Glycosylation of natural human neutrophil gelatinase B and neutrophil gelatinase B-associated lipocalin.
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.
Rudd
P M
PM
Glycobiology Institute, Department of Biochemistry, University of Oxford, U.K.
Mattu
T S
TS
Masure
S
S
Bratt
T
T
Van den Steen
P E
PE
Wormald
M R
MR
Küster
B
B
Harvey
D J
DJ
Borregaard
N
N
Van Damme
J
J
Dwek
R A
RA
Opdenakker
G
G
D000209:Acute-Phase Proteins; D000581:Amidohydrolases / Q000378:metabolism; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D000071068:Lipocalin-2; D054834:Lipocalins; D020780:Matrix Metalloproteinase 9 / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology*; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D015513:Oncogene Proteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000209:Acute-Phase Proteins; D002352:Carrier Proteins; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D054834:Lipocalins; D009844:Oligosaccharides; D015513:Oncogene Proteins; D011134:Polysaccharides; D011518:Proto-Oncogene Proteins; D020780:Matrix Metalloproteinase 9; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase