Gelatinase B is a matrix metalloproteinase (MMP-9) involved in tissue remodeling, development, cancer, and inflammation. Neutrophils produce three major forms of (pro)gelatinase B: 92 kDa monomers, homodimers, and complexes of gelatinase B covalently bound to neutrophil gelatinase B-associated lipocalin (NGAL). In contrast to the case for other proteinases, little information about the glycosylation of any natural human MMP is available. Here, both gelatinase B and NGAL were purified from human

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... peripheral blood neutrophils, and the entire contents of the released N-and O-glycan pools were analyzed simultaneously using recently developed high-performance liquid chromatography-based technology. The results are discussed within the context of the domain structure of gelatinase B and a molecular model of NGAL based on data from this study and the three-dimensional nuclear magnetic resonance (NMR) structure of the protein. More than 95% of the N-linked glycans attached to both gelatinase B and NGAL were partially sialylated, core-fucosylated biantennary structures with and without outer arm fucose. The O-linked glycans, which were estimated to comprise approximately 85% of the total sugars on gelatinase B, mainly consisted of type 2 cores with Gal 1,4GlcNAc (lactosamine) extensions, with or without sialic acid or outer arm fucose. This paper also contains the first report of O-linked glycans attached to NGAL. Although both proteins were isolated from neutrophils and contained O-linked glycans mainly with type 2 cores, the glycans attached to individual serine/threonine residue(s) in NGAL were significantly smaller than those on gelatinase B. In contrast to NGAL, gelatinase B contains a region rich in Ser, Thr, and Pro typical of O-glycosylated mucin-like domains. Remodeling of the extracellular matrix (ECM) 1 is achieved by both de novo synthesis and enzymatic modifications of ECM components. The matrix metalloproteinases (MMPs) (collagenases, stromelysins, matrilysins, gelatinases, and membrane-type metalloproteinases) are a family of enzymes which control this process (1, 2). Among these, gelatinase B (MMP-9, EC 3.4.24.35) is the most complex in terms of protein domain structure. The regulation of expression and activity of gelatinase B is also more complex than those of most other MMPs. For instance, in contrast to gelatinase A, gelatinase B is not produced constitutively by most cells, but its activity is induced by different stimuli depending on the cell type, thus providing a means of raising the local concentration of gelatinolytic activity in response to specific †