Human Α-mannosidase Research Articles

Synthesis, α-mannosidase inhibition studies and molecular modeling of 1,4-imino-ᴅ-lyxitols and their C-5-altered N-arylalkyl derivatives.

A synthesis of 1,4-imino-ᴅ-lyxitols and their N-arylalkyl derivatives altered at C-5 is reported. Their inhibitory activity and selectivity toward four GH38 α-mannosidases (two Golgi types: GMIIb from Drosophila melanogaster and AMAN-2 from Caenorhabditis elegans, and two lysosomal types: LManII from Drosophila melanogaster and JBMan from Canavalia ensiformis) were investigated. 6-Deoxy-DIM was found to be the most potent inhibitor of AMAN-2 (K i = 0.19 μM), whose amino acid sequence and 3D structure of the active site are almost identical to the human α-mannosidase II (GMII). Although 6-deoxy-DIM was 3.5 times more potent toward AMAN-2 than DIM, their selectivity profiles were almost the same. N-Arylalkylation of 6-deoxy-DIM resulted only in a partial improvement as the selectivity was enhanced at the expense of potency. Structural and physicochemical properties of the corresponding inhibitor:enzyme complexes were analyzed by molecular modeling.

Manno-epi-cyclophellitols Enable Activity-Based Protein Profiling of Human α-Mannosidases and Discovery of New Golgi Mannosidase II Inhibitors.

Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAcMan5GlcNAc2 to produce GlcNAcMan3GlcNAc2, the precursor for all complex N-glycans, including the branched N-glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms. Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitol aziridine derivatives enabled activity-based protein profiling of α-mannosidases from both human cell lysate and mouse tissue extracts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.

Traffic of Human α-Mannosidase in Plant Cells Suggests the Presence of a New Endoplasmic Reticulum-to-Vacuole Pathway without Involving the Golgi Complex

The transport of secretory proteins from the endoplasmic reticulum to the vacuole requires sorting signals as well as specific transport mechanisms. This work is focused on the transport in transgenic tobacco (Nicotiana tabacum) plants of a human α-mannosidase, MAN2B1, which is a lysosomal enzyme involved in the turnover of N-linked glycoproteins and can be used in enzyme replacement therapy. Although ubiquitously expressed, α-mannosidases are targeted to lysosomes or vacuoles through different mechanisms according to the organisms in which these proteins are produced. In tobacco cells, MAN2B1 reaches the vacuole even in the absence of mannose-6-phosphate receptors, which are responsible for its transport in animal cells. We report that MAN2B1 is targeted to the vacuole without passing through the Golgi complex. In addition, a vacuolar targeting signal that is recognized in plant cells is located in the MAN2B1 amino-terminal region. Indeed, when this amino-terminal domain is removed, the protein is retained in the endoplasmic reticulum. Moreover, when this domain is added to a plant-secreted protein, the resulting fusion protein is partially redirected to the vacuole. These results strongly suggest the existence in plants of a new type of vacuolar traffic that can be used by leaf cells to transport vacuolar proteins.

Enzyme replacement therapy: Production of human α-mannosidase in transgenic tobacco plants

Enzyme replacement therapy: Production of human α-mannosidase in transgenic tobacco plants

Impaired Lysosomal Trimming of N-Linked Oligosaccharides Leads to Hyperglycosylation of Native Lysosomal Proteins in Mice with α-Mannosidosis

Alpha-mannosidosis is caused by the genetic defect of the lysosomal alpha-d-mannosidase (LAMAN), which is involved in the breakdown of free alpha-linked mannose-containing oligosaccharides originating from glycoproteins with N-linked glycans, and thus manifests itself in an extensive storage of mannose-containing oligosaccharides. Here we demonstrate in a model of mice with alpha-mannosidosis that native lysosomal proteins exhibit elongated N-linked oligosaccharides as shown by two-dimensional difference gel electrophoresis, deglycosylation assays, and mass spectrometry. The analysis of cathepsin B-derived oligosaccharides revealed a hypermannosylation of glycoproteins in mice with alpha-mannosidosis as indicated by the predominance of extended Man3GlcNAc2 oligosaccharides. Treatment with recombinant human alpha-mannosidase partially corrected the hyperglycosylation of lysosomal proteins in vivo and in vitro. These data clearly demonstrate that LAMAN is involved not only in the lysosomal catabolism of free oligosaccharides but also in the trimming of asparagine-linked oligosaccharides on native lysosomal proteins.

Unusual N-Glycan Structures in α-Mannosidase II/IIx Double Null Embryos Identified by a Systematic Glycomics Approach Based on Two-dimensional LC Mapping and Matrix-dependent Selective Fragmentation Method in MALDI-TOF/TOF Mass Spectrometry

alpha-Mannosidase IIx (MX) is an enzyme closely related to alpha-mannosidase II (MII), a key enzyme in N-glycan biosynthesis that catalyzes the first step in conversion of hybrid- to complex-type N-glycans in Golgi apparatus. Recently we generated MII/MX double knock-out mice and found that double nulls completely lack the complex-type N-glycans (Akama, T. O., Nakagawa, H., Wong, N. K., Sutton-Smith, M., Dell, A., Morris, H. R., Nakayama, J., Nishimura, S.-I., Pai, A., Moremen, K. W., Marth, J. D., and Fukuda, M. N. (2006) Essential and mutually compensatory roles of alpha-mannosidase II and alpha-mannosidase IIx in N-glycan processing in vivo in mice. Proc. Natl. Acad. Sci. U. S. A. 103, 8983-8988). In the present study, we determined minor but unusual N-glycan structures found in MII/MX double knock-out mice. We identified such N-glycans by a systematic glycomics approach applying a two-dimensional LC mapping database and matrix-dependent selective fragmentation technique in MALDI-TOF/TOF MS, a highly sensitive and reliable technique that provides specific fragmentations enabling the determination of precise oligosaccharide structures including regioisomers (Kurogochi, M., and Nishimura, S.-I. (2004) Structural characterization of N-glycopeptides by matrix-dependent selective fragmentation of MALDI-TOF/TOF tandem mass spectrometry. Anal. Chem. 76, 6097-6101). Quantitative profiling of all N-glycan structures including minor components from MII/MX nulls, MII nulls, MX nulls, and wild-type mice at embryonic day 15.5 yielded a total of 37 species when structural heterogeneity was reduced by the removal of the sialic acids. Among six unusual N-glycan structures, two glycoforms were novel and were found only in MII/MX double nulls. We characterize such structure as pseudocomplex-type N-glycans. The present study demonstrated that use of the versatile matrix-dependent selective fragmentation method in MALDI-TOF/TOF MS greatly accelerates detailed structural analysis of a trace amount of N-glycans.

Characterization of a Human Core-specific Lysosomal α1,6-Mannosidase Involved in N-Glycan Catabolism

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.

Over-expression of human lysosomal α-mannosidase in mouse embryonic stem cells

α-Mannosidosis is a lysosomal storage disorder characterised by the lysosomal accumulation of mannose-containing oligosaccharides and a range of pathological consequences, caused by a deficiency of the lysosomal enzyme α-mannosidase. One of the major features of α-mannosidosis is progressive neurological decline, for which there is no safe and effective treatment. Implantation of stem cells into the central nervous system has been proposed as a potential therapy for these disorders. We report the construction and characterisation of mouse embryonic stem cell lines for the sustained over-expression of recombinant human lysosomal α-mannosidase (rhαM). Two vectors (involving recombinant human α-mannosidase expression driven by either the chicken β-actin promoter/CMV enhancer or by the elongation factor 1-α promoter) were constructed and used to transfect mouse D3 embryonic stem cells. Selected clonal cell lines were isolated and tested to evaluate their expression of recombinant human α-mannosidase. Stem cell clones transfected with the chicken β-actin promoter/CMV enhancer maintained rhαM expression levels throughout differentiation. This expression was not markedly elevated above background. In contrast, the vector incorporating the elongation factor 1-α promoter facilitated substantial over-expression of α-mannosidase when analysed out to 21 days of differentiation in stably transfected cell lines. The highest expressing cell line was found to qualitatively retain a similar differentiation potential to untransfected cells, and to secrete α-mannosidase that could mediate a reduction in the level of oligosaccharides stored by human α-mannosidosis skin fibroblasts. These results suggest potential for the use of this cell line for investigation of a stem cell therapy approach to treat α-mannosidosis.

Two novel mutations in the gene for human alpha-mannosidase that cause alpha-mannosidosis.

Mutation analysis performed on two Italian patients with alpha-mannosidosis allowed the identification of two new mutations, IVS20-2A>G and 322-323insA. The patients were both homozygous for these mutations. The first mutation causes skipping of exon 21, whereas the second causes a frameshift introducing a stop codon at position 160 of the amino acid sequence.

Expression of Human Lysosomal Alpha-Mannosidase Activity in Transfected Murine Cells and Human Alpha-Mannosidase Deficient Fibroblasts

We studied the human lysosomal alpha-mannosidase (MANB) by expressing the putative cDNA in mammalian cells, using the eucaryotic expression vector pCDE. The construct pCDE-MANB and pSV2-Neo were cotransfected into human α-mannosidase deficient fibroblasts and into a murine cell line and selected by culture in the presence of G418. Six G418 resistant 3T3 clones had increased α-mannosidase activity 2 to 3 times above the controls. Two clones from transfected human fibroblasts showed a 2 fold increase in enzyme activity. The human MANB cDNA gene was demonstrated in the target cells by Southern blot analysis and the expression of the gene was shown by RT-PCR analysis. This study is the first to successfully express the MANB gene in a human and a murine cell line. The results confirm that the putative MANB cDNA encodes the full length of lysosomal α-mannosidase. Molecular characterization of mannosidosis and approaches to gene therapy are now possible using this cDNA.

Structure-activity relationship of swainsonine. Inhibition of human α-mannosidases by swainsonine analogues

The inhibitory properties of a series of synthetic epimers and analogues of swainsonine towards the multiple forms of human alpha-mannosidases were studied in vitro and in cells in culture. Of the five epimers tested, only the 8a-epimer and 8,8a-diepimer of swainsonine were specific and competitive inhibitors (Ki values of 7.5 x 10(-5) and 2 x 10(-6) M respectively) of lysosomal alpha-mannosidases in vitro and induced storage of mannose-rich oligosaccharides in human fibroblasts in culture. The structures of these storage products indicated that processing alpha-mannosidases had also been inhibited. This was consistent with the observed inhibition in vitro of these enzymes by these compounds. In contrast, the 8-epimer, 1,8-diepimer and 2,8a-diepimer of swainsonine had no appreciable effect on any alpha-mannosidases. The corresponding open-chain analogues of swainsonine, namely 1,4-dideoxy-1,4-imino-D-mannitol, of the 8a-epimer, namely 1,4-dideoxy-1,4-imino-D-talitol, and of the 8,8a-diepimer, namely 1,4-dideoxy-1,4-imino-L-allitol, were weaker competitive inhibitors of lysosomal alpha-mannosidase, with Ki values of 1.3 x 10(-5), 1.2 x 10(-4) and 1.2 x 10(-4) M respectively. These analogues also proved less effective at inducing oligosaccharide accumulation and in disturbing glycoprotein processing. These compounds offer the opportunity to determine which alterations in the chirality of the swainsonine molecule affect its inhibitory specificity. A comparison of their biological activities has identified reagents that will be useful for studying steps in the biosynthesis and catabolism of glycoproteins and that may be of potential value in chemotherapy.

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

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.

Specificity studies on α-mannosidases using oligosaccharides from mannosidosis urine as substrates

Oligosaccharides containing terminal non-reducing α(1 → 2)-, α(1 → 3)-, and α(1 → 6)-linked mannose residues, isolated from human and bovine mannosidosis urines were used as substrates to test the specificities of acidic α-mannosidases isolated from human and bovine liver. The enzymes released all the α-linked mannose residues from each oligosaccharide and were most effective on the smallest substrate. Enzyme A in each case was less active on the oligosaccharides than α-mannosidase B 2, even though the apparent K m value for the substrates was the same with each enzyme. The human acidic α-mannosidases were also found to be more active on substrates isolated from human rather than bovine mannosidosis urine. Human α-mannosidase C, which has a neutral pH optimum when assayed with a synthetic substrate, did not hydrolyse any of the oligosaccharides at neutral pH, but was found to be active at an acidic pH.

The relationship between different forms of human α-mannosidase

The tissue distribution and some properties of human α-mannosidase (α- d-mannoside mannohydrolase EC 3.2.1.24) have been studied. The acidic forms of the enzyme were fairly stable, whereas the neutral forms easily lost enzymic activity. The acidic forms were sensitive to neuraminidase but the neutral forms were unaffected. The experiments indicate that the acidic components are closely related to each other, differing only in sialic acid content and possibly conformation. The neutral forms of the enzyme are probably quite different from the acidic forms both in structure and cellular function.

Electrophoretic heterogeneity of human α-mannosidase

Electrophoresis on cellulose acetate of human tissue extracts demonstrated the presence of three bands of α-mannosidase (α- d-mannoside mannohydrolase, EC 3.2.1.24) activity. One band stained at a neutral pH, the two others at an acid pH. The pattern of acid α-mannosidase was different in various tissues.