Expression and functional characterization of human mutant sulfamidase in insect cells.
Mucopolysaccharidosis IIIA (MPS IIIA; Sanfilippo syndrome) is an autosomal recessive lysosomal disorder caused by the deficiency of sulfamidase (EC 3.10.1.1), required for the degradation of the mucopolysaccharide heparan sulfate. The molecular defects of 26 unrelated Spanish MPS IIIA patients were recently reported by our group. Here we describe the heterologous expression, using a baculovirus system, of the cDNAs corresponding to eight out of the 14 mutant alleles present in this patient group and the characterization of the corresponding mutant enzymes. In particular, we expressed the following alleles: p.S66W, p.R74H, p.Q85R, p.R206P, p.L386R, p.R433W, p.R433Q, and c.1079delC (previously named as c.1091delC), and the two variants of the polymorphism p.R456H. The expression of the mutant alleles and the characterization of the corresponding enzymes revealed that their activity was severely compromised. Only mutations p.S66W and p.R206P retained low levels of residual activity. However, Western blot analysis showed in all cases the presence of the expected two forms of the sulfamidase, the precursor and the mature proteins, indicating a normal processing of the mutant enzyme.
Montfort
Magda
M
Departament de Genètica, Universitat de Barcelona, Spain.
Garrido
Elena
E
Hopwood
John J
JJ
Grinberg
Daniel
D
Chabás
Amparo
A
Vilageliu
Lluïsa
L
D000483:Alleles; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005838:Genotype; D006801:Humans; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D009084:Mucopolysaccharidosis III / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D013030:Spain; D018411:Spodoptera
D017931:DNA Primers; D018076:DNA, Complementary; D006867:Hydrolases; C022708:N-sulfoglucosamine sulfohydrolase