Site-specific analysis of N-linked oligosaccharides of recombinant lysosomal arylsulfatase A produced in different cell lines.

TY - JOUR

T1 - Site-specific analysis of N-linked oligosaccharides of recombinant lysosomal arylsulfatase A produced in different cell lines.

AU - Schröder, Stephan

AU - Matthes, Frank

AU - Hyden, Pia

AU - Andersson, Claes

AU - Fogh, Jens

AU - Müller-Loennies, Sven

AU - Braulke, Thomas

AU - Gieselmann, Volkmar

AU - Matzner, Ulrich

PY - 2010

Y1 - 2010

N2 - Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a deficiency of the lysosomal enzyme arylsulfatase A (ASA). Enzyme replacement therapy (ERT) is a therapeutic option for MLD and other lysosomal disorders. This therapy depends on N-linked oligosaccharide-mediated delivery of intravenously injected recombinant enzyme to the lysosomes of patient cells. Because of the importance of N-linked oligosaccharide side chains in ERT, we examined the composition of the three N-linked glycans of four different recombinant ASAs in a site-specific manner. Depending on the culture conditions and the cell line expressing the enzyme, we detected a high variability of the high-mannose-type N-glycans which prevail at all glycosylation sites. Our data show that the composition of the glycans is largely determined by substantial trimming in the medium. The susceptibility for trimming is different for the glycans at the three N-glycosylation sites. Interestingly, which of the glycans is most susceptible to trimming also depends on production conditions. CHO cells cultured under bioreactor conditions yielded recombinant ASA with the most preserved N-glycan structures, the highest mannose-6-phosphate content and the highest similarity to non-recombinant enzyme. Notably, roughly one-third of the N-glycans released from the three glycosylation sites were fucosylated. In the last years, numerous recombinant lysosomal enzymes were used for preclinical ERT trials. Our data show that the oligosaccharide structures were very different in these trials making it difficult to draw common conclusions from the various investigations.

AB - Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a deficiency of the lysosomal enzyme arylsulfatase A (ASA). Enzyme replacement therapy (ERT) is a therapeutic option for MLD and other lysosomal disorders. This therapy depends on N-linked oligosaccharide-mediated delivery of intravenously injected recombinant enzyme to the lysosomes of patient cells. Because of the importance of N-linked oligosaccharide side chains in ERT, we examined the composition of the three N-linked glycans of four different recombinant ASAs in a site-specific manner. Depending on the culture conditions and the cell line expressing the enzyme, we detected a high variability of the high-mannose-type N-glycans which prevail at all glycosylation sites. Our data show that the composition of the glycans is largely determined by substantial trimming in the medium. The susceptibility for trimming is different for the glycans at the three N-glycosylation sites. Interestingly, which of the glycans is most susceptible to trimming also depends on production conditions. CHO cells cultured under bioreactor conditions yielded recombinant ASA with the most preserved N-glycan structures, the highest mannose-6-phosphate content and the highest similarity to non-recombinant enzyme. Notably, roughly one-third of the N-glycans released from the three glycosylation sites were fucosylated. In the last years, numerous recombinant lysosomal enzymes were used for preclinical ERT trials. Our data show that the oligosaccharide structures were very different in these trials making it difficult to draw common conclusions from the various investigations.

KW - Animals

KW - Humans

KW - Cells, Cultured

KW - CHO Cells

KW - Cerebroside-Sulfatase biosynthesis

KW - Chromatography, High Pressure Liquid

KW - Cricetinae

KW - Cricetulus

KW - Lysosomes enzymology

KW - Oligosaccharides analysis

KW - Recombinant Proteins biosynthesis

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

KW - Animals

KW - Humans

KW - Cells, Cultured

KW - CHO Cells

KW - Cerebroside-Sulfatase biosynthesis

KW - Chromatography, High Pressure Liquid

KW - Cricetinae

KW - Cricetulus

KW - Lysosomes enzymology

KW - Oligosaccharides analysis

KW - Recombinant Proteins biosynthesis

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

M3 - SCORING: Zeitschriftenaufsatz

VL - 20

SP - 248

EP - 259

JO - GLYCOBIOLOGY

JF - GLYCOBIOLOGY

SN - 0959-6658

IS - 2

M1 - 2

ER -