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Glycobiology vol 2 no 4 pp. 327-336, 1992
© 1992

research-article

A human lysosomal {alpha}(1->6)-mannosidase active on the branched trimannosyl core of complex glycans

Peter F. Daniel4, James E. Evans, Rita De Gasperi1,3, Bryan Winchester2 and Christopher D. Warren1

Departments of Neurology, Massachusetts General Hospital and Harvard Medical School, and Department of Biomedical Sciences, Shriver Center for Mental Retardation 200 Trapelo Road, Waltham, MA 02254, USA
1Carbohydrate Unit, Lovett Laboratories, Harvard Medical School and Massachusetts General Hospital Charlestown, MA 02129, USA
2Department of Clinical Biochemistry, Institute of Child Health, University of London London WC1N 1EH, UK
3Present address: Department of Neurology, New York University School of Medicine 550 1st Avenue, RR210, New York, NY, USA

4To whom correspondence should be addressed at the Shriver Center

Received on March 12, 1992; accepted on May 8, 1992

Normal human fibroblasts and fibroblasts from a patient with {alpha}-mannosidosis were grown in the presence or absence of 100 µM swainsonine for 7 days. Accumulated oligosaccharides were isolated and analysed by high performance liquid chromatography (HPLC) and methylation analysis. Man{alpha}1->3Manß1->4GlcNAc and Man{alpha}1->2Man{alpha}1->3Manß1->4GlcNAc (where Man is D-mannose and GlcNAc is N-acetyl-D-glucosamine) comprised >80% of the total oligosaccharides in untreated mannosidosis cells. However, Man{alpha}1->6[Man{alpha}1->3]Manß1->4GlcNAc was the major Man3 GlcNAc isomer present after 7 days of swainsonine treatment. No mannose-containing oligosaccharides were detected in control fibroblasts in the absence of swainsonine but, in its presence, oligosaccharides containing 2–9 mannose residues accumulated. Man{alpha}1->6[Man{alpha}1->3] Man{alpha}1->6[Man{alpha}1->3]-Manß1->4GlcNac and Man{alpha}1->6-[Man{alpha}1->3]Manß1->4GlcNac were the major components (67%). Surprisingly, Man{alpha}1->3Manß1->4GlcNAc was only observed in swainsonine-treated control cells during the recovery period after removal of swainsonine. These studies suggest the presence of a second lysosomal {alpha}-mannosidase activity which is unaffected in genetic {alpha}-mannosidosis, but is inhibited by swainsonine. This enzyme would cleave the {alpha}(1->6)-linked mannose residue from branched Man3 GlcNAc to form Man{alpha}1->3Manß1ß4GlcNAc. To confirm this hypothesis, fractions from {alpha}-mannosidosis and control fibroblasts that bound to concanavalin A (ConA)–Sepharose and were eluted with 0.5 M {alpha}-methyl mannoside were incubated at pH 4.0 with Man{alpha}1->6[Man{alpha}1->3]Manß1->4-GlcNac. As anticipated,Man{alpha}l->3Manß1->4GlcNac was the sole product using enzyme from mannosidosis fibroblasts, while the major product from control fibroblasts was Man{alpha}l->6Manß1-->4GlcNAc. This confirmed the presence of a swainsonine-inhibitable {alpha}(1->6)-mannosidase activity unaffected by the disease. The differing substrate specificities of the {alpha}(1->6)-mannosidase and the major lysosomal {alpha}-mannosidase indicate that the {alpha}(1->6)-mannosidase plays an important role in the generation of the oligosaccharides accumulated in {alpha}-mannosidosis patients.

human fibroblasts lysosomes {alpha}(1->6)-mannosidase {alpha}-mannosidosis swainsonine


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