title: Functional characterization of the protein C A267T mutation: evidence for impaired secretion due to defective intracellular transport.
abstract: BACKGROUND
  Activated protein C (PC) is a serine protease that regulates blood coagulation by inactivating coagulation factors Va and VIIIa. PC deficiency is an autosomally inherited disorder associated with a high risk of recurrent venous thrombosis. The aim of the study was to explore the mechanisms responsible for severe PC deficiency in a patient with the protein C A267T mutation by in-vitro expression studies.
  
  
  RESULTS
  Huh7 and CHO-K1 cells were transiently transfected with expression vectors containing wild-type (WT PC) and mutated PC (A267T PC) cDNAs. PC mRNA levels were assessed by qRT-PCR and the PC protein levels were measured by ELISA. The mRNA levels of WT PC and A267T PC were similar, while the intracellular protein level of A267T PC was moderately decreased compared to WT PC. The secretion of A267T PC into the medium was severely impaired. No differences in molecular weights were observed between WT and A267T PC before and after treatment with endo-β-N-acetylglucosaminidase. Proteasomal and lysosomal degradations were examined using lactacystin and bafilomycin, respectively, and revealed that A267T PC was slightly more susceptible for proteasomal degradation than WT PC. Intracellular co-localization analysis indicated that A267T PC was mainly located in the endoplasmic reticulum (ER), whereas WT PC was observed in both ER and Golgi.
  
  
  CONCLUSIONS
  In contrast to what has been reported for other PC mutants, intracellular degradation of A267T PC was not the main/dominant mechanism underlying the reduced intracellular and secretion levels of PC. Our results indicate that the A267T mutation most likely caused misfolding of PC, which might lead to increased retention of the mutated PC in ER.
authors.affiliation: Department of Hematology, Oslo University Hospital, Oslo, Norway.
authors.forename: Lena
authors.initials: L
authors.lastname: Tjeldhorn
authors.forename: Nina
authors.initials: N
authors.lastname: Iversen
authors.forename: Kirsten
authors.initials: K
authors.lastname: Sandvig
authors.forename: Jonas
authors.initials: J
authors.lastname: Bergan
authors.forename: Per Morten
authors.initials: PM
authors.lastname: Sandset
authors.forename: Grethe
authors.initials: G
authors.lastname: Skretting
mesh_terms: D000818:Animals; D001777:Blood Coagulation / Q000235:genetics; D016466:CHO Cells; D025063:Chromosome Disorders / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D009154:Mutation / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011486:Protein C / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000235:genetics; D012008:Recurrence; D012307:Risk Factors; D019076:Transgenes / Q000235:genetics; D020246:Venous Thrombosis / Q000235:genetics / Q000378:metabolism*
chemical_list: D011486:Protein C; D046988:Proteasome Endopeptidase Complex
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