Heparan N-sulfatase: in vitro mutagenesis of potential N-glycosylation sites.
Heparan N-sulfatase cDNA contains five potential N-glycosylation sites at Asn positions 41, 142, 151, 264, and 413. We used site-directed mutagenesis, substituting the codon of asparagine for glutamine, to eliminate selected glycosylation sites and then performed expression studies in COS-7 cells to determine the influence on the catalytic activity, lysosomal targeting, and glycosylation-phosphorylation of the enzyme. Elimination of site 5 did not affect significantly enzyme activity; elimination of sites 2 and 4 gave a partial reduction, while elimination of sites 1 and 3 resulted in drastic reduction of catalytic activity (25 and 14%, respectively, of normal values), indicating that glycosylation of asparagine 41 and asparagine 151 is essential for catalysis and/or enzyme stability. Wild type enzyme produced in the presence of tunicamycin was also inactive, indicating that glycosylation is required for acquisition of enzyme activity and/or for enzyme stability. Metabolic labeling of each mutant cDNA, transiently transfected into COS cells, showed that enzyme from mutants N142Q, N264Q, and N413Q appeared to be properly folded, as judged by its ability to be proteolytically processed to a lower molecular weight form, while enzyme from mutants N41Q and N151Q did not reach lysosomes. These studies confirm that the five glycosylation sites of heparan N-sulfatase are all functional and show that Asn 41 and Asn 151 have a role in protein folding and/or stability.
Di Natale
P
P
Department of Biochemistry and Medical Biotechnologies, University of Naples Federico II, Naples, Italy. dinatale@cds.unina.it
Vanacore
B
B
Daniele
A
A
Esposito
S
S
D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D008360:Mannosephosphates / Q000494:pharmacology; D009084:Mucopolysaccharidosis III / Q000378:metabolism / Q000473:pathology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013429:Sulfatases / Q000235:genetics* / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
D008360:Mannosephosphates; D014415:Tunicamycin; C027693:mannose-6-phosphate; D001216:Asparagine; D013429:Sulfatases; C021346:heparan sulfate sulfatase; D006026:Glycoside Hydrolases