Abstract
Arylsulfatase G (ARSG) is a recently identified lysosomal sulfatase that was shown to be responsible for the degradation of 3-O-sulfated N-sulfoglucosamine residues of heparan sulfate glycosaminoglycans. Deficiency of ARSG leads to a new type of mucopolysaccharidosis, as described in a mouse model. Here, we provide a detailed molecular characterization of the endogenous murine enzyme. ARSG is expressed and proteolytically processed in a tissue-specific manner. The 63-kDa single-chain precursor protein localizes to pre-lysosomal compartments and tightly associates with organelle membranes, most likely the endoplasmic reticulum. In contrast, proteolytically processed ARSG fragments of 34-, 18-, and 10-kDa were found in lysosomal fractions and lost their membrane association. The processing sites and a disulfide bridge between the 18- and 10-kDa chains could be roughly mapped. Proteases participating in the processing were identified as cathepsins B and L. Proteolytic processing is dispensable for hydrolytic sulfatase activity in vitro. Lysosomal transport of ARSG in the liver is independent of mannose 6-phosphate, sortilin, and Limp2. However, mutation of glycosylation site N-497 abrogates transport of ARSG to lysosomes in human fibrosarcoma cells, due to impaired mannose 6-phosphate modification.
Highlights
Lysosomal arylsulfatase G (ARSG) is critical in heparan sulfate degradation
An additional strong immunoreactive band with a molecular mass of ϳ34 kDa was observed in the liver and even more abundant in brain lysates, where overall ARSG protein levels were highest among the evaluated tissues
In this study, detailed biochemical description of the endogenous ARSG enzyme is presented to understand in vivo expression, function, and properties of this enzyme, which is critical for the degradation of 3-O-sulfated glucosamine residues of heparan sulfate
Summary
Lysosomal arylsulfatase G (ARSG) is critical in heparan sulfate degradation. Results: ARSG is differentially expressed, processed, and transported in tissues involving a membrane-associated pre-lysosomal precursor; processing is dispensable for enzymatic activity. Examination of the GAG storage material derived from these KO mice identified it as heparan sulfate, and further detailed analysis of the nonreducing end revealed 3-O-sulfated N-sulfoglucosamine (GlcNS3S) as the natural substrate of ARSG. We classified this new lysosomal storage disease as MPS IIIE, consistent with its function in the lysosomal degradative pathway of glucosamine residues in heparan sulfate. The natural substrate of ARSG is known and its role in the degradation of heparan sulfate was unequivocally shown by our KO approach, no data are available on biochemical properties of the endogenous enzyme, including its expression in tissues, post-translational modifications, or mode of transport to lysosomes. We report on in-depth analysis of tissue expression, proteolytic processing, and nontypical lysosomal transport mechanisms of ARSG
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