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Partial glycosylation at asparagine-2181 of the second C-type domain of human factor V modulates assembly of the prothrombinase complex.

Publication ,  Journal Article
Kim, SW; Ortel, TL; Quinn-Allen, MA; Yoo, L; Worfolk, L; Zhai, X; Lentz, BR; Kane, WH
Published in: Biochemistry
August 31, 1999

Thrombin-activated factor Va exists as two isoforms, factor Va(1) and factor Va(2), which differ in the size of their light chains and their affinity for biological membranes. The heterogeneity of the light chain remained following incubation of factor Va with N-glycanase. However, we found that the factor V C2 domain, which contains a single potential glycosylation site at Asn-2181, was partially glycosylated when expressed in COS cells. To confirm the structural basis for factor Va(1) and factor Va(2), we mutated Asn-2181 to glutamine (N2181Q) and expressed this mutant using a B domain deletion construct (rHFV des B) in COS cells. Thrombin activation of N2181Q released a light chain with mobility identical to that of factor Va(2) on SDS-PAGE. The functional properties of purified N2181Q were similar to those of factor Va(2) in prothrombinase assays carried out in the presence of limiting concentrations of phosphatidylserine. The binding of human factor Va(1) and factor Va(2) to 75:25 POPC/POPS vesicles was also investigated in equilibrium binding assays using proteins containing a fluorescein-labeled heavy chain. The affinity of human factor Va(2) binding to POPC/POPS vesicles was approximately 3-fold higher than that of factor Va(1). These results indicate that partial glycosylation of factor V at asparagine-2181 is the structural basis of the light chain doublet and that the presence of this oligosaccharide reduces the affinity of factor Va for biological membranes.

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Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

August 31, 1999

Volume

38

Issue

35

Start / End Page

11448 / 11454

Location

United States

Related Subject Headings

  • Transfection
  • Thromboplastin
  • Recombinant Proteins
  • Protein Structure, Tertiary
  • Protein Binding
  • Peptide Fragments
  • Mutagenesis, Site-Directed
  • Humans
  • Glycosylation
  • Glycoside Hydrolases
 

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Kim, S. W., Ortel, T. L., Quinn-Allen, M. A., Yoo, L., Worfolk, L., Zhai, X., … Kane, W. H. (1999). Partial glycosylation at asparagine-2181 of the second C-type domain of human factor V modulates assembly of the prothrombinase complex. Biochemistry, 38(35), 11448–11454. https://doi.org/10.1021/bi991275y
Kim, S. W., T. L. Ortel, M. A. Quinn-Allen, L. Yoo, L. Worfolk, X. Zhai, B. R. Lentz, and W. H. Kane. “Partial glycosylation at asparagine-2181 of the second C-type domain of human factor V modulates assembly of the prothrombinase complex.Biochemistry 38, no. 35 (August 31, 1999): 11448–54. https://doi.org/10.1021/bi991275y.
Kim SW, Ortel TL, Quinn-Allen MA, Yoo L, Worfolk L, Zhai X, et al. Partial glycosylation at asparagine-2181 of the second C-type domain of human factor V modulates assembly of the prothrombinase complex. Biochemistry. 1999 Aug 31;38(35):11448–54.
Kim, S. W., et al. “Partial glycosylation at asparagine-2181 of the second C-type domain of human factor V modulates assembly of the prothrombinase complex.Biochemistry, vol. 38, no. 35, Aug. 1999, pp. 11448–54. Pubmed, doi:10.1021/bi991275y.
Kim SW, Ortel TL, Quinn-Allen MA, Yoo L, Worfolk L, Zhai X, Lentz BR, Kane WH. Partial glycosylation at asparagine-2181 of the second C-type domain of human factor V modulates assembly of the prothrombinase complex. Biochemistry. 1999 Aug 31;38(35):11448–11454.
Journal cover image

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

August 31, 1999

Volume

38

Issue

35

Start / End Page

11448 / 11454

Location

United States

Related Subject Headings

  • Transfection
  • Thromboplastin
  • Recombinant Proteins
  • Protein Structure, Tertiary
  • Protein Binding
  • Peptide Fragments
  • Mutagenesis, Site-Directed
  • Humans
  • Glycosylation
  • Glycoside Hydrolases