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

• Project/Area Number: 09670216
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Experimental pathology
• Research Institution: UNIVERSITY OF TSUKUBA
• Principal Investigator: KAMMA Hiroshi Inst.Basic Med.Sciences., UNIVERSITY OF TSUKUBA Assoc.Prof., 基礎医学系, 講師 (10195191)
• Co-Investigator(Kenkyū-buntansha): SATOH Hiroaki Inst.Clinical Medicine, Assoc.Prof., 基礎医学系, 講師 (00261800) ; HORIGUCHI Hisashi Ibaraki Pref.University, Research Assoc., 助手 (30238795)
• Project Period (FY): 1997 – 1998
• Project Status: Completed (Fiscal Year 1998)
• Budget Amount: ¥3,200,000 (Direct Cost: ¥3,200,000); Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000); Fiscal Year 1997: ¥2,200,000 (Direct Cost: ¥2,200,000)
• Keywords: hnRNP A2 / hnRNP B1 / hnRNP B0 / telomere / collagen disease / 一本鎖テロメアDNA / hnRNPA_2 / hnRNPB_1 / hnRNPB_0

Research Abstract

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^<- 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.

Research Products

• [Publications] Kamma H,Horiguchi H,Wan L,Matsui M: "Characterization of the hnRNP A2/B1 Proteins : Tissue-Specific Expression and Novel Isoforms." Exp.Cell.Res. 246・2. 399-411 (1999)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Yazawa T,Kamma H,Fujiwara M,Matsui M.: "Lack of Class II trans-activator causes severe deficiency in HLA-DR expression in small cell lung cancer." J.Pathology. 187・2. 191-199 (1999)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Kamma H,Hroguchi H,Wan L.Matsui M,Fujiwara M,Fujimoto M,Yazawa T and Dreyfuss G: "Molecular characterization of the hnRNP A2/B1 proteins : tissue-specific expression and novel isoforms." Exp Cell Res. 1 ; 246. 399-411 (1999)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Yazawa T,Kamma H.Fujiwara M,Matsui M,Horiguchi H,Ogata T,Satoh H,Fujimoto, Yokoyama K.: "Lack of Class II trans-activator causes severe dificiency in HLA-DR expression in small cell lung cancer." J.Pathology. 187. 191-199 (1999)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Kamma H,Horiguchi H,Wan L.,Matsui M,et al.: "Characterization of the hnRNP A2/B1 Proteins: Tissue-Specific Expression and Novel Isoforms." Exp.Cell.Res. 246・2. 399-411 (1999)
• [Publications] Yazawa T,Kamma H,Fujiwara M,Matsui M,et al.: "Lack of Class II trans-activator causes severe deficiency in HLA-DR expression in small cell lung cancer." J.Pathology. 187・2. 191-199 (1999)
• [Publications] Kamma H., Wan L., Horiguchi H, Matsui M,: "Molecular Characterization of the hnRNP A2/B1 Proteins:Tissue-Specific Expression and Novel Isoforms" J.Cell Biology. (in press). (1998)
• [Publications] Katsuoka F, Kawakami Y, Arai T, Imuta H, Fujiwara M, Kanma H,: "Type II alveolar epithelial cells in lung express receptor for advanced glycation end products(RAGE)gene." Biochem Biophys.Res Commun. 238. 512-516 (1997)
• [Publications] Horiguchi H, Matsui M, Yazawa T, Fujiwara M, Ogata T, Kamma H: "Establishment and characterization of typical primitive neuroectodermal tumor cell line." In Vitro. 5. in press (1998)
• [Publications] Satoh H, Ishikawa H, Fujiwara M, Yamashita YT Ohtsuka M, Ogata T, Hasegawa S, Kamma H.: "Production of cytokeratin 19 fragment by human squamous lung cancer cell lines." Am.J.Respiratory.Cell and Mol.Biology. 16. 1-8 (1997)