Arroyo J, Díez-Orejas R, Alvarez A M, Shaw S, Sánchez-Pérez M
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense, Madrid, Spain.
Immunogenetics. 1994;39(1):40-7. doi: 10.1007/BF00171795.
A collection of HLA-DP mutants was generated, using ICR 191 as the mutagenic agent and resistance to lysis mediated by HLA-DPw2 allospecific cytotoxic T lymphocytes (CTLs) as the selection criterion. These mutants were derived from the HLA haploid lymphoblastoid cell line 45.1. Loss of HLA-DPw2 surface expression accounted for the lack of HLA-DPw2 CTL recognition in all the mutants. However, one of them, 45.EM19, binds to DPw2-specific monoclonal antibodies (mAb) after cell permeabilization. HLA-DPA1 and DPB1 mRNA expression studies permitted the classification of the mutants in four categories: 1) DPA1-negative, DPB1-positive; 2) DPA1-positive, DPB1-negative; 3) DPA1- and DPB1-negative, and 4) DPA1- and DPB1-positive mutants. Mutant 45.EM19 is included in the last group. The cloning and sequencing of the full-length DPA1 (DPA10103) and DPB1 (DPB102012) cDNAs from this mutant showed no changes in the DPA1 sequence compared to the wild-type sequence. However, a frame-shift mutation in the DPB1 gene exon coding for the transmembrane region was detected. The insertion of a guanine nucleotide provokes an extension of the open reading frame, increasing the length of the C-terminal domain and changing the hydropathicity pattern of the transmembrane domain. This change should be responsible for the phenotype of the 45.EM19 mutant.
以ICR 191作为诱变剂，以对HLA - DPw2同种异体特异性细胞毒性T淋巴细胞（CTL）介导的裂解的抗性作为选择标准，构建了一组HLA - DP突变体。这些突变体源自HLA单倍体淋巴母细胞系45.1。所有突变体中HLA - DPw2表面表达的缺失导致了HLA - DPw2 CTL识别的缺乏。然而，其中一个突变体45.EM19在细胞透化后能与DPw2特异性单克隆抗体（mAb）结合。HLA - DPA1和DPB1 mRNA表达研究允许将突变体分为四类：1）DPA1阴性、DPB1阳性；2）DPA1阳性、DPB1阴性；3）DPA1和DPB1均阴性，以及4）DPA1和DPB1均阳性的突变体。突变体45.EM19属于最后一组。对该突变体的全长DPA1（DPA10103）和DPB1（DPB102012）cDNA进行克隆和测序，结果显示与野生型序列相比，DPA1序列没有变化。然而，在编码跨膜区的DPB1基因外显子中检测到一个移码突变。鸟嘌呤核苷酸的插入导致开放阅读框延长，增加了C末端结构域的长度，并改变了跨膜结构域的亲水性模式。这种变化应该是45.EM19突变体表型的原因。
