title: Receptor mediated binding of two glycosylation forms of N-acetylgalactosamine-4-sulphatase.
abstract: The lysosomal storage disorders are a group of inherited metabolic diseases each characterised by a relative or absolute deficiency of one or more of the lysosomal proteins involved in the hydrolysis of glycoconjugates or in the transport of the resulting product. Enzyme replacement therapies are under consideration for a number of these disorders and are based on the in vitro observation that cells from affected patients can be corrected by addition of exogenous enzyme. In this study, two glycosylation variants of the lysosomal enzyme N-acetylgalactosamine-4-sulphatase (4S) (the deficiency of which causes Mucopolysaccharidosis (MPS) type VI, (Maroteaux-Lamy syndrome) were made by expression of 4S cDNA in both wild type chinese hamster ovary (CHO-K1), and Lec1 (N-acetylglucosaminyltransferase I deficient CHO-K1) cells. Differences in the glycosylation pattern of the two enzyme forms were demonstrated with endoglycosidase H and N-glycosidase F digestions. The receptor mediated binding of these two forms of 4S to two cell types, human skin fibroblasts and rat alveolar macrophages, was then analysed. We have shown that both enzyme forms bind to the mannose-6-phosphate receptor on human skin fibroblasts with equal affinity demonstrating that the degree of phosphorylation of mannose residues in the two forms is similar. However, using rat alveolar macrophages, we found that the binding/uptake of the two enzymes differs considerably. These results show that differences in glycosylation of lysosomal enzymes can be an important factor in altering enzyme uptake by different cell types. Thus, producing carbohydrate modification variants in this way may be useful for altering the distribution of exogenous enzyme in vivo.
authors.affiliation: Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women's and Children's Hospital, 72 King William Road, North Adelaide, 5006, South Australia, Australia.
authors.forename: M
authors.initials: M
authors.lastname: Fuller
authors.forename: J J
authors.initials: JJ
authors.lastname: Hopwood
authors.forename: D S
authors.initials: DS
authors.lastname: Anson
mesh_terms: D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002460:Cell Line; D006224:Cricetinae; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D016676:Macrophages, Alveolar / Q000201:enzymology / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009087:Mucopolysaccharidosis VI / Q000201:enzymology / Q000378:metabolism; D020051:N-Acetylgalactosamine-4-Sulfatase / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D017527:Receptor, IGF Type 2 / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014316:Tritium
chemical_list: D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; D014316:Tritium; D020051:N-Acetylgalactosamine-4-Sulfatase