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

Thomas Berg, Ole K. Tollersrud, Steven U. Walkley, Donald Siegel, Øivind Nilssen

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Abstract

α-Mannosidosis is a lysosomal storage disorder that is caused by the deficiency of lysosomal α-mannosidase. Feline α-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 a-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 α-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 α-mannosidases sequences respectively. A 4 bp deletion was identified in an α-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 α-mannosidase activity could be detected in the liver of this cat. A domestic long-haired cat expressing a milder α-mannosidosis phenotype than the Persian cat had a lysosomal α-mannosidase activity of 2% of normal. This domestic long-haired cat did not possess the 4 bp deletion, proving molecular heterogeneity for feline α-mannosidosis.

Original languageEnglish (US)
Pages (from-to)863-870
Number of pages8
JournalBiochemical Journal
Volume328
Issue number3
StatePublished - Dec 15 1997

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Mannosidase Deficiency Diseases
Mannosidases
Felidae
Purification
Cats
Complementary DNA
Mutation
Amino Acids
Peptides
RNA-Directed DNA Polymerase
Reverse Transcriptase Polymerase Chain Reaction
Liver
Amino Acid Sequence
Enzymes
Nonsense Codon
Molecular mass
Fibroblasts
Protein Sorting Signals
DNA Sequence Analysis
Codon

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification of feline lysosomal α-mannosidase, determination of its cDNA sequence and identification of a mutation causing α-mannosidosis in Persian cats. / Berg, Thomas; Tollersrud, Ole K.; Walkley, Steven U.; Siegel, Donald; Nilssen, Øivind.

In: Biochemical Journal, Vol. 328, No. 3, 15.12.1997, p. 863-870.

Research output: Contribution to journalArticle

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