Winter C C, Carreno B M, Turner R V, Koenig S, Biddison W E
Molecular Immunology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
J Immunol. 1991 May 15;146(10):3508-12.
Amino acid substitutions were introduced into the 45 pocket of HLA-A2.1 to determine the potential role of this structurally defined feature of class I molecules in viral peptide and alloantigen presentation. The 45 pocket lies below the alpha 1-domain alpha-helix and is composed of five amino acids, three of which differ between HLA-A2.1 and HLA-B37. These two class I molecules have previously been shown to have largely non-overlapping peptide-binding specificities. Site-directed mutagenesis was used to replace the hydrophobic residues at positions 24, 45, and 67 in the 45 pocket of HLA-A2.1 with the hydrophilic amino acids found in these positions in HLA-B37. Thus, three single amino acid mutants were produced: 24A----S, 45 M----T, and 67V----S. These mutants were transfected into HMy2.C1R cells and assessed for their ability to present influenza virus matrix M1 57-68 peptide and HTLV-I Tax-1 2-25 peptide to HLA-A2.1-restricted, peptide-specific CTL and to present alloantigens to HLA-A2-allospecific CTL lines. Each of these substitutions in the 45 pocket produced a molecule that failed to present the M1 peptide to most M1 peptide-specific CTL lines. In contrast, none of these mutations affected presentation of the Tax-1 peptide to Tax-1-specific CTL lines, which indicates that these mutant HLA-A2 molecules can function in viral peptide presentation. Two of the three substitutions in the 45 pocket resulted in lack of recognition by a subset of HLA-A2 allospecific CTL lines. These results demonstrate that the amino acid side chains in the 45 pocket can strongly influence peptide presentation and suggest that the 45 pocket may play a role in determining peptide-binding specificity.
将氨基酸替换引入HLA - A2.1的45口袋区域，以确定I类分子这一结构特征在病毒肽和同种异体抗原呈递中的潜在作用。45口袋位于α1结构域的α螺旋下方，由五个氨基酸组成，其中三个在HLA - A2.1和HLA - B37之间存在差异。此前已表明这两种I类分子具有基本不重叠的肽结合特异性。采用定点诱变，用HLA - B37中这些位置发现的亲水性氨基酸替换HLA - A2.1的45口袋区域中第24、45和67位的疏水性残基。由此产生了三个单氨基酸突变体：24A----S、45M----T和67V----S。将这些突变体转染到HMy2.C1R细胞中，并评估它们将流感病毒基质M1 57 - 68肽和HTLV - I Tax - 1 2 - 25肽呈递给HLA - A2.1限制性、肽特异性CTL的能力，以及将同种异体抗原呈递给HLA - A2同种特异性CTL系的能力。45口袋区域的每一次替换都产生了一种无法将M1肽呈递给大多数M1肽特异性CTL系的分子。相反，这些突变均未影响Tax - 1肽向Tax - 1特异性CTL系的呈递，这表明这些突变的HLA - A2分子可在病毒肽呈递中发挥作用。45口袋区域的三个替换中有两个导致一部分HLA - A2同种特异性CTL系无法识别。这些结果表明，45口袋区域的氨基酸侧链可强烈影响肽的呈递，并提示45口袋区域可能在决定肽结合特异性方面发挥作用。
