title: [Molecular mechanisms of protein C deficiency caused by C64W and F139V mutations].
abstract: OBJECTIVE
  To study the molecular mechanisms of protein C (PC) deficiency caused by PC gene mutations of C64W, F139V and K150 deletion (K150d).
  
  
  METHODS
  Wild-type and mutant PC cDNA expression plasmids (PCwt, PC C64W, PC F139V, PC K150d) were constructed and transfected into COS-7 cells or CHO cells respectively for in vitro expression study and immunofluorescent assay. Fluorescent real-time PCR was used to detect the expression of PC mRNA, protein degradation inhibition and endo-beta-N-acetylglucosaminidase H (Endo H) digestion experiments to explain the mutant protein degradation pathway and its localizations inside the cells.
  
  
  RESULTS
  PC C64W was not secreted from the cells and was gradually degraded inside the cells. There was partial secretion of PC F139V, most of the protein molecule was not secreted and degraded intracellularly. Mutant PC K150d was secreted normally from the cells. Fluorescent realtime PCR analysis of total mRNA from transfected cells showed no reduction of the mutant PC mRNA expression compared with that of wild-type PC mRNA. Protein degradation inhibition experiments showed that mutants PC C64W and PC F139V were degraded intracellularly through the proteasome pathway. Endo H digestion experiments and immunofluorescence results suggested that mutant PC molecules were located mainly in pre-Golgi apparatus.
  
  
  CONCLUSIONS
  Impaired secretion and degradation intracellularly of the mutants might be the molecular mechanisms of PC deficiency caused by C64W and F139V mutations. K150 deletion mutation might not affect the secretion of the mutant.
authors.affiliation: Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
authors.forename: Rong-Fu
authors.initials: RF
authors.lastname: Zhou
authors.forename: Xiao-Hong
authors.initials: XH
authors.lastname: Cai
authors.forename: Shuang
authors.initials: S
authors.lastname: Xie
authors.forename: Wen-Bin
authors.initials: WB
authors.lastname: Wang
authors.forename: Jing
authors.initials: J
authors.lastname: Dai
authors.forename: Qiu-Lan
authors.initials: QL
authors.lastname: Ding
authors.forename: Yi
authors.initials: Y
authors.lastname: Fang
authors.forename: Fei
authors.initials: F
authors.lastname: Xie
authors.forename: Xue-Feng
authors.initials: XF
authors.lastname: Wang
authors.forename: Hongli
authors.initials: H
authors.lastname: Wang
mesh_terms: D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011486:Protein C / Q000235:genetics*; D020151:Protein C Deficiency / Q000235:genetics*; D014162:Transfection
chemical_list: D011486:Protein C