title: Functional role of O-linked and N-linked glycosylation sites present on the activation peptide of factor X.
abstract: BACKGROUND
  There are two O-linked and two N-linked glycosylation sites on the activation peptide of factor X (FX) involving residues Thr-17, Thr-29, Asn-39 and Asn-49.
  
  
  OBJECTIVE
  The purpose of this study was to explore the contribution of carbohydrates of the FX activation peptide to zymogen recognition by physiological activators.
  
  
  METHODS
  The putative glycosylation sites were substituted individually or in combinations with Ala and mutants were expressed in mammalian cells. The entire activation peptide up to the P3 residue was deleted in another construct.
  
  
  RESULTS
  It was discovered that activation of zymogen mutants by both FVIIa and FIXa on negatively charged phospholipid vesicles has been improved 2-40-fold independent of a cofactor. These mutants were activated with slightly lower catalytic efficiency (k(cat)/K(m)) by FVIIa in the extrinsic Xase complex, though both K(m) and k(cat) constants for mutants were elevated. With the exception of approximately 3-fold improvement in the activation of N49A, the catalytic efficiency of FIXa toward mutants was decreased 2-5-fold in the intrinsic Xase complex.
  
  
  CONCLUSIONS
  The carbohydrate chains of the FX activation peptide play an important role in restricting the specificity of zymogen recognition by both FVIIa and FIXa, thereby preventing the cofactor-independent activation of FX by these proteases. On the other hand, the carbohydrates contribute to the cofactor-dependent recognition of the zymogen by both extrinsic and intrinsic Xase complexes.
authors.affiliation: Edward A. Doisy Department of Biochemistry, Saint Louis University School of Medicine, Saint Louis, MO 63104, USA.
authors.forename: L
authors.initials: L
authors.lastname: Yang
authors.forename: C
authors.initials: C
authors.lastname: Manithody
authors.forename: A R
authors.initials: AR
authors.lastname: Rezaie
mesh_terms: D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D015949:Factor IXa / Q000378:metabolism; D015942:Factor VIIa / Q000378:metabolism; D005170:Factor X / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D004792:Enzyme Precursors; D000939:Epitopes; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005170:Factor X; D015942:Factor VIIa; D015949:Factor IXa; D015951:Factor Xa; D003546:Cysteine Endopeptidases; C030910:cancer procoagulant
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