Ikematsu H, Ichiyoshi Y, Schettino E W, Nakamura M, Casali P
Department of Pathology, New York University School of Medicine, New York 10016.
J Immunol. 1994 Feb 1;152(3):1430-41.
In some patients with insulin-dependent (type I) diabetes mellitus (IDDM), autoantibodies to insulin are present at diagnosis. After initiation of the treatment with not only animal but also human insulin, anti-insulin, mainly IgG, autoantibodies become a major component of the autoimmune response in virtually all IDDM patients. Their structure, however, is still relatively unknown. We analyzed the structure of the VH and V kappa segments of three human IgG mAb derived from three IDDM patients. The sequences of VH genes of two IgG, mAb13 and mAb48, were 98.3 and 96.6% identical with those of the H11 and 1.9III genes (VHIII family), respectively. The sequence of the VH gene of the third IgG, mAb49, was 98.6% identical with that of the 51p1 gene (VHI family). All three IgG mAb used V kappa III segments. The V kappa III gene sequences of mAb13 and mAb49 were 97.9 and 98.9% identical, respectively, to that of the kv3g gene; the mAb48 V kappa gene sequence was 96.5% identical to that of the kv328 gene. The VH and/or V kappa segments of these anti-insulin IgG mAb are similar to Ig V genes expressed in the fetal, and adult normal and autoimmune B cell repertoires. The nucleotide differences displayed by the three anti-insulin IgG mAb VH gene sequences, when compared with those of the closest reported germ-line genes, were concentrated in the CDR (6.2 x 10(-2) and 0.8 x 10(-2) difference/base in CDR and FR, respectively; p < 0.01, chi 2 test), and yielded a significantly higher putative replacement (R) to silent (S) mutation ratio in the CDR (12.0) than in the framework (0.2). The concentration of nucleotide differences in the CDR and their high R:S putative mutation ratios were consistent with the hypothesis that these expressed VH genes underwent a process of somatic mutation and Ag-driven clonal selection. That such differences constituted somatic point-mutations was formally proved in IgG mAb13, by differentially targeted PCR amplification and Southern blot hybridization of the mAb13-producing cell line DNA. The putative germ-line gene that gave rise to the expressed VH segment was cloned using genomic DNA from PMN of the same patient whose B cells were used for the generation of this mAb. Overall, in the anti-insulin IgG mAb VH and V kappa III genes, the (putative and verified) somatic point-mutations yielded 27 amino acid replacements, of which 14 nonconserved. Four of these resulted in positively charged residues, three Arg and one His.(ABSTRACT TRUNCATED AT 400 WORDS)
在一些胰岛素依赖型（I型）糖尿病（IDDM）患者中，诊断时就存在胰岛素自身抗体。在用动物胰岛素以及人胰岛素开始治疗后，抗胰岛素自身抗体（主要是IgG）几乎在所有IDDM患者中成为自身免疫反应的主要成分。然而，它们的结构仍相对未知。我们分析了源自三名IDDM患者的三个人IgG单克隆抗体的VH和Vκ片段的结构。两种IgG单克隆抗体mAb13和mAb48的VH基因序列分别与H11和1.9III基因（VHIII家族）的序列有98.3%和96.6%的同一性。第三种IgG单克隆抗体mAb49的VH基因序列与51p1基因（VHI家族）的序列有98.6%的同一性。所有三种IgG单克隆抗体都使用VκIII片段。mAb13和mAb49的VκIII基因序列分别与kv3g基因的序列有97.9%和98.9%的同一性；mAb48的Vκ基因序列与kv328基因的序列有96.5%的同一性。这些抗胰岛素IgG单克隆抗体的VH和/或Vκ片段类似于在胎儿、成人正常和自身免疫性B细胞库中表达的Ig V基因。与最接近的已报道种系基因相比，三种抗胰岛素IgG单克隆抗体VH基因序列显示的核苷酸差异集中在互补决定区（CDR）（CDR和框架区中每碱基的差异分别为6.2×10⁻²和0.8×10⁻²；p<0.01，卡方检验），并且在CDR中产生的推定置换（R）与沉默（S）突变率（12.0）显著高于框架区（0.2）。CDR中核苷酸差异的集中及其高R:S推定突变率与这些表达的VH基因经历体细胞突变和抗原驱动的克隆选择过程的假设一致。通过对产生mAb13细胞系DNA进行差异靶向PCR扩增和Southern印迹杂交，在IgG单克隆抗体mAb13中正式证明了这种差异构成体细胞点突变。使用来自同一患者多形核白细胞的基因组DNA克隆了产生所表达VH片段的推定种系基因，该患者的B细胞用于产生此单克隆抗体。总体而言，在抗胰岛素IgG单克隆抗体的VH和VκIII基因中，（推定的和已证实的）体细胞点突变产生了27个氨基酸置换，其中14个是非保守的。其中四个导致带正电荷的残基，三个精氨酸和一个组氨酸。
