Heparan N‐sulfatase: cysteine 70 plays a role in the enzyme catalysis and processing

Abstract

Sulfatases are members of a highly conserved family of enzymes that catalyze the hydrolysis of sulfate ester bonds from a variety of substrates. The functional correlation reflects a high degree of amino acid sequence similarity along the entire length, in particular in the active site where the C(X)PSR consensus sequence is present. Cysteine undergoes an important co-or post-translation modification essential for the accomplishment of catalytic activity : conversion in formylglycine. In this work, the cysteine of heparan N-sulfatase (NS) was replaced either by a serine (C70S) or by a methionine (C70M) using sitedirected mutagenesis. C70S and C70M mutant cDNAs were expressed and analyzed in COS cells; both mutations caused a loss of NS activity; however, while C70S showed a normal precursor form undergoing processing to a reduced mature form within the lysosomes, C70M was poorly synthesized and formed a complex with the molecular chaperone immunoglobulin binding protein. ß

Analysis of Adiponectin Gene and Comparison of Its Expression in Two Different Pig Breeds

“…Ten microgram of purified serum were lysed in Laemmli buffer with and without dithiothreitol 10 mmol/l (28). The gel was exposed to high‐performance autoradiography film (Amersham Biosciences, Milan, Italy), digitalized with a scanner (1,200 dpi) and analyzed by densitometry with the Jasc Paint Shop Pro 7.00 software.…”

A homozygous founder missense variant in arylsulfatase G abolishes its enzymatic activity causing atypical Usher syndrome in humans

“…Lack of processing was observed earlier also for ARSG-p.C84A (unpublished data) and also for active site mutants of two other human sulfatases. 27,28 While this maturation of ARSG is dispensable for enzymatic activity in vitro, the observed lack of in vivo processing, which relies on the lysosomal proteases cathepsin B and L, 16 suggests that ARSG-p.D45Y is improperly transported to the lysosomes. To conclude, the p.D45Y-mutation, apart from blocking the catalytic activity, clearly affects maturation and probably also trafficking of the enzyme to its target organelle.…”

Analysis of Sanfilippo A gene mutations in a large pedigree

“…7). We have recently demonstrated, using site-directed mutagenesis, that cysteine 70 plays a role in enzyme catalysis and processing (8).…”