The International Society for Mannosidosis & Related Diseases, Inc. presents
the road so far:
the tromso mannosidosis group
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by Dag Malm Senior Consultant,
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as published in Pathways
Spring 2000 edition (vol. 1, no 3)
see also our comprehensive section on Alpha-Mannosidosis
Editor’s note: the Tromso Mannosidosis Group, located in Norway, has developed much of the current knowledge about alpha mannosidosis. With this issue one of it’s founders relates the story so far.
When the Research Group in Norway started, very little was known about mannosidosis. Although the clinical symptoms and diagnostic techniques were known, information about the enzyme (amino acid sequence or structure), the gene (base pair sequence and organization) and the disease causing mutations were not.
Prof. Ole Kristian Tollersrud had much experience in isolating and purifying lysosomal enzymes since he had studied aspartylglucosaminuria a disease similar to mannosidosis. It started with the purchase of 80 kg cattle kidney at the butchers shop which was used to purify mannosidosis.
After grinding the kidneys, mannosidase was purified to homogeneity by using alcohol and so called salt fractionation, cellulose chromatography, gel filtration, concanavalin A-Sepharose affinity chromatography, heat treatment at 70 degrees C and preparative SDS/polyacrylamide-gel electrophoresis. These are standard chemical techniques. The 80 kg of ox kidneys yielded 5 mg purified mannosidase enzyme with a high specific activity. The molecular mass was determined by electrophoresis and mannosidase was found to be composed of five non- identical subunits joined by strong non-covalent forces and the molecular masses was determined by SDS/polyacrylamide-gel electrophoresis. The enzyme, which basically is a protein, was found to have several sugar chains bound to it. The enzyme was further characterized with its pH optimum, meaning the acidity where it works as best.
Now that the group had the purified enzyme, the next step was to make antibodies to mannosidase by injecting the enzyme in rabbits which then formed antibodies. These could be used in the characterization of where enzymes are transported in the body after injection and the nature of the “residual activity” which is measured to 1-8% in patients. Some enzyme was sent to CERN to analyze its molecular structure by bombardment with atomic particles.
The next leap was to find and sequence…….
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