Oligosaccharides of recombinant mouse gelatinase B variants

• Published in: Biochimica et biophysica acta 1998-11, Vol.1425 (3), p.587-598
• Main Authors: Van den Steen, Philippe, Rudd, Pauline M., Proost, Paul, Martens, Erik, Paemen, Liesbet, Küster, Bernhard, van Damme, Jo, Dwek, Raymond A., Opdenakker, Ghislain
• Format: Article
• Language: English
• Subjects: Acetaldehyde - analogs & derivatives; Animals; Chromatography, High Pressure Liquid; Collagenases - biosynthesis; Collagenases - chemistry; Collagenases - genetics; Gelatinase B; Glycoside Hydrolases; Glycosylation; Hemopexin - chemistry; Mass spectrometry; Matrix Metalloproteinase 9; Mice; Mutation; Normal phase-high performance liquid chromatography; Oligosaccharides - chemistry; Pichia - metabolism; Pichia pastoris expression; Polysaccharides - chemistry; Polysaccharides - metabolism; Recombinant Proteins - chemistry; Sequence Analysis
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/S0304-4165(98)00113-5

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 (Man 8GlcNAc 2 to Man 15GlcNAc 2). Analysis of the hydrazine-released sugars showed that the O-linked oligosaccharides contained α1-2, α1-3 or α1-6 linked mannoses. These results were confirmed by lectin blot analysis of intact and glycosidase-treated enzyme variants.