Oligosaccharides of recombinant mouse gelatinase B variants.
Gelatinase B (matrix metalloproteinase-9, MMP-9) contains three N-glycosylation sites and a Ser/Thr/Pro-rich type V collagen domain with repetitive attachment sites for O-linked sugars. Recombinant mouse gelatinase B was expressed in the yeast Pichia pastoris and the N-linked oligosaccharides of the truncated glycoprotein variants were analysed by in gel enzymatic release followed by mass spectrometry and normal phase HPLC. This technology, despite of the limiting amount of material, allowed the analysis of the formula of N- and O-linked sugars of the different glycoprotein variants. The 112/99- and 88-kDa gelatinase B forms each contained an oligomannose series (Man8GlcNAc2 to Man15GlcNAc2). Analysis of the hydrazine-released sugars showed that the O-linked oligosaccharides contained alpha1-2, alpha1-3 or alpha1-6 linked mannoses. These results were confirmed by lectin blot analysis of intact and glycosidase-treated enzyme variants.
Van den Steen
P
P
Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
Rudd
P M
PM
Proost
P
P
Martens
E
E
Paemen
L
L
Küster
B
B
van Damme
J
J
Dwek
R A
RA
Opdenakker
G
G
D000079:Acetaldehyde / Q000031:analogs & derivatives; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D017364:Collagenases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006026:Glycoside Hydrolases; D006466:Hemopexin / Q000737:chemistry; D020780:Matrix Metalloproteinase 9; D051379:Mice; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry*; D010843:Pichia / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017421:Sequence Analysis
D009844:Oligosaccharides; D011134:Polysaccharides; D011994:Recombinant Proteins; C049826:2-methoxyacetaldehyde; D006466:Hemopexin; D006026:Glycoside Hydrolases; D017364:Collagenases; D020780:Matrix Metalloproteinase 9; D000079:Acetaldehyde