Taylor V C, Sims M, Brett S, Field M C
Glaxo-Wellcome Medical Research Laboratories, Stevenage, Herts., UK.
Biochem J. 1997 Mar 15;322 ( Pt 3)(Pt 3):919-25. doi: 10.1042/bj3220919.
The CD52 antigen is a lymphocyte glycoprotein with an extremely short polypeptide backbone and a single N-linked glycan, and it is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Treatment of rheumatoid arthritis patients with CAMPATH-1H, a humanized monoclonal antibody against CD52, resulted, in a small number of cases, in the appearance and persistence of CD52-negative T cells. Similarly, CD52-negative B cells emerged following in vitro treatment of a CD52-positive human B cell line with CAMPATH-1H. Both the B and T CD52-negative cells were also found to be defective in surface expression of other GPI-anchored proteins. Biochemical analysis revealed a severe defect in the synthesis of a mature GPI precursor in both the B and T cell lines. Therefore the phenotype of these CD52-negative B and T cells closely resembles that of lymphocytes from patients with paroxysmal nocturnal haemoglobinuria (PNH), in which the first step of the GPI-biosynthetic pathway, i.e. synthesis of GlcNAc-phosphatidylinositol, is blocked. In all cases studied to date, this defect maps to a mutation of the phosphatidylinositolglycan class A (PIG-A) structural gene. We therefore amplified the PIG-A gene from both the GPI-negative B and T cells by PCR and determined the nucleotide sequence. No differences from the wild-type sequence were detected; therefore a classical PNH mutation cannot be responsible for the GPI-biosynthesis defect in these cell lines. Significantly, the GPI-negative phenotype of the B cells was reversible upon separation of the positive and negative cells, resulting in a redistribution to a mixed population with either CD52-positive or -negative cells, whereas populations of 100% CD52-negative T cells were stably maintained during culture. Therefore, whereas the GPI-biosynthesis deficiency in the T cell lines may be due to a mutation in another gene required by the GPI-biosynthetic pathway, the reversible nature of this block in the B cell lines suggests a less direct cause, possibly an alteration in a regulatory factor. Overall, these data demonstrate that the PNH phenotype can be generated without a mutation in the PIG-A structural gene, and thereby identify a novel mechanism for the development of GPI deficiency.
CD52抗原是一种淋巴细胞糖蛋白，其多肽主链极短且仅有一个N-连接聚糖，通过糖基磷脂酰肌醇（GPI）锚定连接于细胞膜。用抗CD52的人源化单克隆抗体CAMPATH-1H治疗类风湿关节炎患者，在少数病例中导致了CD52阴性T细胞的出现和持续存在。同样，用CAMPATH-1H对CD52阳性的人B细胞系进行体外处理后，出现了CD52阴性B细胞。还发现B和T CD52阴性细胞在其他GPI锚定蛋白的表面表达上也存在缺陷。生化分析显示，B细胞系和T细胞系中成熟GPI前体的合成均存在严重缺陷。因此，这些CD52阴性B细胞和T细胞的表型与阵发性夜间血红蛋白尿（PNH）患者的淋巴细胞表型极为相似，在PNH患者中，GPI生物合成途径的第一步，即GlcNAc-磷脂酰肌醇的合成被阻断。在迄今为止研究的所有病例中，这种缺陷都定位在磷脂酰肌醇聚糖A类（PIG-A）结构基因的突变上。因此，我们通过PCR从GPI阴性B细胞和T细胞中扩增了PIG-A基因并测定了核苷酸序列。未检测到与野生型序列的差异；因此，经典的PNH突变不可能是这些细胞系中GPI生物合成缺陷的原因。值得注意的是，如果将阳性和阴性细胞分离，B细胞的GPI阴性表型是可逆的，会重新分布为既有CD52阳性细胞又有CD52阴性细胞的混合群体，而在培养过程中100% CD52阴性T细胞群体则稳定维持。因此，虽然T细胞系中的GPI生物合成缺陷可能是由于GPI生物合成途径所需的另一个基因突变所致，但B细胞系中这种阻断的可逆性表明原因不那么直接，可能是调节因子发生了改变。总体而言，这些数据表明，在没有PIG-A结构基因突变的情况下也可产生PNH表型，从而确定了一种新的GPI缺陷发生机制。
