INTERMEDIATE GOLGI α-D-MANNOSIDASE ACTIVITY IN FIBROBLASTS FROM PATIENTS WITH MANNOSIDOSIS AND MUCOLIPIDOSIS II AND III

Abstract

Summary: We have examined extracts of fibroblasts from patients with mannosidosis, mucolipidosis (ML) II, ML III, and normal controls for α-D-mannosidase activity against 4-methylumbelliferyl-α-D-mannopyranoside to test for the presence of the “intermediate” pH S.5 enzyme activity that has been called the “Golgi mannosidase.” Fibroblast extracts were prepared by sonication and sedimented to separate membrane-associated activities from cytosolic and lysosomal α-D-mannosidases. Membranes were extracted by salt washes (0.4 M NaCl) to desorb the lysosomal enzymes that sertimented with membranes. The α-D-mannosidase activity remaining with membranes showed many properties described for the Golgi mannosidase including: 1) an “intermediate” pH optimum (pH 5.5–6.0), 2) activity with the synthetic 4-methylumbelliferyl substrate, 3) lack of inhibitability by 200 mM methyl- α-D-mannopyranoside, and 4) partial resistance to solubilization by salt washing, and to a single extraction with the non-ionic detergent Triton X-100.

This intermediate activity was the major α-D-mannosidase activity remaining in membranes from normal fibroblasts following sedimentation and salt washing, and was the major component of the α-D-mannosidase activity in extracts of fibroblasts from patients with mannosidosis, ML II, and ML III, in all of which, the lysosomal “acid” α-D-mannosidase was greatly reduced.

The specific activity of intermediate α-D-mannosidase in membranes from fibroblasts from patients with mannosidosis, ML II, and ML III was not reduced compared to that of fibroblasts from controls. These studies provided no evidence to support the prior suggestion of a deficiency of “Golgi mannosidase” in ML II or ML III.

Speculation: Although these studies argue againct a deficiency of “Golpi mannosidase” in rucolipidosis (ML) II and ML III, other evidence suggests that there is an oligosaccharide processing abnormality affecting the oligosaccharides on acid hydrolases secreted by ML II fibroblasts. This abnormality is inferred from the presence of complex-type oligosaccharide chains rather than high-mannose type chains on I-cell secretion enzymes. We suggest that this abnormality can be explained by the primary defect in I-cell disease, failure to phosphorylate the high-mannose oligosaccharide chains of newly synthesized ML II acid hydrolases. The mannose6-phosphate recognition markers normally bind receptors and effect segregation of acid hydrolases from secretory products of the endoplasmic reticulum. We suggest that the phosphate also prevents processing of the protscted high-mannose oligosaccharide chains by mannosidases, which participate in the conversion of high-mannose type to complex type oligosaccharides that are typical of secretory glycoproteins. Absence of the phosphate would allow the unprotected highmannose chains on I-cell enzymes to be processed to complex-type oligosaccharides in the Golgi apparatus before the enzymes are secreted, which would explain the properties of I-cell secretion enzymes.

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