Predominant role of N-terminal residue of nonamer peptides in their binding to HLA-B* 5101 molecules
Predominant role of N-terminal residue of nonamer peptides in their binding to HLA-B* 5101 molecules
复制标题
九聚肽 N 端残基在与 HLA-B* 5101 分子结合中的主要作用
作者：
T. Sakaguchi;M. Ibe;K. Miwa;S. Yokota;Katsuaki Tanaka;C. Schönbach;Masafumi Takiguchi
化学合成的含初级锚残基的多肽与HLA I类分子结合的研究进展（Ruppert等人，1993;帕克等人，1994; Scho Bennbach等，1995，1996）已经表明，不仅初级锚残基对于主要组织相容性复合体（MHC）I类肽结合是关键的，而且二级残基在MHC I类肽结合中起重要作用。我们最近的研究（Scho Bennbach等，1995，1996）已经证明，除了在位置2（P2）和C-末端的两个初级锚残基之外，次级锚残基可以通过统计分析来鉴定。然而，如Udaka和同事（1995）所示，还有其他方法来分析二级锚对MHC I类肽结合的影响。在以前使用HLA-B*5101稳定化测定的研究中（菊池等，1996），我们未能通过统计分析阐明HLA-B*5101结合肽的二级锚残基的作用，因为HLAB*5101结合肽稳定HLA-B*5101分子的能力非常弱。因此，我们通过如下延长孵育时间来改进HLA-B *5101结合肽的稳定化测定：将在26 ℃下培养18-24小时的RMA-S-B*5101细胞与各种浓度的肽一起在26 ℃下孵育3小时，然后在37 ℃下孵育3小时。然后用TP 25.99 HLA I类α3结构域特异性单克隆抗体（mAb）（D 'Urso et al. 1991：田边et al. 1992）和羊/小鼠特异性IG抗体的FITC-偶联IgG对细胞进行染色。通过使用FACScan测量细胞的平均线性荧光强度（MFI）。通过从用TP 25.99 mAb染色的肽负载细胞的MFI中减去未用肽负载且用TP 25.99 mAb染色的细胞的MFI来获得相对MFI。肽的亲和力由半最大结合水平（BL 50）表示，其是产生半最大平均荧光强度的肽浓度。根据BL 50将结合肽分为三类：高结合剂（BL 50 # 10-4 M）、中等结合剂（10 - 45 BL 50 # 10-3 M）和低结合剂（10-3 M 5 BL 50）。高、中、低和非结合分别为3、2、1和0级，并计算平均结合等级（MBR）。我们测试了127个在P2（Pro、Ala和Gly）和P9（Ile、瓦尔、Leu和Met）处携带锚残基的九聚体肽（Sakaguchi等，1997），所述九聚体肽选自人免疫缺陷病毒-1的SF 2株（Sanchez-Pescador等，1985）和丙型肝炎病毒蛋白的JT株（Tanaka等，1992）的序列。通过改进的稳定化测定，这些肽的MBR从0.22增加到0.42（Sakaguchi等人，1997）。随后，我们对这些九聚体肽进行了分析，以确定有助于HLA-B*5101-肽相互作用的二级锚残基。在MannWhitney U-检验中计算每个非一级锚位置（位置1、3、4、5、6、7和8）处的每个氨基酸或氨基酸组的结合肽和MBR的频率（表1）。发现P1处的芳香族（Tyr、Phe、Trp和His）和脂肪族（Leu、瓦尔、Ile和Met）疏水残基对肽与HLA-B*5101分子的结合有积极作用（P50.01）。同样，P6的小氨基酸Ala和Gly显著增强与HLA-B*5101分子的结合（P50.05）。相比之下，在P1时观察到Gly和Ala对肽与HLAB*5101分子结合的负面影响（p50.05）。为了确认二级锚残基的作用，测试了在P1和P6处突变的肽的结合。NPPIPVGEI的P1位上的Asn被Tyr和Leu取代后，与HLA-B*5101分子的结合增强。此外，LPCRIKQII的P1处的两个突变，Tyr和瓦尔为Leu不影响T结合。坂口？M.伊部？C.申巴赫？M. Takiguchi（）东京大学医学科学研究所肿瘤生物学系，日本东京108港区白金台4-6-1
Previous studies on the binding of HLA class I molecules to chemically synthesized peptides carrying primary anchor residues (Ruppert et al. 1993; Parker et al. 1994; Scho ̈nbach et al. 1995, 1996) have shown that not only are primary anchor residues critical for major histocompatibility complex (MHC) class I peptide binding, but also that secondary residues play an important role in MHC class I peptide binding. Our recent studies (Scho ̈nbach et al. 1995, 1996) have demonstrated that in addition to two primary anchor residues at position 2 (P2) and the C-terminus, secondary anchor residues can be identified by statistical analysis. However, there are other methods to analyze the effect of secondary anchors on MHC class I peptide binding, as shown by Udaka and co-workers (1995). In a previous study using the HLA-B*5101 stabilization assay (Kikuchi et al. 1996), we failed to clarify the role of secondary anchor residues of HLA-B*5101 binding peptides by statistical analysis because the ability of HLAB*5101 binding peptides to stabilize HLA-B*5101 molecules is very weak. We therefore improved the stabilization assay for HLA-B*5101 binding peptides by extending the incubation time as follows: RMA-S-B*5101 cells cultured at 26°C for 18–24 h were incubated at 26 °C for 3 h with various concentrations of peptides followed by a 3 h incubation at 37°C. The cells were then stained with TP25.99 HLA class Iα3 domain specific monoclonal antibody (mAb) (D’Urso et al. 1991: Tanabe et al. 1992) and FITC-conjugated IgG of sheep mouse-specific Ig antibodies. The mean linear fluorescence intensity (MFI) of the cells was measured by using a FACScan. The relative MFI was obtained by subtracting the MFI of cells not l aded with peptide and stained with TP25.99 mAb from the MFI of peptide-loaded cells stained with TP25.99 mAb. The affinity of a peptide was represented by the halfmaximal binding level (BL50) which is the peptide concentration yielding the half-maximal mean fluorescence intensity. Binding peptides were classified according to the BL50 into three categories: high binder (BL 50 # 10–4 M), medium binder (10 –45 BL50 # 10–3 M), and low binder (10–3 M 5 BL50). High, medium, low, and nonbinders were given ranks 3, 2, 1, and 0, respectively, and the mean binding rank (MBR) was calculated. We tested 127 nonamer peptides (Sakaguchi et al. 1997) carrying the anchor residues at P2 (Pro, Ala, and Gly) and P9 (Ile, Val, Leu and Met) which were selected from the sequence of the SF2 strain of human immunodeficiency virus-1 (Sanchez-Pescador et al. 1985) and the JT strain of hepatitis C virus protein (Tanaka et al. 1992). The MBR of these peptides increased from 0.22 to 0.42 by the improved stabilization assay (Sakaguchi et al. 1997). Subsequently we conducted an analysis of these nonamer peptides to determine secondary anchor residues which contribute to the HLA-B*5101-peptide interaction. The frequency of binding peptides and the MBR was calculated for each amino acid or groups of amino acids at each non-primary anchor position (positions 1, 3, 4, 5, 6, 7, and 8) in a MannWhitney U-test (Table 1). Positive effects on the peptide binding to HLA-B*5101 molecules were found for aromatic (Tyr, Phe, Trp, and His) and aliphatic (Leu, Val, Ile, and Met) hydrophobic residues at P1 ( P50.01). Likewise, small amino acids, Ala and Gly at P6, significantly enhanced the binding to HLA-B*5101 molecules ( P50.05). In contrast, negative effects on the peptide binding to HLAB*5101 molecules were observed for Gly and Ala at P1 (p50.05). In order to confirm the effect of the secondary anchor residues, the binding of peptides mutated at P1 and P6 was tested. The substitution of hydrophobic residues Tyr and Leu for Asn at P1 of NPPIPVGEI increased the binding to HLA-B*5101 molecules. Moreover, two mutations, Tyr and Val for Leu at P1 of LPCRIKQII did not affect the binding T. Sakaguchi? M. Ibe ? C. Schönbach? M. Takiguchi ( ) Department of Tumor Biology, Institute of Medical Science, University of Tokyo, Shirokanedai 4-6-1, Minato-ku, Tokyo 108, Japan
与 HLA I 类 α 3 结构域反应的单克隆抗体识别的单态决定簇的结构和功能分析。
DOI：
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发表时间：
1992
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
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作者：
Tanabe,M;Sekimata,M;Ferrone,S;Takiguchi,M
通讯作者：
Takiguchi,M