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

Gelatinase B is a matrix metalloproteinase (MMP-9) expressed under strict c ontrol by many cell types including neutrophils, monocytes, macrophages, an d tumor cells. MMP-9 is a key mediator in the physiological maintenance of the extracellular matrix both in tissue remodeling and development, while u ncontrolled enzyme activity contributes to pathologies such as cancer and i nflammation. Neutrophils release MMP-9 from granules in response to IL-8 st imulation. 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 doma in, which is expected to be heavily O-glycosylated. Indeed, approximately 8 5% of the total sugars on human neutrophil MMP-9 are O-linked. This paper p resents 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 initi al structural assignments were confirmed using HPLC with online MS/MS fragm entation analysis. Twelve sugars were identified that contained from two to nine monosaccharide residues. Most of these contained type 2 core structur es with Gal beta1-4GlcNAc (N-acetyl lactosamine) extensions, with or withou t sialic acid or fucose. The O-glycans were modeled using the oligosacchari de 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 gelat inase B is located on a loop remote from the active site. Fourteen potentia l O-glycosylation sites are multiply presented on this loop of 52 amino aci ds. Many of the O-glycans identified contain terminal galactose residues th at may provide recognition epitopes. Importantly, heavy glycosylation of th is loop region, absent in gelatinase A, has considerable implications for t he domain organization of MMP-9.