Berg T, Tollersrud O K, Walkley S U, Siegel D, Nilssen O
Department of Medical Genetics, Institute of Clinical Medicine, University of Tromso, 9037 Tromso, Norway.
Biochem J. 1997 Dec 15;328 ( Pt 3)(Pt 3):863-70. doi: 10.1042/bj3280863.
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.
α-甘露糖苷贮积症是一种溶酶体贮积病，由溶酶体α-甘露糖苷酶缺乏引起。猫α-甘露糖苷贮积症是一种特征明确的动物模型，用于研究溶酶体贮积病的病理和治疗方面。我们在此报告猫肝脏溶酶体α-甘露糖苷酶的纯化及其cDNA序列的测定。活性酶由三种多肽组成，分子量分别为72、41和12 kDa，通过非共价力结合。猫溶酶体α-甘露糖苷酶的cDNA序列是从皮肤成纤维细胞mRNA获得的逆转录酶PCR产物中确定的。推导的氨基酸序列包含72 kDa和41 kDa肽段的N端序列。这表明该酶作为单链前体合成，具有一个50个氨基酸的推定信号肽，其后是一个957个氨基酸的多肽链，该多肽链被切割成成熟酶的三种多肽。推导的氨基酸序列与人类和牛溶酶体α-甘露糖苷酶序列的同一性分别为81.1%和83.2%。通过对逆转录酶PCR产物进行DNA测序，在一只受α-甘露糖苷贮积症影响的波斯猫中鉴定出一个4 bp的缺失。该缺失导致从密码子583处发生移码，并在密码子645处提前终止。在这只猫的肝脏中未检测到溶酶体α-甘露糖苷酶活性。一只表现出比波斯猫更温和的α-甘露糖苷贮积症表型的家养长毛猫，其溶酶体α-甘露糖苷酶活性为正常水平的2%。这只家养长毛猫没有4 bp的缺失，证明了猫α-甘露糖苷贮积症存在分子异质性。
