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Partial sequence of the purified protein confirms the identity of cDNA coding for human lysosomal alpha-mannosidase B.

ABSTRACT: Human lysosomal alpha-mannosidase has been purified by a simple and rapid method in sufficient quantities for the analysis of its subunit composition and partial protein sequencing. Analysis of the N-terminal residues of the 30 kDa polypeptide has enabled us to confirm the identity of the recently cloned cDNA that was tentatively identified as that of lysosomal alpha-mannosidase [Nebes and Schmidt (1994) Biochem. Biophys. Res. Commun. 200, 239-245] and to locate the position of this polypeptide within the total deduced amino acid sequence. This finding will therefore provide a firm foundation for the characterization of alpha-mannosidosis mutations.

SUBMITTER: Emiliani C

PROVIDER: S-EPMC1136369 | biostudies-other | 1995 Jan

REPOSITORIES: biostudies-other

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Intracellular transport of human lysosomal alpha-mannosidase and alpha-mannosidosis-related mutants.

Project description:Human LAMAN (lysosomal a-mannosidase) was synthesized as a 120 kDa precursor in transfected COS cells [African-green-monkey kidney cells], which was partly secreted as a single-chain form and partly sorted to the lysosomes being subsequently cleaved into three peptides of 70, 40 and 15 kDa respectively. Both the secreted and the lysosomal forms contained endo H (endoglucosidase H)-resistant glycans, suggesting a common pathway through the trans-Golgi network. A fraction of LAMAN was retained intracellularly as a single-chain endo H-sensitive form, probably in the ER (endoplasmic reticulum). The inherited lack of LAMAN causes the autosomal recessive storage disease a-mannosidosis. To understand the biochemical consequences of the disease-causing mutations, 11 missense mutations and two in-frame deletions were introduced into human LAMAN cDNA by in vitro mutagenesis and the resulting proteins were expressed in COS cells. Some selected mutants were also expressed in Chinese-hamster ovary cells. T355P (Thr355Pro), P356R, W714R, R750W and L809P LAMANs as well as both deletion mutants were misfolded and arrested in the ER as inactive single-chain forms. Six of the mutants were transported to the lysosomes, either with less than 5% of normal specific activity (H72L, D196E/N and R220H LAMANs) or with more than 30% of normal specific activity (E402K LAMAN). F320L LAMAN resulted in much lower activity in Chinese-hamster ovary cells when compared with COS cells. Modelling into the three-dimensional structure revealed that the mutants with highly reduced specific activities contained substitutions of amino acids involved in the catalysis, either co-ordinating Zn2+ (His72 and Asp196), stabilizing the active-site nucleophile (Arg220) or positioning the active-site residue Asp319 (Phe320).

| S-EPMC1133862 | biostudies-literature

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

Project description: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.

| S-EPMC1218998 | biostudies-other

Characterization of a human core-specific lysosomal {alpha}1,6-mannosidase involved in N-glycan catabolism.

Project description:In humans and rodents, the lysosomal catabolism of core Man(3)GlcNAc(2) N-glycan structures is catalyzed by the concerted action of several exoglycosidases, including a broad specificity lysosomal alpha-mannosidase (LysMan), core-specific alpha1,6-mannosidase, beta-mannosidase, and cleavage at the reducing terminus by a di-N-acetylchitobiase. We describe here the first cloning, expression, purification, and characterization of a novel human glycosylhydrolase family 38 alpha-mannosidase with catalytic characteristics similar to those established previously for the core-specific alpha1,6-mannosidase (acidic pH optimum, inhibition by swainsonine and 1,4-dideoxy-1,4-imino-d-mannitol, and stimulation by Co(2+) and Zn(2+)). Substrate specificity studies comparing the novel human alpha-mannosidase with human LysMan revealed that the former enzyme efficiently cleaved only the alpha1-6mannose residue from Man(3)GlcNAc but not Man(3)GlcNAc(2) or other larger high mannose oligosaccharides, indicating a requirement for chitobiase action before alpha1,6-mannosidase activity. In contrast, LysMan cleaved all of the alpha-linked mannose residues from high mannose oligosaccharides except the core alpha1-6mannose residue. alpha1,6-Mannosidase transcripts were ubiquitously expressed in human tissues, and expressed sequence tag searches identified homologous sequences in murine, porcine, and canine databases. No expressed sequence tags were identified for bovine alpha1,6-mannosidase, despite the identification of two sequence homologs in the bovine genome. The lack of conservation in 5'-flanking sequences for the bovine alpha1,6-mannosidase genes may lead to defective transcription similar to transcription defects in the bovine chitobiase gene. These results suggest that the chitobiase and alpha1,6-mannosidase function in tandem for mammalian lysosomal N-glycan catabolism.

| S-EPMC1351102 | biostudies-literature

Characterization of human liver alpha-D-mannosidase purified by affinity chromatography.

Project description:Human liver acidic alpha-D-mannosidase was purified 1400-fold by a relatively short procedure incorporating chromatography on concanavalin A-Sepharose and affinity chromatography on Sepharose 4B-epsilon-aminohexanoylmannosylamine. In contrast with the acidic enzymic activity the neutral alpha-mannosidase did not bind to the concanavalin A-Sepharose so the two types of alpha-mannosidase could be separated at an early stage in the purification. The only significant glycosidase contaminant after affinity chromatography on the mannosylamine ligand was alpha-L-fucosidase, which was selectively removed by affinity chromatography on the corresponding fucosylamine ligand. The final preparation was free of other glycosidase activities. The pI of the purified enzyme was increased from 6.0 to 6.45 on treatment with neuraminidase. Although the pI and the mol.wt. (220 000) suggested that alpha-mannosidase A had been purified selectively, ion-exchange chromatography on DEAE-cellulose indicated that the preparation consisted predominantly of alpha-mannosidase B. This discrepancy is discussed in relation to the basis of the multiple forms of human alpha-mannosidase. The purified enzyme completely removed the alpha-linked non-reducing terminal mannose from a trisaccharide isolated from the urine of a patient with mannosidosis. A comparison of the activity of the pure enzyme towards the natural substrate and synthetic substrates suggests that the same enzymic activity is responsible for hydrolysing all the substrates. These results validate the use of synthetic substrates for determining the mannosidosis genotype. They are also further evidence that mannosidosis is a lysosomal storage disease resulting from a deficiency of acidic alpha-mannosidase.

| S-EPMC1172625 | biostudies-other

Deficiency of alpha-mannosidase in Angus cattle. An inherited lysosomal storage disease.

Project description:A disease of Angus cattle previously known as pseudolipidosis has been shown to be an inherited lysosomal storage disease, in which an oligosaccharide containing mannose and glucosamine is the storage substance. Diseased animals have a near-absolute deficiency of the lysosomal enzyme, alpha-mannosidase, whereas heterozygotes have a partial deficiency of this enzyme. The condition is analogous to the human disease known as mannosidosis.

| S-EPMC1173571 | biostudies-other

The core-specific lysosomal alpha(1-6)-mannosidase activity depends on aspartamidohydrolase activity.

Project description:The substrate specificity of the core-specific rat liver lysosomal alpha(1-6)-mannosidase was investigated using mannosylated oligosaccharides and glycoasparagines. Hydrolysis of Man(alpha 1-6) linkage hydrolysis was demonstrated to follow the action of endoglycosidases, namely aspartyl-N-acetyl-beta-D-glucosaminidase and endo-N-acetyl-beta-D-glucosaminidase. The results are discussed with respect to the nature of the carbohydrate materials stored in the tissues and excreted in the urine from patients suffering from aspartylglucosaminuria and fucosidosis.

| S-EPMC1137856 | biostudies-other

Inhibition of lysosomal alpha-mannosidase by swainsonine, an indolizidine alkaloid isolated from Swainsona canescens.

Project description:An indolizidine alkaloid (swainsonine) was isolated from the plant Swainsona canescens. Swainsonine is a specific and potent inhibitor of alpha-mannosidase (EC 3.2.1.24) and when administered to animals produces a phenocopy of the genetically based lysosomal storage disease, mannosidosis. Evidence is presented to suggest that swainsonine is a reversible active site-directed inhibitor of lysosomal alpha-mannosidase.

| S-EPMC1162258 | biostudies-other

Expression, purification and preliminary crystallographic analysis of Drosophila melanogaster lysosomal ?-mannosidase.

Project description:The lysosomal ?-mannosidases are class II mannosidases that belong to glycoside hydrolase family 38 and play an important role in the degradation of asparagine-linked carbohydrates of glycoproteins. Based on peptide similarity to human and bovine lysosomal mannosidase (LM), recombinant ?-mannosidase from Drosophila melanogaster (dLM408) was cloned and heterologously expressed in Pichia pastoris. The recombinant form of dLM408 designed for structural analysis lacks the transmembrane domain and was crystallized using standard vapour-diffusion and counter-diffusion techniques. The crystals grew as flat plates and as tetragonal bipyramids, respectively. The plate-shaped crystals exhibited the symmetry of space group P2(1)2(1)2(1) and diffracted to a minimum d-spacing of 3.5?Å.

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cDNA sequence analysis confirms that the etiologic agent of callitrichid hepatitis is lymphocytic choriomeningitis virus.

Project description:Callitrichid hepatitis is an infection of New World primates caused by an arenavirus, currently referred to as callitrichid hepatitis virus, that is closely related to lymphocytic choriomeningitis virus (LCMV). We have cloned and sequenced the GP-C gene of callitrichid hepatitis virus and found that the cDNA sequence is 84 to 86% identical to those of the GP-C genes of LCMV strains Armstrong and WE, while the deduced amino acid sequence is 95 to 96% identical to those of the GP-C gene products of the same strains. This high degree of similarity indicates that the etiologic agent of callitrichid hepatitis is in fact LCMV. The wide geographic distribution of callitrichid hepatitis outbreaks in the United States serves as a reminder that LCMV is also a human pathogen whose public health implications are not well understood.

| S-EPMC188719 | biostudies-other

Essential and mutually compensatory roles of {alpha}-mannosidase II and {alpha}-mannosidase IIx in N-glycan processing in vivo in mice.

Project description:Many proteins synthesized through the secretory pathway receive posttranslational modifications, including N-glycosylation. alpha-Mannosidase II (MII) is a key enzyme converting precursor high-mannose-type N-glycans to matured complex-type structures. Previous studies showed that MII-null mice synthesize complex-type N-glycans, indicating the presence of an alternative pathway. Because alpha-mannosidase IIx (MX) is a candidate enzyme for this pathway, we asked whether MX functions in N-glycan processing by generating MII/MX double-null mice. Some double-nulls died between embryonic days 15.5 and 18.5, but most survived until shortly after birth and died of respiratory failure, which represents a more severe phenotype than that seen in single-nulls for either gene. Structural analysis of N-glycans revealed that double-nulls completely lack complex-type N-glycans, demonstrating a critical role for at least one of these enzymes for effective N-glycan processing. Recombinant mouse MX and MII showed identical substrate specificities toward N-glycan substrates, suggesting that MX is an isozyme of MII. Thus, either MII or MX can biochemically compensate for the deficiency of the other in vivo, and either of two is required for late embryonic and early postnatal development.

| S-EPMC1474017 | biostudies-literature

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