title: Factor X Shanghai and disruption of translocation to the endoplasmic reticulum.
abstract: OBJECTIVE
  Most secreted proteins, including coagulation factor X (FX), are synthesized with a signal peptide, which is necessary for targeting the nascent polypeptide into the endoplasmic reticulum. Characterization of naturally occurring mutations may provide insights into the functional roles of the amino acids in the signal peptide.
  
  
  METHODS
  A 52-year old male patient with type I FX deficiency was studied. Mutations were searched for by FX gene (F10) sequencing. The wild-type and the mutant FX proteins were expressed in transfected cells and then immunological assays were performed. Pulse-chase experiments and cell-free expression studies were conducted to determine the cellular fate of the mutant FX molecules.
  
  
  RESULTS
  The patient we studied was homozygous for a substitution of arginine for serine at codon -30 in the signal sequence of F10. Immunoassays detected low FX antigen levels in both the conditioned media and lysates of the cells expressing the mutant protein. Pulse-chase analysis showed that only trace amounts of the mutant FX protein were detectable in the conditioned media, and that the mutant molecules did not accumulate inside the cells either. The results of cell-free expression studies showed that although the transcription and translation of the mutant construct were normal, no post-translational processing, such as N-linked glycosylation, occurred in the presence of microsomes.
  
  
  CONCLUSIONS
  These findings suggest that substitution of a neutral polar amino acid, serine by arginine, in the hydrophobic core of FX signal peptide severely impairs the ability of the protein to enter the endoplasmic reticulum and results in FX deficiency.
authors.affiliation: Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Second Medical University, PR China.
authors.forename: Wen-Bin
authors.initials: WB
authors.lastname: Wang
authors.forename: Qi-Hua
authors.initials: QH
authors.lastname: Fu
authors.forename: Jun
authors.initials: J
authors.lastname: Yin
authors.forename: Wen-Man
authors.initials: WM
authors.lastname: Wu
authors.forename: Qiu-Lan
authors.initials: QL
authors.lastname: Ding
authors.forename: Rong-Fu
authors.initials: RF
authors.lastname: Zhou
authors.forename: Yi-Qun
authors.initials: YQ
authors.lastname: Hu
authors.forename: Xue-Feng
authors.initials: XF
authors.lastname: Wang
authors.forename: Zhen-Yi
authors.initials: ZY
authors.lastname: Wang
authors.forename: Hong-Li
authors.initials: HL
authors.lastname: Wang
mesh_terms: D019943:Amino Acid Substitution; D002474:Cell-Free System; D002478:Cells, Cultured; D003241:Consanguinity; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005170:Factor X / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005171:Factor X Deficiency / Q000235:genetics* / Q000378:metabolism; D006471:Gastrointestinal Hemorrhage / Q000209:etiology; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008297:Male; D008875:Middle Aged; D020125:Mutation, Missense; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C512564:factor X Shanghai; D005170:Factor X