Glycosylation of Natural Human Neutrophil Gelatinase B and Neutrophil Gelatinase B-Associated Lipocalin

Abstract

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 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 Galbeta1,4GlcNAc (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.

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.…”

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).…”

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

“…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).…”