O-Glycan Analysis of Natural Human Neutrophil Gelatinase B Using a Combination of Normal Phase- HPLC and Online Tandem Mass Spectrometry: Implications for the Domain Organization of the Enzyme
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- 作者：Taj S. Mattu ; Louise Royle ; Jim Langridge ; Mark R. Wormald ; Philippe E. Van den Steen ; Jo Van Damme ; Ghislain Opdenakker ; David J. Harvey ; Raymond A. Dwek ; and Pauline M. Rudd
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：December 26, 2000
- 年：2000
- 卷：39
- 期：51
- 页码：15695 - 15704
- 全文大小：314K
- 年卷期：v.39,no.51(December 26, 2000)
- ISSN：1520-4995
文摘
Gelatinase B is a matrix metalloproteinase (MMP-9) expressed under strict control by manycell types including neutrophils, monocytes, macrophages, and tumor cells. MMP-9 is a key mediator inthe physiological maintenance of the extracellular matrix both in tissue remodeling and development,while uncontrolled enzyme activity contributes to pathologies such as cancer and inflammation. Neutrophilsrelease MMP-9 from granules in response to IL-8 stimulation. Human MMP-9 has three potential N-linkedglycosylation sites and contains a Ser/Pro/Thr rich domain, known as the type V collagen-like domain,which is expected to be heavily O-glycosylated. Indeed, approximately 85% of the total sugars on humanneutrophil MMP-9 are O-linked. This paper presents the detailed analysis of picomole amounts of theseO-glycans using a novel HPLC-based strategy for O-glycan analysis that provides linkage and arm specificinformation in addition to monosaccharide sequence. The initial structural assignments were confirmedusing HPLC with online MS/MS fragmentation analysis. Twelve sugars were identified that containedfrom two to nine monosaccharide residues. Most of these contained type 2 core structures with Gal
hars/beta2.gif" BORDER=0 ALIGN="middle">1-4GlcNAc (N-acetyl lactosamine) extensions, with or without sialic acid or fucose. The O-glycans weremodeled using the oligosaccharide structural database. On the basis of the structure of gelatinase A (MMP-2), a model of MMP-9 suggests that the type V collagen-like domain in gelatinase B is located on a loopremote from the active site. Fourteen potential O-glycosylation sites are multiply presented on this loopof 52 amino acids. Many of the O-glycans identified contain terminal galactose residues that may providerecognition epitopes. Importantly, heavy glycosylation of this loop region, absent in gelatinase A, hasconsiderable implications for the domain organization of MMP-9.
hars/beta2.gif" BORDER=0 ALIGN="middle">1-4GlcNAc (N-acetyl lactosamine) extensions, with or without sialic acid or fucose. The O-glycans weremodeled using the oligosaccharide structural database. On the basis of the structure of gelatinase A (MMP-2), a model of MMP-9 suggests that the type V collagen-like domain in gelatinase B is located on a loopremote from the active site. Fourteen potential O-glycosylation sites are multiply presented on this loopof 52 amino acids. Many of the O-glycans identified contain terminal galactose residues that may providerecognition epitopes. Importantly, heavy glycosylation of this loop region, absent in gelatinase A, hasconsiderable implications for the domain organization of MMP-9.