Heparan N-sulfatase: cysteine 70 plays a role in the enzyme catalysis and processing.
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 site-directed 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.
Daniele
A
A
Department of Biochemistry and Medical Biotechnologies, Medical School, University of Naples Federico II, Via Pansini 5, 80131, Naples, Italy. daniele@cds.unina.it
Di Natale
P
P
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism; D002384:Catalysis; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D008715:Methionine / Q000235:genetics; D018832:Molecular Chaperones / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013429:Sulfatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D017931:DNA Primers; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D012333:RNA, Messenger; D008715:Methionine; D013429:Sulfatases; C021346:heparan sulfate sulfatase; D003545:Cysteine