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

新的hnRNPA2/B1蛋白亚型是利用单抗在体内组织学研究中发现的，它在睾丸组织中特异表达，命名为hnnRNP B0^&lt；a/b&gt；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交替剪接产生的A2、BI、B0^&lt；a&gt；和B0^&lt；b&gt；4种异构体进行了克隆、测序和序列分析。为了研究A2/B1异构体与端粒DNA的相互作用，利用pET-11c系统在大肠杆菌中表达了它们的重组蛋白，并通过柱层析分离纯化了它们的重组蛋白。凝胶迁移率分析表明，重组hnRNP B0和B1与单链TEL-DNA具有较高的亲和力(B0^b和B1的Kd分别为2.15×10~(-1)和1.75×10~(-1)…)。分别是7&GT；M更多)。用Biacore_&lt；TM&gt；系统进一步分析了它们的结合动力学。B0b蛋白从端粒单链DNA中释放的速度Kd2(8.4X10；-5；S)比B1(0.109 S)慢。此外，与Bob结合的ssTEL-DNA增加了它们的亲和力。对微球菌核酸酶的抗性和BO^b加速了端粒酶反应。我们认为hnRNP B0^b是哺乳动物端粒功能蛋白的候选者。最近有报道称，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，我们还利用重组A2/B1蛋白对类风湿疾病患者的自身抗体进行了研究，发现抗A2/B1抗体在类风湿疾病、系统性红斑狼疮和PSS患者中都有升高，我们打算从衰老和癌变的角度进一步研究与端粒维持相关的生理功能，以及抗A2/B1抗体在自身免疫性疾病中的病理学意义。较少

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