Synthesis and Processing of Arylsulfatase A in Human Skin Fibroblasts

Abstract

Biosynthesis of arylsulfatase A in normal and mutant human fibroblasts was studied by growing cells in the presence of L-[4,5-3H]leucine or [2-3H]mannose, isolation of labeled arylsulfatase A by immune precipitation and visualization of electrophoretically separated polypeptides by fluorography. Arylsulfatase A was synthesized as a precursor with a mean apparent molecular mass of 62 kDa [kilodaltons]. Intracellularly the precursor was converted into a 60.5 kDa polypeptide within a chase period of 1-7 days. The 60.5 kDa product in polyacrylamide corresponded to one of 2 polypeptides present in arylsulfatase A isolated from human placenta. In fibroblasts from a patient with metachromatic leukodystrophy no immune precipitable polypeptides of arylsulfatase A were detected. In normal fibroblasts < 10% of the precursor of arylsulfatase A was secreted into the medium, whereas in mucolipidosis II fibroblasts and in control fibroblasts grown in the presence of NH4 Cl up to 90% of the precursor of arylsulfatase A, appeared in the medium and remained there without change in the apparent molecular mass for at least 7 days. Arylsulfatase A polypeptides appear to contain 2 carbohydrate side chains. In about 90% of the polypeptides both side chains are cleaved by endo-.beta.-N-acetylglucosaminidase H, whereas in the remaining chains 1 of the 2 oligosaccharides is not cleaved.