1*1104 and eluted the DR-bound peptides as described in Sect. 2.4.The peptide pools were separated by reverse phase HPLC and the obtained separation profiles are shown in Fig. 5. The separation profiles of the peptide material eluted from DR dimers with either a DRl31*1101 or a DRBl"1104 P chain are clearly different. In particular, with the DRB1*1104 specificity an additional peak is observed around fraction 21. This indicates that there is a difference between both alleles with respect to bound peptides which have a tyrosine residue available for iodination. Thus, the unique difference at residue 86 of the DRB1*1101 and DRl31*1104 molecules appears to be sufficient for the selection of (partially) different peptide populations.

m

16

20

25

30

36

Figure 5. Reverse-phase HPLC separation of DR-bound peptides. Peptides were eluted from DR dimers isolated from cell lines expressing either the DRB1*1101 or the DRBl*1104 gene product. The peptides were separated by reverse phase HPLC and fractions of 1 ml were counted in a gamma counter. The DR molecules were derived from the cell surface-iodinated cell lines SPO-010 (DRB1*1101) and JP (DRB1*1104). Both cell lines coexpress the DRB3*0202 gene product. In both reverse-phase HPLC runs an unlabeled synthetic peptide was added as a control. This peptide was monitored by absorption at 214 nm and eluted at the same position in both profiles.

4 Discussion Recently three new DRBl alleles have been defined that differ from known alleles by only a Valss to Glyg6change [14, 151. Ten Valss/glyss allelic pairs are now known that differ only at that position [14, 151 and various previous studies have shown that the dimorphism at position 86 is directly involved in the binding and presentation of immunogenic peptides [16-191 and in the generation of allodeterminants [19-241. In particular it has been shown that Valss does not favor the binding andor presentation of certain immunogenic peptides that can bind to and be presented by the related Gly%ontaining allele [16, 17, 191. Although at present it cannot be ruled out that theVaYGly dimorphism is directly responsible for the observed differences in stability between Val- and Gly-containing DR dimers, it is known that the SDS stable conformation of class I1 dimers is only observed after peptide binding [3,4,7, 81. We, therefore, feel that it is more likely that Valss- and Glyss-containing DRaP dimers select (partially) different sets of peptides resulting in the preferential formation of stable and unstable af3 dimers, respectively. Indeed, we have observed a difference between iodinated peptide pools isolated from DRB1*1101 and DRB1*1104 f3 chain-bearing class I1 dimers. Further experiments, however, characterizing the nature of peptides bound to Val and Gly allelic gene products, will be necessary to reveal the structural constraints which dictate specific binding of peptides to one of these diallelic molecules. Because of the differential peptide binding ([16-181 this study) it is likely that theVaYGly dimorphism influences the antigen presentation capacity of MHC class I1 molecules. This may be reflected by the observation that the Vals6 and Glyg6 allelic gene products are not equally present in a random population. For example, in a random pool of 992

1350

E A. W. Verreck, A. Terjmijtelen and F. Koning

individuals tested (*), the DRB1*1401 (ValM)fl chain was expressed by 48 out of 51 DR14 positive individuals and the DRB1*0301 p-chain by 191out of 197 DR3-positive individuals. In contrast, the DREJ1*0101 (Gly%) fl chain was found in 179out of 206 DR1-positive individuals in this pool.Therefore, depending on the DR specificity,either the Vals or the Glys allelic form is favored. This may be the result of the ability of these molecules t o bind and present immunological relevant peptides. In conclusion, evidence is accumulating that the Valss and Gly86 allelic gene products select (partially) different peptide populations and this is likely to be the molecular basis for the observed differences in SDS-stability of HLA-DR dimers. It remains to be determined if effective antigen presentation is dependent on the stability of the class I1 dimers. We thank the Drs. I. Schreuder, U! van Schooten, M . Kast, II v.d. Elsen, R. de Vries,J. DIAmaro, H . S. de Koster, E. Goulmy and J. U! Dr$hout for critically reading the manuscript. U! Verduyn and J. Drabbels for oligotyping, J. DiAmaro for help with statistical analysis and Eurotransplant for making the oligotyping data available. Received December 2, 1992; in revised form February 25, 1993.

5 References 1 Springer, T. A., Kaufman, J. A., Terhorst, C. and Strominger, J. L., Nature 1977. 268: 213. 2 Cresswell, I?, Eur. J. Immunol. 1977. 7: 636. 3 Mellins, E., Smith, L., Arp, B., Cotner,T., Celis, E. and Pious, D.. Nature 1990. 343: 71.

*

The HLA class I1 phenotype of 992 Eurotransplant donors was determined by routine class I1 oligotyping in the tissue typing section of our department. The group of donors consisted of 60 % males and 40 % females. Age distribution was as follows: 10 % older than 50,56 YO between 21 and 50,34 % between 1 and 20.

Eur. J. Immunol. 1993. 23: 1346-1350

4 Germain, R. N. and Hendrix, L. R., Nature 1991.353: 134. 5 Sadegh-Nasseri, S. and Germain, R. N., Nature 1991.353: 167. 6 Lanzavecchia,A., Reid, I? A. and Watts, C., Nature 1992.357: 249. 7 Stem, L. J. and Wiley, D. C., Cell 1992. 68: 465. 8 Neefjes, J. J. and Ploegh, H. L., EMBO J. 1992.1I: 411. 9 Lew, A. M., Maloy, W. L., Koning, F.,Valas, R. and Coligan, J. E., J. Immunol. 1987. 138: 807. 10 Koning, F., Maloy,W. L. and Coligan, J. E., Eur. J. Immunol. 1990. 20: 299. 11 Koning, F., Schreuder,G. M. Th., Giphart, M. J. and Bruning, J. W., Hum. Immunol. 1984. 9: 221. 12 Koning, F., Schreuder, G. M. Th., Bontrop, R., deVries, I?, Giphart, M. J., Xrmijtelen, A. and Bruning, J. W., Dis. Markers 1984. 2: 75. 13 Rebai, N., Malissen, B., Dieres, M., Acolla, R. S., Corte, G. and Mawas, C., Eur. J. Immunol. 1983. 13: 106. 14 Apple, R. J. and Erlich, H. A., Tissue Antigens 1992. 40: 69. 15 Petersdorf, E. W., Smith, A. G., Martin, I? J. and Hansen, J. A., Tissue Antigens 1992. 40: 267. 16 Krieger, J. I., Karr, R. W., Grey, H. M.,Yu,W.-Y, O’Sullivan, D., Batovsky, L., Zheng, Z.-L., Colon, S. M., Gaeta, F. C. A., Sidney, J., Albertson, M., del Guercio, M.-E, Chesnut, R. W. and Sette, A., J. Immunol. 1991. 146: 2331. 17 Bush, R., Hill, C. M., Hayball, J. D., Lamb, J. R. and Rothbard, J. B., J. Immunol. 1991. 147: 1292. 18 Ong, B., Willcox, N., Wordsworth, I !, Beeson, D. ,Vincent, A., Altmann, D., Lanchbury, J. S. S., Harcourt, G. C., Bell, J. I. and Newsom-Davis, J., Proc. Natl. Acad. Sci. USA 1991. 88: 7343. 19 Demotz, S., Barbey, C., Corradin, G., Amoroso, A. and Lanzavecchia, A., Eur. J. Immunol. 1993. 23: 425. 20 Eckels, D. D., Geiger, M. J., Sell, T. W. and Gorski, J. A., Hum. Immunol. 1990.27: 240. 21 Lang, B., Navarrete, C., LoGalbo, I? R., Nepom, G. T., Silver, J., Winchester, R. J. and Gregersen, I? K., Hum. Immunol. 1990. 27: 378. 22 Zeliszewski, D.,Tiercy, J.-M., Dorval, I., Kaplan, C., Mach, B. and Sterkers, G., Hum. Immunol. 1990. 28: 345. 23 Johnson, A. H.,Tang,T. F., Cowel1,V.and Hurley, C. K., Hum. Immunol. 1991.32: 46. 24 de Koster, H. S., van Rood, J. J. and Rrmijtelen, A., Eur. J. Immunol. 1992. 22: 1531. 25 Marsh, S. G. E. and Bodmer, J. G., Immunogenetics 1991.33: 321. 26 The WHO Nomenclature Committee for factors of the HLA system, Immunogenetics 1992. 36: 135.