Molecular cloning,expression,and chromosomal localization of a human tubulointerstitial nephritis antigen

肾小管间质性肾炎抗原（Tubulointerstitial nephritis antigen，TIN-ag）是一种细胞外基质基础蛋白，最初被鉴定为抗肾小管基底膜（antitubular basement membrane，TBM）抗体介导的间质性肾炎（interstitial nephritis，TIN）的靶抗原。进一步的研究表明，TIN-ag在肾小管发生中起作用，并且TIN-ag在遗传性肾小管间质疾病如青少年肾单位肾痨中有缺陷。我们先前分离和表征54 dKa糖蛋白为TIN-ag。最近已经鉴定了编码兔和小鼠TIN-ag的cDNA。本研究克隆了人TIN-ag同源基因的cDNA，并测定了其核苷酸序列（登录号：AB 022277，DDBJ核苷酸序列数据库）。推导的氨基酸序列（476 aa）显示存在信号肽（1- 18 aa）、称为卵泡抑素模块的半胱氨酸残基、六个潜在的糖基化位点和一个ATP/GTP结合位点。同源性分析表明，该基因与兔和小鼠的TIN-ag同源性均为约85%，与C.优雅人TIN-ag的氨基端含有与细胞外基质分子相似的氨基酸序列，羧基端含有与半胱氨酸蛋白酶组织蛋白酶家族相似的氨基酸序列。北方印迹分析显示该分子在人成人和胎儿肾组织中特异性表达。使用识别人TIN-ag的单克隆抗体，在用人TIN-ag cDNA转染的培养的COS-1细胞中鉴定蛋白质表达（约50 kDa）。将人TIN-ag定位于染色体6p 11。2-12通过荧光原位杂交。这些结果为进一步了解TIN-ag分子提供了依据，也为儿童肾单位营养不良的基因分析提供了线索。

Tubulointerstitial nephritis antigen (TIN-ag) is an extracellular matrix base ment protein which was originally identified as a target antigen involved in anti-tubular basement membrane (TBM) antibody-mediated interstitial nephritis(TIN). Further investigations elucidated that TIN-ag plays a role in renal tubulogenesis and that TIN-ag is defected in hereditary tubulointerstitial disorder such as juvenile nephronophthisis. We previously isolated and characterized 54 dKa glycoprotein as TIN-ag. cDNA encoding rabbit and mouse TIN-ag has recently been identified. In the present study, the cDNA of the human honologue of TIN-ag was cloned and its nucleotid sequence was determined (accession No.AB022277, the DDBJ nucleotide sequence database). Deduced amino acid sequence (476aa) exhibited the presence of a signal peptide (1-18aa), cysteine residues termed follistatinmodule, six potential glycosylation sites, and an ATP/GTP-binding site. Homology search revealed 〜 85% homology with both rabbit and mouse TIN-ag, and also some (〜40%) similarity with C. elegans. Human TIN-ag contained a sequence similar to several classes of extracelular matrix molecules in amino terminal region and to cathepsin family of cysteine proteinases in the carboxyl terminal region. Northern blot analysis revealed exclusive expression of this molecule in human adult and fetal kidney tissues. Using a monoclonal antibody recognizing human TIN-ag, protein expression (〜50 kDa) was identified in cultured COS-1 cells transfected with human TIN-ag cDNA. The human TIN-ag was mapped to chromosome 6p11. 2-12 by fluorescence in situ hybridization. These results may privide further evidence for understanding TIN-ag molecule and clues for gene analysis of juvenile nephronophthisis.