PURIFICATION OF BOVINE LYSOSOMAL ALPHA-MANNOSIDASE, CHARACTERIZATION OF ITS GENE AND DETERMINATION OF 2 MUTATIONS THAT CAUSE ALPHA-MANNOSIDOSIS

Bovine kidney lysosomal a-mannosidase was purified to homogeneity and the gene was cloned. The gene was organized in 24 exons that spanned 1 6 kb and its corresponding cDNA contained an open reading frame of 299 7 bp beginning from a putative ATG start codon. The deduced amino acid sequence contained a signal peptide of 50 amino acids adjactent to a protein sequence of 949 amino acids that was cleaved into five peptide s in the mature enzyme; starting with the peptide derived from the N-t erminal part of this precursor, their molecular masses were 35/38 (pep tide a), 11/13 (peptide b), 22 (peptide c), 38 (peptide d) and 13/15 k Da (peptide e). Variation in the degree of N-glycosylation accounts fo r molecular mass heterogeneities of peptides a, b and e. Peptides a, b and c were disulphide-linked. A T961-->C transition, resulting in Phe 321-->Leu substitution, was identified in the cDNA of alpha-mannosidos is-affected Angus cattle. In affected Galloway cattle, a G662-->A tran sition that causes Arg221-->His substitution was identified. Phe321 an d Arg221 are conserved among the a-mannosidase class-2 family, indicat ing that the substitutions resulted from disease-causing mutations in these breeds.