RESUMO
Recombinant baculoviruses encoding truncated HLA-A*0101 and HLA-A*0201 class I heavy chains have been isolated and used to infect lepidopteran cells. Proteins overexpressed in this system were glycosylated, and consisted of 282 amino acid residues after signal sequence cleavage. These class I heavy chains could fold into their native conformation in the presence of recombinant human beta2-microglobulin expressed in Escherichia coli and a synthetic peptide library of nonamers bound to resin-support beads. Reconstitution into native ternary complexes was detected using a conformation specific monoclonal antibody followed by isolation and sequencing of the bound peptides. The motifs obtained for HLA-A1.1 and HLA-A2.1 peptides are similar although more extensive than those derived from sequencing endogenous peptides. This approach selects peptides which form very stable complexes regardless of whether these peptides are generated under physiological conditions, thereby providing unique supplementary data for predicting and designing CTL epitopes. This method is based solely on peptide binding to the class I molecule and is therefore independent of any constraints imposed by endogenous intracellular processing or transport systems. A comparison of the two motifs provides an opportunity to distinguish between the requirements of binding from those arising as a function of intracellular processing or transport. Our findings are not consistent with a recent report suggesting that constraints on the COOH termini of these peptides can be attributed to the effects of either intracellular processing or transport. We find that the carboxy termini in the class I peptides analyzed to date mimic the endogenous data, suggesting that residues in this position contribute to binding affinity.
