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

Abstract

Gelatinase B is a matrix metalloproteinase (MMP-9) expressed under strict control by many cell types including neutrophils, monocytes, macrophages, and tumor cells. MMP-9 is a key mediator in the physiological maintenance of the extracellular matrix both in tissue remodeling and development, while uncontrolled enzyme activity contributes to pathologies such as cancer and inflammation. Neutrophils release MMP-9 from granules in response to IL-8 stimulation. Human MMP-9 has three potential N-linked glycosylation 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 human neutrophil MMP-9 are O-linked. This paper presents the detailed analysis of picomole amounts of these O-glycans using a novel HPLC-based strategy for O-glycan analysis that provides linkage and arm specific information in addition to monosaccharide sequence. The initial structural assignments were confirmed using HPLC with online MS/MS fragmentation analysis. Twelve sugars were identified that contained from two to nine monosaccharide residues. Most of these contained type 2 core structures with Galbeta1-4GlcNAc (N-acetyl lactosamine) extensions, with or without sialic acid or fucose. The O-glycans were modeled 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 loop remote from the active site. Fourteen potential O-glycosylation sites are multiply presented on this loop of 52 amino acids. Many of the O-glycans identified contain terminal galactose residues that may provide recognition epitopes. Importantly, heavy glycosylation of this loop region, absent in gelatinase A, has considerable implications for the domain organization of MMP-9.

Gelatinase-mediated migration and invasion of cancer cells

“…MMP-2 and MMP-9 are highly similar enzymes in many respects, but significant differences exist in the regulation of expression, glycosylation, proenzyme activation and substrate selectivity. For example, MMP-2 is a 72-kDa nonglycosylated protein, whereas the 92-kDa MMP-9 contains two N-glycosylated sites in the prodomain and the catalytic domain (Kotra et al, 2002) and a number of O-linked glycans (Mattu et al, 2000;Rudd et al, 1999). Furthermore, MMP-9 exists in plasma as a monomer, complexed with neutrophil lipocalin and as a dimer, whereas MMP-2 is strictly monomeric.…”

“…MMP-9 has additionally a unique collagen V-like insertion between the catalytic domain and the C-terminal domain. The function of this insertion is unknown, but it contains most of the O-linked glycans of MMP-9 (Mattu et al, 2000;Rudd et al, 1999). The hemopexin/vitronectin-like C-terminal domain is responsible for multiple protein-protein interactions.…”

The Hemopexin and O-Glycosylated Domains Tune Gelatinase B/MMP-9 Bioavailability via Inhibition and Binding to Cargo Receptors

“…Various models of the OG domain were generated to link the N-and C-terminal domains. The fully extended model of OG domain was built using InsightII and then restrained simulated annealing was used to generate structures of an average extension of 2.6 Å per residue (10). The extended model of OG domain allowing for the disulfide bond between the OG domain and the hemopexin domain was generated in a similar way, but in addition fixing the distance between Cys 468 and the C-terminal residue of the OG domain.…”

“…Structures of N-and O-linked sugars were determined for natural MMP-9 from human neutrophils (9,10). Little information is available concerning the structures or functions of glycosylation of other MMPs, except that in MT1-MMP, the O-linked glycans on the linker peptide between the active site and the hemopexin domain are essential for the binding of TIMP-2 (11).…”

Novel MMP-9 Substrates in Cancer Cells Revealed by a Label-free Quantitative Proteomics Approach

“…However, the complex of pro-MMP-9 with TIMP-1 involves binding of the inhibitor to the hemopexin-like domain (18). The glycosylation of the hinge region affects activity indirectly through influences on cell surface binding and internalization (19,20). Different cell lines catalyze different glycosylation patterns (21).…”