Subcellular distribution in rat liver of a novel broad‐specificity (α1 → 2, α1 → 3 and α1 → 6) mannosidase active on oligomannose glycans

Open Access
Publisher Website
Google Scholar
Abstract
Recently, the purification to homogeneity was reported of a novel broad‐specificity α‐mannosidase from rat liver microsomal membranes [P. Bonay and R. C. Hughes (1991) Eur. J. Biochem. 197, 229–238]. The enzyme catalyzed the ordered removal of α1 → 2‐, α1 → 3‐ and α1 → 6‐linked mannose residues from MannGlcNAc oligosaccharide substrates where n= 4–9. We now show by subcellular fractionation and immunocytochemistry that the novel mannosidase is present in the endoplasmic reticulum, Golgi apparatus and endosomes. Enzyme activity is enriched in a heavy Golgi membrane fraction and to lesser extent in an intermediate density Golgi membrane fraction containing GlcNAc transferase I activity and in a ‘late’ endosomal fraction. Low levels of enzyme activity were detectable in endoplasmic reticulum membranes and in ‘early’ endosomes but not in receptor‐enriched and ligand‐free endosomes. Assays of enzymic activity using Golgi membrane fractions in the absence and presence of Triton X‐100 showed that the active site of the enzyme faces the lumen of the membrane vesicles. Antibodies directed against the purified mannosidase showed no immunological cross‐reaction to known endoplasmic reticulum and Golgi mannosidases. Conversely, the purified mannosidase was not recognized by antibodies directed against endoplasmic reticulum mannosidase nor Golgi mannosidase IA.