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

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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