Miyazato H, Yoshioka K, Hino S, Aya N, Matsuo S, Suzuki N, Suzuki Y, Sinohara H, Maki S
Department of Pediatrics, Kinki University School of Medicine, Osaka-sayama, Japan.
Autoimmunity. 1994;18(4):259-65. doi: 10.3109/08916939409009527.
Our previous studies showed that 54 kD and 48 kD tubular basement membrane (TBM) proteins were the major form of the target antigen involved in anti-TBM antibody-mediated tubulo-interstitial nephritis in humans. In those studies, we isolated the 54 kD glycoprotein (named gp54) from collagenase-digested bovine TBM. NH2-terminal amino acid sequencing indicated that gp54 represented a newly defined glycoprotein. In this study, we further characterized the target antigen, using mouse monoclonal antibodies to gp54 and polyclonal anti-gp54 peptide antibody. Two monoclonal antibodies (H79 and H80) were established, and they reacted, by immunofluorescence, predominantly with the proximal TBM of humans, rabbits, and Wistar, Sprague-Dawley, and Brown-Norway rats, but not with that of Lewis rats. They were also fixed by blotting intensely to the 54 kD component and weakly to the 48 kD component of collagenase-digested human TBM. In vivo transfer of H79 to Wistar rats showed extensive linear binding of mouse IgG to the TBM and the basal membrane of the small intestine; however, no pathologic changes were seen by light microscopy. The anti-gp54 peptide antibody reacted with both the 54 kD and 48 kD TBM components of human TBM. mRNA was prepared from rabbit kidneys, and fractionated to enrich mRNA encoding the 54 kD and 48 kD peptides. On in vitro translation experiments with the mRNA fraction, the 54 kD and 48 kD peptides were immunoprecipitated with anti-gp54 antibodies. These findings indicate that the 54 kD and 48 kD components are encoded with different mRNA, but that they share the same antigenic epitope.(ABSTRACT TRUNCATED AT 250 WORDS)
我们之前的研究表明，54 kD和48 kD的肾小管基底膜（TBM）蛋白是人类抗TBM抗体介导的肾小管间质性肾炎中靶抗原的主要形式。在那些研究中，我们从胶原酶消化的牛TBM中分离出了54 kD糖蛋白（命名为gp54）。氨基末端氨基酸测序表明gp54代表一种新定义的糖蛋白。在本研究中，我们使用针对gp54的小鼠单克隆抗体和多克隆抗gp54肽抗体进一步对靶抗原进行了表征。建立了两种单克隆抗体（H79和H80），通过免疫荧光，它们主要与人、兔以及Wistar、Sprague-Dawley和Brown-Norway大鼠的近端TBM发生反应，但与Lewis大鼠的近端TBM不反应。它们通过印迹法也与胶原酶消化的人TBM的54 kD成分强烈结合，与48 kD成分弱结合。将H79体内转移至Wistar大鼠显示小鼠IgG广泛线性结合至TBM和小肠基底膜；然而，光学显微镜下未见病理变化。抗gp54肽抗体与人TBM的54 kD和48 kD TBM成分均发生反应。从兔肾制备mRNA，并进行分级分离以富集编码54 kD和48 kD肽的mRNA。在用该mRNA级分进行的体外翻译实验中，54 kD和48 kD肽被抗gp54抗体免疫沉淀。这些发现表明，54 kD和48 kD成分由不同的mRNA编码，但它们共享相同的抗原表位。（摘要截短至250字）
