A difference in the specificities of human liver N-acetyl-beta-hexosaminidases A and B detected by their activities towards glycosaminoglycan oligosaccharides.
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ABSTRACT:
SUBMITTER: Bearpark TM
PROVIDER: S-EPMC1185871 | biostudies-other | 1978 Sep
REPOSITORIES: biostudies-other
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Project description:Human N-acetyl-beta-hexosaminidases As and P are probably sialylated since they are susceptible to neuraminidase attack. twhen infused into the circulation of a rat they are removed more slowly than the non-sialylated forms of the enzyme from tissue and urine.
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Project description:Glycosaminoglycans (GAGs) are a physio- and pharmacologically highly relevant class of complex saccharides, possessing a linear sequence and strongly acidic character. Their repetitive linear core makes them seem structurally simple at first glance, yet differences in sulfation and epimerization lead to an enormous structural diversity with only a few GAGs having been successfully characterized to date. Recent infrared action spectroscopic experiments on sulfated mono- and disaccharide ions show great promise. Here, we assess the potential of two types of gas-phase action spectroscopy approaches in the range from 1000 to 1800 cm-1 for the structural analysis of complex GAG oligosaccharides. Synthetic tetra- and pentasaccharides were chosen as model compounds for this benchmark study. Utilizing infrared multiple photon dissociation action spectroscopy at room temperature, diagnostic bands are largely unresolved. In contrast, cryogenic infrared action spectroscopy of ions trapped in helium nanodroplets yields resolved infrared spectra with diagnostic features for monosaccharide composition and sulfation pattern. The analysis of GAGs could therefore significantly benefit from expanding the conventional MS-based toolkit with gas-phase cryogenic IR spectroscopy. Graphical abstract.
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Project description:We recently discovered that human neutrophils express immunomodulatory glycoproteins carrying unusual and highly truncated paucimannosidic N-glycans (Man1-3GlcNAc2Fuc0-1), but their biosynthesis remains elusive. Guided by the well-characterized truncation pathway in invertebrates and plants in which the N-acetyl-β-D-hexosaminidase (Hex) isoenzymes catalyze paucimannosidic protein (PMP) formation, we here set out to test if the homologous human Hex α and β subunits encoded by HEXA and HEXB drive a similar truncation pathway in human neutrophils. To this end, we performed quantitative glycomics and glycoproteomics of several CRISPR-Cas9-edited Hex-disrupted neutrophil-like HL-60 mutants (HEXA-KO and HEXB-KO) and matching unedited cell lines. Hex disruption was validated using next-generation sequencing, enzyme-linked immunosorbent assay (ELISA), quantitative proteomics and Hex activity assays. Excitingly, all Hex-disrupted mutants displayed significantly reduced levels of paucimannosylation, particularly Man2-3GlcNAc2Fuc1, relative to unedited HL-60 suggesting that both HEXA and HEXB contribute to PMP formation via a hitherto unexplored truncation pathway in neutrophils. Quantitative N-glycomics indeed demonstrated reduced utilization of a putative noncanonical truncation pathway in favor of the canonical elongation pathway in all Hex-disrupted mutants relative to unedited controls. Quantitative glycoproteomics recapitulated the truncation-to-elongation switch in all Hex-disrupted mutants and showed a greater switch for N-glycoproteins cotrafficking with Hex to the azurophilic granules of neutrophils such as myeloperoxidase. Finally, we supported the Hex-PMP relationship by documenting that primary neutrophils isolated from an early-onset Sandhoff disease patient (HEXB-/-) displayed dramatically reduced paucimannosylation relative to neutrophils from an age-matched unaffected donor. We conclude that both human Hex α and β mediate PMP formation via a putative noncanonical truncation pathway in neutrophils.
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Project description:β-N-acetyl-D-hexosaminidases have attracted significant attention due to their crucial role in diverse physiological functions including antibacterial synergists, pathogen defense, virus infection, lysosomal storage, and protein glycosylation. In particular, the GH3 β-N-acetyl-D-hexosaminidase of V. cholerae (VcNagZ), human GH20 β-N-acetyl-D-hexosaminidase B (HsHexB), and human GH84 β-N-acetyl-D-hexosaminidase (hOGA) are three important representative glycosidases. These have been found to be implicated in β-lactam resistance (VcNagZ), lysosomal storage disorders (HsHexB) and Alzheimer's disease (hOGA). Considering the profound effects of these three enzymes, many small molecule inhibitors with good potency and selectivity have been reported to regulate the corresponding physiological functions. In this paper, the best-known inhibitors PUGNAc and two of its derivatives (N-valeryl-PUGNAc and EtBuPUG) were selected as model compounds and docked into the active pockets of VcNagZ, HsHexB, and hOGA, respectively. Subsequently, molecular dynamics simulations of the nine systems were performed to systematically compare their binding modes from active pocket architecture and individual interactions. Furthermore, the binding free energy and free energy decomposition are calculated using the MM/GBSA methods to predict the binding affinities of enzyme-inhibitor systems and to quantitatively analyze the contribution of each residue. The results show that PUGNAc is deeply-buried in the active pockets of all three enzymes, which indicates its potency (but not selectivity) against VcNagZ, HsHexB, and hOGA. However, EtBuPUG, bearing branched 2-isobutamido, adopted strained conformations and was only located in the active pocket of VcNagZ. It has completely moved out of the pocket of HsHexB and lacks interactions with HsHexB. This indicates why the selectivity of EtBuPUG to VcNagZ/HsHexB is the largest, reaching 968-fold. In addition, the contributions of the catalytic residue Asp253 (VcNagZ), Asp254 (VcNagZ), Asp175 (hOGA), and Asp354 (HsHexB) are important to distinguish the activity and selectivity of these inhibitors. The results of this study provide a helpful structural guideline to promote the development of novel and selective inhibitors against specific β-N-acetyl-D-hexosaminidases.
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Project description:Secretions of parasitic worms (helminths) contain a wide collection of immunomodulatory glycoproteins with the potential to treat inflammatory disorders, like autoimmune diseases. Yet, the identification of single molecules that can be developed into novel biopharmaceuticals is hampered by the limited availability of native parasite-derived proteins. Recently, pioneering work has shown that helminth glycoproteins can be produced transiently in Nicotiana benthamiana plants while simultaneously mimicking their native helminth N-glycan composition by co-expression of desired glycosyltransferases. However, efficient "helminthization" of N-glycans in plants by glyco-engineering seems to be hampered by the undesired truncation of complex N-glycans by β-N-acetyl-hexosaminidases, in particular when aiming for the synthesis of N-glycans with antennary GalNAcβ1-4GlcNAc (LacdiNAc or LDN). In this study, we cloned novel β-hexosaminidase open reading frames from N. benthamiana and characterized the biochemical activity of these enzymes. We identified HEXO2 and HEXO3 as enzymes responsible for the cleavage of antennary GalNAc residues of N-glycans on the model helminth glycoprotein kappa-5. Furthermore, we reveal that each member of the HEXO family has a distinct specificity for N-glycan substrates, where HEXO2 has strict β-galactosaminidase activity, whereas HEXO3 cleaves both GlcNAc and GalNAc. The identification of HEXO2 and HEXO3 as major targets for LDN cleavage will enable a targeted genome editing approach to reduce undesired processing of these N-glycans. Effective knockout of these enzymes could allow the production of therapeutically relevant glycoproteins with tailor-made helminth N-glycans in plants.
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Project description:Bovine brain tissue was extracted and the 50 000g supernatant was separated by electrophoresis, DEAE-Sephadex chromatography and gel filtration on Sephadex G-200 and Bio-Gel P-200. The electrophoretic separation showed that the beta-N-acetyl-D-hexosaminidases (hexosaminidases) of bovine brain tissue were composed of four different fractions. Two fractions (A and B) exerted both glucosaminidase and galactosaminidase activity, a third fraction (C) showed only glucosaminidase activity, whereas a fourth form (D) with specificity towards the galactosaminide moiety was found to be present. DEAE-Sephadex chromatography at pH 7.0 showed that the B form was eluted with the void volume, whereas the A and D forms could be eluted in one peak by raising that salt concentration. The C form could not be detected in the eluate. Gel filtration on Sephadex G-200 showed that the B, A and D forms had almost equal molecular weights. In this case also the C form could not be detected in the column eluates. Gel filtration on Bio-Gel P-200 revealed that the C form was eluted with the void volume.
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Project description:Two isoenzymes of N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) (Hex A and Hex B) from human seminal plasma were purified to homogeneity with specific activities of 26 and 60 units/mg of protein respectively. N-Acetyl-beta-D-glucosaminidase activity was inseparable from N-acetyl-beta-D-galactosaminidase activity in both Hex A and Hex B by various conventional chromatographic procedures. Although Km values of N-acetyl-beta-glucosaminidase activity of Hex A and Hex B were similar (1.33 mM), those of N-acetyl-beta-galactosaminidase activity were 0.14 mM for Hex A and 0.40 mM for Hex B. However, pH optima and temperature optima were identical for N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities of both isoenzymes; Hex A was far more heat-sensitive than Hex B. Thiol-reactive compounds such as silver salts, mercuric salts, p-chloromercuribenzoate and thimerosal strongly inhibited the N-acetyl-beta-glucosaminidase activities of both isoenzymes. GSH protected the enzyme activities from inactivation caused by these reagents, confirming the presence of thiol groups at the active centres. Inhibitions of N-acetyl-beta-glucosaminidase activities of both isoenzymes by metal salts and organic anions were comparable; acetate and arsenite were effective inhibitors for both isoenzymes. In contrast, inhibitions of N-acetyl-beta-glucosaminidase activities of the two isoenzymes by iodoacetic acid, iodoacetamide and ethylmaleimide were not comparable; Hex B was more susceptible to inhibition by these agents at 20 mM concentration. The N-acetyl-beta-glucosaminidase activities of both isoenzymes are strongly inhibited, in decreasing order, by N-acetyl-galactosamine, mannosamine, disaccharic acid lactone, N-acetylglucosamine and gluconolactone. The Ki values of the N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities for N-acetylhexosamines and results from mixed-substrate kinetics indicated that the activities for the two substrates are located at different sites in Hex A and at the same site in Hex B. The Mr values of Hex A and Hex B were determined to be 195,000 and 210,000 respectively by gel filtration through Sephadex G-200. SDS/polyacrylamide-gel electrophoresis revealed that Hex A and Hex B are each composed of four subunits corresponding to Mr about 50,000 each. No further polypeptide chain was obtained after reduction and alkylation of Hex A and Hex B with 10 mM-dithiothreitol and 10 mM-iodoacetamide.
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Project description:Yeast is an integral part of mammalian microbiome, and like commensal bacteria, has the potential of being harnessed to influence immunity in clinical settings. However, functional specificities of yeast-derived immunoregulatory molecules remain elusive. Here we find that while under steady state, β-1,3-glucan-containing polysaccharides potentiate pro-inflammatory properties, a relatively less abundant class of cell surface polysaccharides, dubbed mannan/β-1,6-glucan-containing polysaccharides (MGCP), is capable of exerting potent anti-inflammatory effects to the immune system. MGCP, in contrast to previously identified microbial cell surface polysaccharides, through a Dectin1-Cox2 signaling axis in dendritic cells, facilitates regulatory T (Treg) cell induction from naïve T cells. Furthermore, through a TLR2-dependent mechanism, it restrains Th1 differentiation of effector T cells by suppressing IFN-γ expression. As a result, administration of MGCP display robust suppressive capacity towards experimental inflammatory disease models of colitis and experimental autoimmune encephalomyelitis (EAE) in mice, thereby highlighting its potential therapeutic utility against clinically relevant autoimmune diseases.
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