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

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 one of these secreted enzymes, beta-N-acetyl-d-hexosaminidase A (EC 3.2.1.52) were investigated. Normal and mucolipidosis III 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 sulphate/polyacrylamide-slab-gel electrophoresis showed that both enzymes had similar subunit patterns consisting of apparent mol.wts. of 68000, 60000-58000, 55000 and 29000. 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. Our results indicate that 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-molecular-weight subunits from the cell.

Molecular forms of β-hexosaminidase and cathepsin D in serum and urine of healthy subjects and patients with elevated activity of lysosomal enzymes

“…213 lipidosis-II fibroblasts (for interpretation see Hasilik & von Figua, 1981). Our observations on the shift in the oligosaccharide pattern are consistent with a report on f,-hexosaminidase A (Kress et al, 1982), and contrast with another onf,-hexosaminidase B (Hirani et al, 1982), from mucolipidosis-III urine.…”

Lysosomal Enzymes and Their Receptors

“…Bi-and triantennary oligo saccharides comprising about 80% of the total oligosaccharides were found in human J3-glucocerebrosidase (58). It should be pointed out that this hydrolase is an example of a membrane-associated lysosomal enzyme (59 (64,(68)(69)(70)(71). This striking change results from a defect in these mutant cells in the phos phorylation of high-mannose oligosaccharides (see below).…”

Isolation and characterization of a precursor form of lysosomal α‐glucosidase from human urine

“…This may also be true for a-fucosidase [26]. Besides the precursor forms, mature forms of the B-hexosaminidase subunits are found in normal urine [24]. Miller et al therefore suggested [24] that P-hexosaminidase can be secreted into the urine via two pathways: a 'lysosomal' pathway leading to secretion of the mature form of the enzyme, and a 'nonlysosomal' pathway leading to direct secretion of the precursor form.…”

“…The secretion of lysosomal enzymes is unrelated to that of the cytosolic enzyme lactate dehydrogenase, suggesting that the lysosomal enzymes found in the urine are not derived from exfoliated cells but are actively secreted. It has been shown by Miller and coworkers [24,25] that normal urine contains forms of B-hexosaminidase A and B with subunits of higher molecular mass than those present in placental and liver forms of the enzyme, and corresponding in size to those of the precursor forms of the subunits found in the culture medium of fibroblasts. This may also be true for a-fucosidase [26].…”