Abstract
The specificity of peptide binding by human leukocyte antigen (HLA) class I molecules was investigated in a cell-free direct-binding assay. Peptides were assessed for binding to HLA-A2 and HLA-B27 by measuring the formation of heterotrimeric HLA complexes that consisted of iodinated beta 2-microglobulin, HLA heavy chain fragments isolated from the Escherichia coli cytoplasm, and peptide. In this system, no detectable HLA heavy chain-beta 2-microglobulin complexes were formed unless appropriate peptides were intentionally added to the reconstitution solution. Analysis with monoclonal antibodies demonstrated that these heterotrimeric complexes were correctly folded. Five nonhomologous peptides, known to form complexes with HLA-A2 or HLA-B27 from T-cell functional studies, were tested for their capacity to bind to HLA-A2 and HLA-B27 using the reconstitution assay. Four of the peptides bound to the appropriate class I molecule only. One peptide and some (but not all) substitution analogs of it bound to both HLA-A2 and HLA-B27. The effect of peptide length on binding to HLA-B27 was studied, and it was found that the optimal length was 9 or 10 amino acid residues; however, one peptide that bound to HLA-B27 was 15 amino acids long. All peptides that bound to HLA-B27 in the direct-binding assay also competed with antigenic peptides for binding to HLA-B27 on the surface of intact cells, as determined by a standard cytotoxic T-lymphocyte functional assay. Thus, we conclude that HLA-A2 and HLA-B27 bind distinct but partially overlapping sets of peptides and that, at least in vitro, the assembly of HLA heavy chain-beta 2-microglobulin complexes requires specific peptides.
Highlights
The specificity of peptide binding by human leukocyte antigen (HLA) class I molecules was investigated in a cell-free direct-binding assay
The effect of peptide length on binding to Human leukocyte antigen (HLA)-B27 was studied, and it wasfound that the optimal length was 9 or 10amino acid residues; one peptide that bound to HLA-B27 was 15 amino acids long
It is clear from ing toHLA-B27 on the surfaceof intact cells, as deter- these studies that the majority of endogenous peptides are mined by a standard cytotoxic T-lymphocyte func- eight or nine amino acids in length and that particular setional assay
Summary
Vol 267, No 6, h u e of March 15, pp. 5451-5459,1992 Printed in U.S.A. RECONSTITUTION OF HLA-A2AND HLA-B27 HEAVY CHAIN/&-MICROGLOBULIN COMPLEXES REQUIRESSPECIFICPEPTIDES*. PZm,at least for HLA-B27, peptides of either 9 or 10 amino HLA Reconstitution-Each preparation of the HLA heavy chain acids were optimal This system provides a facile assay for assessing the specificity of peptide binding to HLA class I molecules in the absence of competing endogenous peptides and independent of any peptide-loading enzymes or proteases. Antibody Binding Assay-Reconstituted HLA/P,m/peptide preparations were purified bygel filtration.Fractionscontaining HLA complex ( X 0 0 cpm) were mixedwith 2 pl of the antibody to be tested (total volume of 20-100 pl), incubated for 1h a t 0 "C, centrifuged for 2 min, and injected ontotheHPLC column as described above. Competitive Inhibition Assay-Generation of HLA-A2-restricted cally when the 272-amino acid HLA heavy chain was isolated from cultures harboring either pDHl(lane4 ) or p4037 (lane 5 ) , indicating that the same complex was formed from the products of both expression plasmids. Label shifted from a peak centered a t fraction 17(Mr= 12,000, corresponding to Pzm alone) to a peak centered at fraction 13
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