Purification of feline lysosomal alpha-mannosidase, determination of its cDNA sequence and identification of a mutation causing alpha-mannosidosis in Persian cats

Biochem J. 1997 Dec 15;328 ( Pt 3)(Pt 3):863-70. doi: 10.1042/bj3280863.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Blotting, Western
  • Cats
  • Cattle
  • Cloning, Molecular
  • Codon
  • DNA Mutational Analysis
  • DNA, Complementary
  • Disease Models, Animal
  • Frameshift Mutation
  • Humans
  • Liver / enzymology
  • Lysosomes / enzymology*
  • Mannosidases / chemistry*
  • Mannosidases / deficiency
  • Mannosidases / genetics*
  • Mannosidases / isolation & purification
  • Molecular Sequence Data
  • Mutation*
  • Protein Conformation
  • Protein Processing, Post-Translational / genetics
  • Sequence Deletion
  • Sequence Homology, Nucleic Acid
  • alpha-Mannosidase
  • alpha-Mannosidosis / enzymology
  • alpha-Mannosidosis / genetics*

Substances

  • Codon
  • DNA, Complementary
  • Mannosidases
  • alpha-Mannosidase

Associated data

  • GENBANK/AF010191
  • GENBANK/AF010192