Addition of  1-6 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

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.

β1,6-Branched Oligosaccharides Are Increased in Lymph Node Metastases and Predict Poor Outcome in Breast Carcinoma

“…Addition of h1,6-branched oligosaccharides at Asn 772 in the serine protease domain of matriptase played a pivotal role in its stability and resistance against trypsin (35). Matriptase plays important roles in cell migration, extracellular matrix degradation, and the activation of singlechain urokinase-plasminogen activator and hepatocyte growth factor (36). In breast carcinoma, matriptase expression was associated with poor patient outcome (37).…”

Coexpression of β1,6-N-Acetylglucosaminyltransferase V Glycoprotein Substrates Defines Aggressive Breast Cancers with Poor Outcome

“…In particular, GnT-V glycosylation of matriptase and lamp-1 inhibits their degradation and prolongs their localization and activity at the cell membrane (12,17,40). Matriptase potentiates the binding of hepatocyte growth factor to the Met receptor, which promotes tumor proliferation and motility (9,17,34). The stabilization of lamp-1 results in increased extracellular matrix degradation (12).…”

“…Matriptase is an epithelialderived serine protease that regulates cell motility and growth via the hepatocyte growth factor pathway (9,10). Lamp-1 is expressed on cell and lysosome membranes and is important in lysosomal trafficking, matrix degradation, and cell adhesion (11,12).…”

TAILS N-terminomics and proteomics reveal complex regulation of proteolytic cleavage by O-glycosylation

“…Nglycans on subtilisin and matripase have been shown to protect these proteins from proteolytic cleavage (16)(17)(18). Similarly, dense mucin-type or O-N-Acetylgalactosamine (O-GalNAc) Oglycosylation (herein 'O-glycosylation') has long been proposed to non-specifically shield proteins from degradation in the extracellular space (19).…”