CLONING, EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE HUMAN BROAD-SPECIFICITY LYSOSOMAL ACID ALPHA-MANNOSIDASE

We have cloned and expressed two cDNAs encoding the human lysosomal al pha-mannosidase (EC 3.2.1.24) by RT-PCB of human spleen mRNk This enzy me is required for the degradation of N-Linked carbohydrates during gl ycoprotein catabolism in eucaryotic cells, The shorter of the two cDNA s (3 kilobases (kb)) was found to encode an open reading frame of 2964 base pairs and, when expressed in Pichia pastoris, was found to encod e an enzyme that could cleave high mannose oligosaccharides, oligosacc harides isolated from alpha-mannosidosis fibroblasts, and p-nitropheny l-alpha-D-mannopyranoside substrates, In addition, the Pichia-expresse d enzyme was inhibited by swainsonine, and had a pH optimum, K-m, and V-max characteristic of the enzyme purified previously from human live r. 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 enco ded a truncated coding region, indicating that it resulted from altern ate transcript splicing, No alpha-mannosidase activity could be detect ed in Pichia transformants containing this coding region, indicating t hat it did not encode a functional enzyme, Antiserum raised to the rec ombinant 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 t ested, including alpha-mannosidosis fibroblasts, while minor transcrip ts of 3.6 kb were also present in several adult tissues, Human chromos ome mapping of the mannosidase gene confirmed that the functional gene maps to the MANB locus on chromosome 19, Sequence comparisons were ma de to preciously published human cDNA sequences encoding a putative ly sosomal alpha-mannosidase (Nebes, V, L., and Schmidt, M. C. (1994) Bio chem. Biophys. Res, Commun. 200, 289-245) and several differences were found relative to the functional lysosomal alpha-mannosidase encoded by the 3-kb spleen cDNA.