In vitro mutagenesis of HLA-B27. Substitution of an unpaired cysteine residue in the alpha 1 domain causes loss of antibody-defined epitopes.
In vitro mutagenesis of HLA-B27. Substitution of an unpaired cysteine residue in the alpha 1 domain causes loss of antibody-defined epitopes.
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DOI：
10.1172/jci113708
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发表时间：
1988-09
期刊：
影响因子：
--
通讯作者：
J. Taurog;F. El-Zaatari
中科院分区：
文献类型：
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作者：
J. Taurog;F. El-Zaatari
血清学鉴定为HLA-B27的HLA I类分子与强直性脊柱炎和相关人类疾病高度相关。所有已知的HLA-B27氨基酸序列在位置67处含有半胱氨酸残基;没有其他公开的HLA I类序列在α 1结构域的高变区内含有半胱氨酸，其从氨基酸残基63-84延伸。为了研究该半胱氨酸残基在HLA-B27抗原结构中的作用，我们分离了编码HLA-B27.1亚型分子的基因组克隆，并进行了阿托伐他汀定向诱变，将67位的半胱氨酸转化为酪氨酸。当转染到小鼠L细胞中时，野生型和Cys 67-Tyr 67突变体B27基因都指导与单态抗HLA I类抗体W 6/32反应的分子的合成和表面表达。然而，只有用野生型B27基因转染的L细胞与抗B27抗体ME 1反应;用突变体B27转染的L细胞与该抗体完全不反应。用从HLA-B27和HLA-A2基因产生的杂合外显子的实验产生了与ME 1表位到B27 α 1结构域的映射一致的结果。第二种抗B27抗体GS145.2也显示与Cys 67-Tyr 67突变体的结合显著降低。这些研究证明了HLA-B27抗原结构中独特的Cys 67残基的重要性。
The HLA class I molecules identified serologically as HLA-B27 are highly associated with ankylosing spondylitis and related human disorders. All known HLA-B27 amino acid sequences contain a cysteine residue at position 67; no other published HLA class I sequence contains a cysteine within the hypervariable region of the alpha 1 domain, which extends from amino acid residues 63-84. To investigate the role of this cysteine residue in the antigenic structure of HLA-B27, we isolated a genomic clone encoding a molecule of the HLA-B27.1 subtype and performed oligonucleotide-directed mutagenesis to convert the cysteine at position 67 to a tyrosine. When transfected into mouse L cells, both the wild-type and Cys67----Tyr67 mutant B27 genes directed the synthesis and surface expression of molecules reactive with the monomorphic anti-HLA class I antibody W6/32. However, only the L cells transfected with the wild-type B27 gene reacted with the anti-B27 antibody ME1; L cells transfected with the mutant B27 were completely unreactive with this antibody. Experiments with hybrid exons created from the HLA-B27 and HLA-A2 genes yielded results consistent with the mapping of the ME1 epitope to the B27 alpha 1 domain. A second anti-B27 antibody, GS145.2, also showed markedly reduced binding to the Cys67----Tyr67 mutant. These studies document the importance of the unique Cys67 residue in the antigenic structure of HLA-B27.