Studies in Molecular Pathogenesis of Inherited Coagulation Disorders

1999 Fiscal Year Final Research Report Summary

• Project/Area Number: 10672173
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Laboratory medicine
• Research Institution: Toyama Medical and Pharmaceutical University
• Principal Investigator: OZAWA Tetsuo Toyama Medical and Pharmaceutical University, University Hospital, Assistant Professor, 附属病院, 助手 (80262525)
• Project Period (FY): 1998 – 1999
• Keywords: antithrombin deficiency / endoplasmic reticulum / quality control system / mutant protein / intracellular degradation

Research Abstract

Antithrombin (AT) is a major physiological regulator of the activated blood coagulation factors, and hereditary deficiency of AT is an important autosomal dominant thrombophilic disorder. In 1997, we identified a novel single nucleotide substitution of T to C at nt. 2747 of the antithrombin gene (AT Morioka) causing the phenotype of type I deficiency. This mutation predicts an amino acid replacement of Cys (TGT)95 to Arg (CGT) and subsequent loss of one(Cys21-Cys95) of the three disulfide bonds in an AT molecule. This conformational change of the variant molecule was thought to be the most probable cause of the protein defect. In order to elucidate the molecular pathogenesis, I have established stable -expressing CHO cell lines of normal human AT and AT Morioka mutant and characterized the molecular defect in the mutant allele. There was no difference in the transcription levels of AT mRNA estimated by RT-PCR methods between normal and the mutant allele. However, no mutant AT protein was detected in culture medium, whereas the wild-type protein was secreted into medium. Pulse-chase experiments showed that in AT Morioka allele the quantity of synthesized AT is normal, but that the mutant molecule is accumulated within cells because of an intracellular transport block between endoplasmic reticulum and trans-Golgi apparatus. Interestingly, about 90% of the mutant molecule retained within cells without degradation 9 hours after pulse-labeling. Immunogold electron microscopic studies revealed that the mutant protein was localized within unique intracytoplasmic vesicular structure. The manner of transportation defect and intracellular accumulation of AT Morioka molecule doesn't resemble any previous observations in other defective AT mutants. These data lead me to propose a new cellular mechanism of misfolded protein treatment, the details of which are still obscure.