Study of physiological function and pathological significance of hnRNP A2/B1 proteins.

利用单克隆抗体对hnRNPA 2/B1蛋白进行了体内组织学研究，发现了一种新的hnRNPA 2/B1蛋白亚型，命名为a hnnRNPB 0 ^< a/B >。通过对hnRNP A2/B1基因中负责蛋白质-核苷酸和蛋白质-蛋白质相互作用的外显子2和外显子9的选择性剪接，克隆并测序了A2、BI、B 0 ^和B 0 ^四种蛋白质异构体< a >< b >。利用pET-11 c系统在大肠杆菌中表达它们的重组蛋白，并通过连续步骤的柱层析纯化至接近均一。Southwestern分析表明，各亚型对ssTEL-DNA的亲和力为A2= B 0 ^< a >&lt;&lt;B1* B 0 ^b。电泳迁移率变动分析表明，重组hnRNP B 0、< b >B1与ssTEL-DNA具有高亲和力的特异性结合（Kd分别为2.15 × 10 ~（-1）<-7>M和1.75 × 10 ~（-1）M）。 ...更多信息 7&gt; M）。使用BIAcore_ system进一步分析它们的结合动力学<TM>。B 0 b蛋白从端粒ssDNA中释放的kd 2（8.4 × 10 ~（-1）S ~（-1））<-5><-1>比B1蛋白（0.109 S ~（-1<-1>））慢。此外，与Bob结合的ssTEL-DNA增加了它们之间的相互作用。抗微球菌核酸酶消化，BO^B促进端粒酶反应。我们认为hnRNP B 0 ^b是哺乳动物端粒功能蛋白的候选者。近年来有报道称自身免疫性疾病患者体内存在抗A2/B1抗体。为了阐明A2/B1蛋白的病理学意义，我们还使用重组A2/B I蛋白研究了类风湿性疾病患者的自身抗体，并证明了抗A2/B 1抗体在RA、SLE和PSS病例中升高。我们打算从衰老和致癌的观点进一步研究与端粒维持相关的生理功能。抗A2/B1抗体在自身免疫性疾病中的病理意义。少

Novel isoforms of hnRNPA2/B1 proteins which are tissue-specifically expressed in the testis and named as a hnnRNP B0^< a/b > were discovered by in vivo histological survey using monoclonal antibodies. Four isoforms, that are A2, BI, B0^< a > and B0^< b > proteins were cloned, sequenced, and turned out to be generated from the hnRNP A2/B 1 gene by alternative splicing of exons 2 and 9 which are responsible for protein-nucleotide and protein-protein interaction.In order to study the interaction of A2/B 1 isoforms to telomeric DNA, their recombinant proteins were expressed in Escherichia coli using pET- 11c system and purified to near homogeneity by successive steps of column chromatography. Southwestern analysis revealed the affinity of the isoforms for ssTEL-DNA was order of A2=B0^< a ><<B1*B0^b. By electrophoretic mobility shift assay showed that recombinant hnRNP B0^< b > and B1 bind specifically to ssTEL-DNA with high affinity (Kd of B0^b and B1 were 2.15 X 10^<-7> M and 1.75 X 10^<- … More 7> M, respectively). Their binding kinetics were further analyzed using BIAcore_<TM> system. B0b protein was demonstrated to release from telomeric ssDNA with a slower kd2 (8.4 X 10^<-5> S^<-1>) than that of B1 (0.109 S^<-1>). In addit ion, ssTEL-DNA associating with Bob increased their. resistance to digestion by micrococcal nuclease, and BO^b accelerated telomerase reaction. We propose that hnRNP B0^b is a candidate of functional mammalian telomeric protein. Recently it has been reported that the patients of autoimmune diseases have anti-A2/B1 antibodies. In order to clarify a pathological significance of A2/B1 proteins, we also studied autoantibodies of patients with rheumatoid diseases using recombinant A2/B I proteins, and demonstrated anti-A2/B 1 antibodies are raised in the RA, SLE and PSS cases.We intend to further investigate the physiological function related to the telomere maintenance from the viewpoint of senescence and carcinogenesis, and also the pathological significance of anti- A2/B1 antibodies in the autoimmune disease. Less