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Related Experiment Videos

Post-translational modifications of recombinant human tissue factor.

L R Paborsky1, R J Harris

  • 1Department of Cardiovascular Research, Genentech, Inc., South San Francisco, CA 94080.

Thrombosis Research
|December 1, 1990
PubMed
Summary

Recombinant human tissue factor (rTF) is a glycoprotein with specific N-linked glycosylation sites. Its amino acid sequence and disulfide bonds were confirmed, providing key structural insights.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Glycobiology

Background:

  • Recombinant human tissue factor (rTF) is a crucial protein in the coagulation cascade.
  • Understanding its post-translational modifications, like glycosylation, is vital for its function and therapeutic applications.

Purpose of the Study:

  • To characterize the glycosylation pattern of recombinant human tissue factor (rTF).
  • To confirm the amino acid sequence and identify disulfide bonds in rTF.

Main Methods:

  • Purification of rTF from transfected mammalian cells.
  • Analysis of oligosaccharide attachment sites (N-linked and O-linked).
  • Tryptic mapping and Fast Atom Bombardment Mass Spectrometry (FAB-MS) for sequence and disulfide bond confirmation.

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Main Results:

  • rTF is an N-linked glycoprotein, lacking O-linked oligosaccharides.
  • Two extracellular N-linked sites are fully glycosylated; one is ~90% utilized with complex-type oligosaccharides.
  • A potential N-linked site in the cytoplasmic domain is unglycosylated.
  • Tryptic mapping confirmed the amino acid sequence.
  • FAB-MS identified disulfide bonds between Cys-49/Cys-57 and Cys-186/Cys-209.

Conclusions:

  • The study elucidates the specific glycosylation profile of rTF.
  • Key structural features, including disulfide bonds, have been experimentally verified.
  • This detailed characterization provides a foundation for understanding rTF function and engineering.