a-Mannosidosis (MIM 248500) is an autosomal recessive lysosomal storage disorder resulting from deficient activity of lysosomal alpha-mannosidase (LAMAN) (EC 3.2.1.24). The disease is characterized by massive intracellular accumulation of mannose-rich oligosaccharides with resulting mental retardation, hearing loss, immune deficiency and skeletal changes. We report here the purification and characterization of human placenta LAMAN. The enzyme is synthesized as a single-chain precursor which is processed into three glycopeptides of 70, 42 and 15 kDa. The 70 kDa peptide is further partially proteolysed into three more peptides that are joined by disulfide bridges. The laman cDNA sequence was assembled from overlapping fragments obtained by PCR on human fibroblast and human lung cDNA. The deduced amino acid sequence contains a putative signal peptide of 48 amino acids followed by a polypeptide sequence of 962 amino acids. Northern blot analyses revealed a single transcript of approximately 3.5 kb present in all tissues examined but at varying levels. Two affected siblings of Palestinian origin were homozygous for a mutation that causes a His-->Leu replacement at a position which is conserved among class 2 alpha-mannosidases from several species.

Bovine kidney lysosomal α‐mannosidase was purified to homogeneity and the gene was cloned. The gene was organized in 24 exons that spanned 16kb 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 adjactent 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°C transition, resulting in Phe321→Leu substitution, was identified in the cDNA of α‐mannosidosis‐affected Angus cattle. In affected Galloway cattle, a G662→A transition that causes Arg221→His substitution was identified. Phe321 and Arg221 are conserved among the α‐mannosidase class‐2 family, indicating that the substitutions resulted from disease‐causing mutations in these breeds.