Oligosaccharides of recombinant mouse gelatinase B variants

Philippe Van Den Steen; Pauline M. Rudd; Paul Proost; Erik Martens; Liesbet Paemen; Bernhard Küster; Jo Van Damme; Raymond A. Dwek; Ghislain Opdenakker

doi:10.1016/S0304-4165(98)00113-5

Oligosaccharides of recombinant mouse gelatinase B variants

Philippe Van Den Steen, Pauline M. Rudd, Paul Proost, Erik Martens, Liesbet Paemen, Bernhard Küster, Jo Van Damme, Raymond A. Dwek, Ghislain Opdenakker

Research output: Contribution to journal › Article › peer-review

28 Scopus citations

Abstract

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 α1-2, α1-3 or α1-6 linked mannoses. These results were confirmed by lectin blot analysis of intact and glycosidase-treated enzyme variants. Copyright (C) 1998 Elsevier Science B.V.

Original language	English
Pages (from-to)	587-598
Number of pages	12
Journal	Biochimica et Biophysica Acta - General Subjects
Volume	1425
Issue number	3
DOIs	https://doi.org/10.1016/S0304-4165(98)00113-5
State	Published - 27 Nov 1998
Externally published	Yes

Keywords

Gelatinase B
Glycosylation
Mass spectrometry
Normal phase-high performance liquid chromatography
Pichia pastoris expression

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10.1016/S0304-4165(98)00113-5

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@article{e9b4b8ab60cf491e87873de6cf14575a,

title = "Oligosaccharides of recombinant mouse gelatinase B variants",

abstract = "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 α1-2, α1-3 or α1-6 linked mannoses. These results were confirmed by lectin blot analysis of intact and glycosidase-treated enzyme variants. Copyright (C) 1998 Elsevier Science B.V.",

keywords = "Gelatinase B, Glycosylation, Mass spectrometry, Normal phase-high performance liquid chromatography, Pichia pastoris expression",

author = "{Van Den Steen}, Philippe and Rudd, {Pauline M.} and Paul Proost and Erik Martens and Liesbet Paemen and Bernhard K{\"u}ster and {Van Damme}, Jo and Dwek, {Raymond A.} and Ghislain Opdenakker",

note = "Funding Information: The present study was supported by the General Savings and Retirement Fund (A.S.L.K.), the Belgian Cancer Association, the Geconcerteerde OnderzoeksActies and the Fund for Scientific Research (F.W.O.-Vlaanderen). Ph.V.d.S. is research assistant of the F.W.O. and P.P. is postdoctoral assistant of the F.W.O. Financial support from the DAAD (Grant D/94/14920 to B.K.) is also gratefully acknowledged. The NP-HPLC and mass spectrometry analyses were done in the facilities of the Department of Biochemistry at the University of Oxford. We thank Professor Edwin Southern for the use of the Perceptive MS facility, and Ilse Van Aelst for excellent technical help.",

year = "1998",

month = nov,

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doi = "10.1016/S0304-4165(98)00113-5",

language = "English",

volume = "1425",

pages = "587--598",

journal = "Biochimica et Biophysica Acta - General Subjects",

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TY - JOUR

T1 - Oligosaccharides of recombinant mouse gelatinase B variants

AU - Van Den Steen, Philippe

AU - Rudd, Pauline M.

AU - Proost, Paul

AU - Martens, Erik

AU - Paemen, Liesbet

AU - Küster, Bernhard

AU - Van Damme, Jo

AU - Dwek, Raymond A.

AU - Opdenakker, Ghislain

N1 - Funding Information: The present study was supported by the General Savings and Retirement Fund (A.S.L.K.), the Belgian Cancer Association, the Geconcerteerde OnderzoeksActies and the Fund for Scientific Research (F.W.O.-Vlaanderen). Ph.V.d.S. is research assistant of the F.W.O. and P.P. is postdoctoral assistant of the F.W.O. Financial support from the DAAD (Grant D/94/14920 to B.K.) is also gratefully acknowledged. The NP-HPLC and mass spectrometry analyses were done in the facilities of the Department of Biochemistry at the University of Oxford. We thank Professor Edwin Southern for the use of the Perceptive MS facility, and Ilse Van Aelst for excellent technical help.

PY - 1998/11/27

Y1 - 1998/11/27

N2 - 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 α1-2, α1-3 or α1-6 linked mannoses. These results were confirmed by lectin blot analysis of intact and glycosidase-treated enzyme variants. Copyright (C) 1998 Elsevier Science B.V.

AB - 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 α1-2, α1-3 or α1-6 linked mannoses. These results were confirmed by lectin blot analysis of intact and glycosidase-treated enzyme variants. Copyright (C) 1998 Elsevier Science B.V.

KW - Gelatinase B

KW - Glycosylation

KW - Mass spectrometry

KW - Normal phase-high performance liquid chromatography

KW - Pichia pastoris expression

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U2 - 10.1016/S0304-4165(98)00113-5

DO - 10.1016/S0304-4165(98)00113-5

M3 - Article

C2 - 9838222

AN - SCOPUS:0031730071

SN - 0304-4165

VL - 1425

SP - 587

EP - 598

JO - Biochimica et Biophysica Acta - General Subjects

JF - Biochimica et Biophysica Acta - General Subjects

IS - 3

ER -

Oligosaccharides of recombinant mouse gelatinase B variants

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Keywords

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