The core-specific lysosomal alpha(1-6)-mannosidase activity depends on aspartamidohydrolase activity.
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ABSTRACT:
SUBMITTER: Haeuw JF
PROVIDER: S-EPMC1137856 | biostudies-other | 1994 Feb
REPOSITORIES: biostudies-other
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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.
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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).
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Project description:The lysosomal α-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 α-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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Project description:Human alpha-mannosidase activity (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) from tissues and cultured skin fibroblasts was separated by gel electrophoresis into a neutral, cytoplasmic form (alpha-mannosidase A) and two closely related acidic, lysosomal components (alpha-mannosidase B). Human mannosidosis, an inherited glycoprotein storage disorder, has been associated with severe deficiency of both lysosomal alpha-mannosidase B molecular forms. Chromosome assignment of the gene coding for human alpha-mannosidase B (MANB) has been determined in human-mouse and human-Chinese hamster somatic cell hybrids. The human alpha-mannosidase B phenotype showed concordant segregation with the human enzyme glucosephosphate isomerase (GPI) (D-glucose-6-phosphate ketolisomerase, EC 5.3.1.9) but discordant segregation with 30 other enzyme markers representing 20 linkage groups. The glucose-phosphate isomerase gene has been assigned to chromosome 19 in man. This MANB-GPI linkage and confirming chromosome studies demonstrate assignment of the alpha-mannosidase B structural gene to chromosome 19 in man. Since mannosidosis is believed to result from a structural defect in alpha-mannosidase B, these findings suggest that the mannosidosis mutation is located on chromosome 19 in man.
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Project description:Extensive mannose trimming of nascent glycoprotein N-glycans signals their targeting to endoplasmic reticulum-associated degradation (ERAD). ER mannosidase I (ERManI) and the EDEM protein family participate in this process. However, whether the EDEMs are truly mannosidases can be addressed only by measuring mannosidase activity in vitro. Here, we reveal EDEM1 and EDEM2 mannosidase activities in vitro. Whereas ERManI significantly trims free N-glycans, activity of the EDEMs is modest on free oligosaccharides and on glycoproteins. However, mannosidase activity of ERManI and the EDEMs is significantly higher on a denatured glycoprotein. The EDEMs associate with oxidoreductases, protein disulfide isomerase, and especially TXNDC11, enhancing mannosidase activity on glycoproteins but not on free N-glycans. The finding that substrate unfolded status increases mannosidase activity solves an important conundrum, as current models suggest general slow mannose trimming. As we show, misfolded or unfolded glycoproteins are subject to differentially faster trimming (and targeting to ERAD) than well-folded ones.
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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.
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Project description:Oligomannose-type glycans on HIV-1 gp120 form a patch that is targeted by several broadly neutralizing antibodies (bnAbs) and that therefore is of interest to vaccine design. However, attempts to elicit similar oligomannose-specific bnAbs by immunizing with oligomannosidic glycoconjugates have only been modestly successful so far. A common assumption is that eliciting oligomannose-specific bnAbs is hindered by B cell tolerance, resulting from the presented oligomannosides being sensed as self molecules. Here, we present data, along with existing scientific evidence, supporting an additional, or perhaps alternate, explanation: serum mannosidase trimming of the presented oligomannosides in vivo. Mannosidase trimming lessens the likelihood of eliciting antibodies with capacity to bind full-sized oligomannose, which typifies the binding mode of existing bnAbs to the oligomannose patch. The rapidity of the observed trimming suggests the need for immunization strategies and/or synthetic glycosides that readily avoid or resist mannosidase trimming upon immunization and can overcome possible tolerance restrictions.
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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.
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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.
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Project description:Class I alpha-mannosidases (glycoside hydrolase family GH47) play key roles in the maturation of N-glycans and the ER-associated degradation of unfolded glycoproteins. The 1.95 A resolution structure of a fungal alpha-1,2-mannosidase in complex with the substrate analogue methyl-alpha-D-lyxopyranosyl-(1',2)-alpha-D-mannopyranoside (LM) shows the intact disaccharide spanning the -1/+1 subsites, with the D-lyxoside ring in the -1 subsite in the 1C4 chair conformation, and provides insight into the mechanism of catalysis. The absence of the C5' hydroxymethyl group on the D-lyxoside moiety results in the side chain of Arg407 adopting two alternative conformations: the minor one interacting with Asp375 and the major one interacting with both the D-lyxoside and the catalytic base Glu409, thus disrupting its function. Chemical modification of Asp375 has previously been shown to inactivate the enzyme. Taken together, the data suggest that Arg407, which belongs to the conserved sequence motif RPExxE, may act to modulate the activity of the enzyme. The proposed mechanism for modulating the activity is potentially a general mechanism for this superfamily.
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