title: Purification of feline lysosomal alpha-mannosidase, determination of its cDNA sequence and identification of a mutation causing alpha-mannosidosis in Persian cats.
abstract: alpha-Mannosidosis is a lysosomal storage disorder that is caused by the deficiency of lysosomal alpha-mannosidase. Feline alpha-mannosidosis is a well-characterized animal model used for studying pathological and therapeutic aspects of lysosomal storage disorders. We here report the purification of feline liver lysosomal alpha-mannosidase and determination of its cDNA sequence. The active enzyme consisted of three polypeptides, with molecular masses of 72, 41 and 12 kDa, joined by non-covalent forces. The cDNA sequence of feline lysosomal alpha-mannosidase was determined from reverse transcriptase PCR products obtained from skin fibroblast mRNA. The deduced amino acid sequence contained the N-terminal sequences of the 72 and 41 kDa peptides. This indicated that the enzyme is synthesized as a single-chain precursor with a putative signal peptide of 50 amino acids followed by a polypeptide chain of 957 amino acids, which is cleaved into the three polypeptides of the mature enzyme. The deduced amino acid sequence was 81.1 and 83.2% identical with the human and bovine lysosomal alpha-mannosidases sequences respectively. A 4 bp deletion was identified in an alpha-mannosidosis-affected Persian cat by DNA sequencing of reverse transcriptase PCR products. The deletion resulted in a frame shift from codon 583 and premature termination at codon 645. No lysosomal alpha-mannosidase activity could be detected in the liver of this cat. A domestic long-haired cat expressing a milder alpha-mannosidosis phenotype than the Persian cat had a lysosomal alpha-mannosidase activity of 2% of normal. This domestic long-haired cat did not possess the 4 bp deletion, proving molecular heterogeneity for feline alpha-mannosidosis.
authors.affiliation: Department of Medical Genetics, Institute of Clinical Medicine, University of Tromso, 9037 Tromso, Norway.
authors.forename: T
authors.initials: T
authors.lastname: Berg
authors.forename: O K
authors.initials: OK
authors.lastname: Tollersrud
authors.forename: S U
authors.initials: SU
authors.lastname: Walkley
authors.forename: D
authors.initials: D
authors.lastname: Siegel
authors.forename: O
authors.initials: O
authors.lastname: Nilssen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002415:Cats; D002417:Cattle; D003001:Cloning, Molecular; D003062:Codon; D004252:DNA Mutational Analysis; D018076:DNA, Complementary; D004195:Disease Models, Animal; D016368:Frameshift Mutation; D006801:Humans; D008099:Liver / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology*; D008361:Mannosidases / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational / Q000235:genetics; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D043323:alpha-Mannosidase; D008363:alpha-Mannosidosis / Q000201:enzymology / Q000235:genetics*
chemical_list: D003062:Codon; D018076:DNA, Complementary; D008361:Mannosidases; D043323:alpha-Mannosidase