Addition of beta1-6 GlcNAc branching to the oligosaccharide attached to Asn 772 in the serine protease domain of matriptase plays a pivotal role in its stability and resistance against trypsin

Glycobiology. 2004 Feb;14(2):139-46. doi: 10.1093/glycob/cwh013. Epub 2003 Oct 9.

Abstract

beta1-6 GlcNAc branching, a product of N-acetylglucosaminyltransferase V (GnT-V), is a key structure that is associated with malignant transformations and cancer metastasis. Although a number of reports concerning tumor metastasis-related glycoproteins that contain beta1-6 GlcNAc branching have appeared, the precise function of beta1-6 GlcNAc branching on glycoproteins remains to be elucidated. We previously reported on the importance of beta1-6 GlcNAc branching on matriptase in terms of proteolytic degradation in tumor metastasis. We report here that matriptase purified from GnT-V transfectant (beta1-6 GlcNAc matriptase) binds strongly to L4-PHA, which preferentially recognizes beta1-6 GlcNAc branches of tri- or tetraantennary sugar chains, indicating that the isolated matriptase contains beta1-6 GlcNAc branching. The beta1-6 GlcNAc matriptase was resistant to autodegradation, as well as trypsin digestion, compared with matriptase purified from mock-transfected cells. Furthermore, N-glycosidase-F treatment of beta1-6 GlcNAc matriptase greatly reduced its resistance to degradation. An analysis of matriptase mutants that do not contain potential N-glycosylation sites clearly shows that the beta1-6 GlcNAc branching on N-glycans attached to Asn 772 in the serine protease domain plays a major role in trypsin resistance. This is the first example of a demonstration of a direct relationship between beta1-6 GlcNAc branching and a biological function at the protein level.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • COS Cells
  • Cell Line, Tumor
  • Culture Media, Serum-Free
  • Glycosylation
  • Humans
  • Lectins / metabolism
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Neoplasm Metastasis
  • Oligosaccharides / chemistry
  • Serine Endopeptidases / chemistry*
  • Serine Endopeptidases / genetics
  • Serine Endopeptidases / metabolism
  • Transfection
  • Trypsin / pharmacology*

Substances

  • Culture Media, Serum-Free
  • Lectins
  • Oligosaccharides
  • Serine Endopeptidases
  • matriptase
  • ST14 protein, human
  • Trypsin