Mucolipidosis III β-N-acetyl-d-hexosaminidase A. Purification and properties

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Abstract
Mucolipidosis III acid hydrolases possess an altered carbohydrate recognition marker needed for their lysosomal localization. As a result of this alteration, a portion of these enzymes is secreted from the cell to the extracellular spaces. The structural changes that may have occurred to 1 of these secreted enzymes, .beta.-N-acetyl-D-hexosaminidase A, were investigated. Normal and mucolipidosis III [human] urinary .beta.-N-acetyl-D-hexosaminidase A were purified to apparent homogeneity by using affinity [Sepharose-2-acetamido-N-(.epsilon.-aminocaproyl)-2-deoxy-.beta.-D-glucopyranosylamine] and ion-exchange (DEAE- and CM-cellulose) chromatography. Sodium dodecyl sulfate/polyacrylamide-slab-gel electrophoresis showed that both enzymes had similar subunit patterns consisting of apparent MW of 68,000, 60,000-58,000, 55,000 and 29,000. Differences, however, were noted in the relative proportions of the protein bands where the normal urinary .beta.-N-acetyl-D-hexosaminidase A contained predominantly the smaller subunits, whereas the mucolipidosis III enzyme had a predominance of the larger subunits. The binding of mucolipidosis III .beta.-N-acetyl-D-hexosaminidase A to Ricinus communis lectin and concanavalin A with and without endo-.beta.-N-acetyl-D-glucosaminidase H treatment indicated that the mutation leads to a modification of a portion of the normally occurring high-mannose-type oligosaccharide units to the complex-type. This was further supported by carbohydrate compositional analysis, which revealed a mannose/galactose ratio of 2.1 for the mucolipidosis III .beta.-N-acetyl-D-hexosaminidase A compared with a ratio of 3.5 for the normal enzyme. As a result of their inability to be properly localized to the lysosome the majority of the mucolipidosis III lysosomal hydrolase high-mannose oligosaccharide units are further processed to the complex-type before secretion of predominantly higher-MW subunits from the cell.