Related Experiment Videos
Cloning, expression, purification, and characterization of the human broad specificity lysosomal acid
1Complex Carbohydrate Research Center and the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA. moremen@bscr.uga.edu
Abstract:
We have cloned and expressed two cDNAs encoding the human lysosomal alpha-mannosidase (EC 3.2.1.24) by RT-PCR of human spleen mRNA. This enzyme is required for the degradation of N-linked carbohydrates during glycoprotein catabolism in eucaryotic cells. The shorter of the two cDNAs (3 kilobases (kb)) was found to encode an open reading frame of 2964 base pairs and, when expressed in Pichia pastoris, was found to encode an enzyme that could cleave high mannose oligosaccharides, oligosaccharides isolated from alpha-mannosidosis fibroblasts, and p-nitrophenyl-alpha-D-mannopyranoside substrates. In addition, the Pichia-expressed enzyme was inhibited by swainsonine, and had a pH optimum, Km, and Vmax characteristic of the enzyme purified previously from human liver. The second, larger RT-PCR product (3.6 kb) was found to contain an insertion and a deletion relative to the 3-kb spleen amplimer and encoded a truncated coding region, indicating that it resulted from alternate transcript splicing. No alpha-mannosidase activity could be detected in Pichia transformants containing this coding region, indicating that it did not encode a functional enzyme. Antiserum raised to the recombinant product of the 3-kb alpha-mannosidase cDNA immunoprecipitated lysosomal alpha-mannosidase activity from human fibroblast extracts. Northern blots identified a 3-kb RNA transcript in all human tissues tested, including alpha-mannosidosis fibroblasts, while minor transcripts of 3.6 kb were also present in several adult tissues. Human chromosome mapping of the mannosidase gene confirmed that the functional gene maps to the MANB locus on chromosome 19. Sequence comparisons were made to previously published human cDNA sequences encoding a putative lysosomal alpha-mannosidase (Nebes, V. L., and Schmidt, M. C. (1994) Biochem. Biophys. Res. Commun. 200, 239-245) and several differences were found relative to the functional lysosomal alpha-mannosidase encoded by the 3-kb spleen cDNA.