Purification of bovine lysosomal alpha-mannosidase, characterization of its gene and determination of two mutations that cause alpha-mannosidosis

Bovine kidney lysosomal alpha-mannosidase was purified to homogeneity and the gene was cloned. The gene was organized in 24 exons that spanned 16 kb and its corresponding cDNA contained an open reading frame of 2997 bp beginning from a putative ATG start codon. The deduced amino acid sequence contained a signal peptide of 50 amino acids adjacent to a protein sequence of 949 amino acids that was cleaved into five peptides in the mature enzyme; starting with the peptide derived from the N-terminal part of this precursor, their molecular masses were 35/38 (peptide a), 11/13 (peptide b), 22 (peptide c), 38 (peptide d) and 13/15 kDa (peptide e). Variation in the degree of N-glycosylation accounts for molecular mass heterogeneities of peptides a, b and e. Peptides a, b and c were disulphide-linked. A T961 leads to C transition, resulting in Phe321 leads to Leu substitution, was identified in the cDNA of alpha-mannosidosis-affected Angus cattle. In affected Galloway cattle, a G662 leads to A transition that causes Arg221 leads to His substitution was identified. Phe321 and Arg221 are conserved among the alpha-mannosidase class-2 family, indicating that the substitutions resulted from disease-causing mutations in these breeds.