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.
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.
Ihara
Shinji
S
Department of Biochemistry, Osaka University Graduate School of Medicine, B1, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan.
Miyoshi
Eiji
E
Nakahara
Susumu
S
Sakiyama
Haruhiko
H
Ihara
Hideyuki
H
Akinaga
Ayumi
A
Honke
Koichi
K
Dickson
Robert B
RB
Lin
Chen-Yong
CY
Taniguchi
Naoyuki
N
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D016895:Culture Media, Serum-Free; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009362:Neoplasm Metastasis; D009844:Oligosaccharides / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology*
D016895:Culture Media, Serum-Free; D037102:Lectins; D009844:Oligosaccharides; D012697:Serine Endopeptidases; C485595:matriptase; C485596:ST14 protein, human; D014357:Trypsin