Glycobiology Advance Access published online on October 28, 2009
Glycobiology, doi:10.1093/glycob/cwp171
Site specific analysis of N-linked oligosaccharides of recombinant lysosomal arylsulfatase A produced in different cell lines
1 Rheinische Friedrich-Wilhelms Universität, Institut für Biochemie und Molekularbiologie, Nussallee 11, D-53115 Bonn, Germany
2 Zymenex A/S, Roskildevej 12 C, DK-3400 Hillerød, Denmark and Dalernum 13, S-18170 Lidingö, Sweden
3 Research Center Borstel, Leibniz Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany
4 Children's Hospital, University of Hamburg, Martinistr. 52, D-20246 Hamburg, Germany
* to whom correspondence should be addressed: Tel: +49-228-735046. Fax: +49-228-732416. E-mail: matzner{at}ibmb.uni-bonn.de
corresponding author: Ulrich Matzner, PhD, Nussallee 11, 53115 Bonn, Germany. Tel: +49-228-735046. Fax: +49-228-732416. E-mail: matzner{at}ibmb.uni-bonn.de
Received on April 28, 2009; accepted on October 24, 2009
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.
Key words: arylsulfatase A / enzyme replacement therapy / mannose 6-phosphate / metachromatic leukodystrophy