N-acetylglucosamine Oligomers Research Articles

Proteome Analysis of Serum Purified Using Solanum tuberosum and Lycopersicon esculentum Lectins.

Serum and plasma exhibit a broad dynamic range of protein concentrations, posing challenges for proteome analysis. Various technologies have been developed to reduce this complexity, including high-abundance depletion methods utilizing antibody columns, extracellular vesicle enrichment techniques, and trace protein enrichment using nanobead cocktails. Here, we employed lectins to address this, thereby extending the scope of biomarker discovery in serum or plasma using a novel approach. We enriched serum proteins using 37 different lectins and subjected them to LC-MS/MS analysis with data-independent acquisition. Solanum tuberosum lectin (STL) and Lycopersicon esculentum lectin (LEL) enabled the detection of more serum proteins than the other lectins. STL and LEL bind to N-acetylglucosamine oligomers, emphasizing the significance of capturing these oligomer-binding proteins when analyzing serum trace proteins. Combining STL and LEL proved more effective than using them separately, allowing us to identify over 3000 proteins from serum through single-shot proteome analysis. We applied the STL/LEL trace-protein enrichment method to the sera of systemic lupus erythematosus model mice. This revealed differences in >1300 proteins between the systemic lupus erythematosus model and control mouse sera, underscoring the utility of this method for biomarker discovery.

Crystal structures of human immune protein FIBCD1 suggest an extended binding site compatible with recognition of pathogen-associated carbohydrate motifs

Fibrinogen C-domain containing-1 (FIBCD1) is an immune protein proposed to be involved in host recognition of chitin on the surface of pathogens. As FIBCD1 readily binds to acetylated molecules, we have determined the high-resolution crystal structures of a recombinant fragment of the FIBCD1 C-terminal domain complexed with small N-acetyl-containing ligands to determine the mode of recognition. All ligands bind at the conserved N-acetyl binding site (S1) with galactose and glucose-derived ligands rotated 180⁰ with respect to each other. One subunit of a native structure derived from protein expressed in mammalian CHO cells binds glycosylation from a neighbouring subunit, in an extended binding site. Across the various structures, the primary S1 binding pocket is occupied by N-acetyl-containing ligands or acetate, with N-acetyl, acetate or a sulfate ion in an adjacent pocket S1(2). Inhibition binding studies of N-acetylglucosamine oligomers, (GlcNAc)n, n=1,2,3,5,11, via ELISA along with Microscale Thermophoresis affinity assays indicate a strong preference of FIBCD1 for longer N-acetylchitooligosaccharides. Binding studies of mutant H396A, located beyond the S1(2) site, showed no significant difference to wildtype but K381L, within the S1(2) pocket, blocked binding to the model ligand acetylated bovine serum albumin suggesting that this pocket may have functional importance in FIBCD1 ligand binding. The binding studies, alongside structural definition of diverse N-acetyl monosaccharide binding in the primary S1 pocket and of additional, adjacent binding pockets, able to accommodate both carbohydrate and sulfate functional groups, suggests a versatility in FIBCD1 to recognise chitin oligomers and other pathogen-associated carbohydrate motifs across an extended surface.

Enhancement of microalgal biomass, lipid production and biodiesel characteristics by mixotrophic cultivation using enzymatically hydrolyzed chitin waste

There is a growing interest for the mixotrophic cultivation of microalgae using sustainable natural carbon sources. Chitin from shrimp waste was hydrolyzed using chitinase from Trichoderma asperellum and was exploited for the mixotrophic cultivation of microalgae. Chitin saccharification was optimized using Box-Behnken design to produce 50.41% (w/w) of reducing sugars using chitin, 0.83% (w/v), chitinase, 2.92 (v/v), temperature, 40 °C and time, 4.48 h. The biomass productivity of Tetradesmus obliquus and Aphanocapsa sp. was promoted to 39.1 and 40.57 mg L−1 day−1 in the presence of 0.5 g L−1 of N-acetylglucosamine oligomers (GlcNAc), which was estimated to be ∼1.9 and ∼2-folds higher than the autotrophic conditions, respectively. Modified Logistic kinetic model confirmed higher growth rates and shortened lag-time of the mixotrophic cultures. The lipid productivity of both microalgae species was >1.5-times higher than the autotrophic conditions. Nitrate removal from the culture medium was found to be an effective strategy to increase both lipid content and lipid productivity for T. obliquus, but this strategy was not effective for Aphanocapsa sp. Fatty acid analysis revealed an increase of saturated fatty acids under mixotrophic cultivation for both microalgae species. Additionally, several biodiesel evaluation parameters indicated superior characteristics for the mixotrophic culture compared to the autotrophic conditions. Accordingly, mixotrophic growth of microalgae using GlcNAc as a new, sustainable carbon and nitrogen source opens a new avenue for biofuel production with an effective waste conversion into valuable commodities.

Kinetic Characterization of a Putatively Chitin-Active LPMO Reveals a Preference for Soluble Substrates and Absence of Monooxygenase Activity.

Enzymes known as lytic polysaccharide monooxygenases (LPMOs) are recognized as important contributors to aerobic enzymatic degradation of recalcitrant polysaccharides such as chitin and cellulose. LPMOs are remarkably abundant in nature, with some fungal species possessing more than 50 LPMO genes, and the biological implications of this diversity remain enigmatic. For example, chitin-active LPMOs have been encountered in biological niches where chitin conversion does not seem to take place. We have carried out an in-depth kinetic characterization of a putatively chitin-active LPMO from Aspergillus fumigatus (AfAA11B), which, as we show here, has multiple unusual properties, such as a low redox potential and high oxidase activity. Furthermore, AfAA11B is hardly active on chitin, while being very active on soluble oligomers of N-acetylglucosamine. In the presence of chitotetraose, the enzyme can withstand considerable amounts of H2O2, which it uses to efficiently and stoichiometrically convert this substrate. The unique properties of AfAA11B allowed experiments showing that it is a strict peroxygenase and does not catalyze a monooxygenase reaction. This study shows that nature uses LPMOs for breaking glycosidic bonds in non-polymeric substrates in reactions that depend on H2O2. The quest for the true substrates of these enzymes, possibly carbohydrates in the cell wall of the fungus or its competitors, will be of major interest.

Characterization of the glycans involved in sea urchin Paracentrotus lividus reversible adhesion

Sea urchins have hundreds of specialized adhesive organs, the tube feet, which play a key role in locomotion, substrate attachment and food capture. Tube feet are composed by two functional units: a proximal cylindrical stem that is mobile and flexible, attached to a distal flattened disc that produces adhesive secretions. Oral tube feet discs possess a specialized duo-glandular epidermis that produces adhesive and de-adhesive secretions, enabling strong but reversible adhesion to the substrate. Due to the growing interest in biomimetic adhesives, several studies have been carried out to characterize sea urchin adhesives, and up to date, it has been shown that it is composed by proteins and glycans. The protein fraction has been the subject of several studies, that pin-pointed several adhesion-related candidates. Contrastingly, little is known about the glycans that compose sea urchin adhesives. This study aims at contributing to this topic by focusing on the characterization of the glycosidic fraction of the adhesive secreted by the sea urchin Paracentrotus lividus (Lamarck, 1816), using a battery of 22 lectins, applied to 3 complementary techniques. Our results show that five lectins label exclusively the disc adhesive epidermis and simultaneously the secreted adhesive, being, therefore, most likely relevant for sea urchin adhesion. In addition, it was possible to determine that the glycosidic fraction of the adhesive is composed by a high molecular weight glycoprotein containing N-acetylglucosamine oligomers.

Characterization of a novel exo-chitosanase, an exo-chitobiohydrolase, from Gongronella butleri

An exo-chitosanase was purified from the culture filtrate of Gongronella butleri NBRC105989 to homogeneity by ammonium sulfate precipitation, followed by column chromatography using CM-Sephadex C-50 and Sephadex G-100. The enzyme comprised a monomeric protein with a molecular weight of approximately 47,000 according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited optimum activity at pH 4.0, and was stable between pH 5.0 and 11.0. It was most active at 45°C, but was stable at temperatures below 30°C. The enzyme hydrolyzed soluble chitosan and glucosamine (GlcN) oligomers larger than tetramers, but did not hydrolyze N-acetylglucosamine (GlcNAc) oligomers. To clarify the mode of action of the enzyme, we used thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) to investigate the products resulting from the enzyme-catalyzed hydrolysis of chitosan and N1-acetylchitohexaose [(GlcN)5-GlcNAc] with a GlcNAc residue at the reducing end. The results indicated that the enzyme is a novel exo-type chitosanase, exo-chitobiohydrolase, that releases (GlcN)2 from the non-reducing ends of chitosan molecules. Analyses of the hydrolysis products of partially N-acetylated chitooligosaccharides revealed that the enzyme cleaves both GlcN-GlcNAc and GlcNAc-GlcN bonds in addition to GlcN-GlcN bonds in the substrate.

Mode of action and specificity of a chitinase from unicellular microalgae, Euglena gracilis.

Euglena gracilis is a unicellular microalga showing characteristics of both plants and animals, and extensively used as a model organism in the research works of biochemistry and molecular biology. Biotechnological applications of E. gracilis have been conducted for production of numerous important compounds. However, chitin-mediated defense system intensively studied in higher plants remains to be investigated in this microalga. Recently, Taira et al. (Biosci Biotechnol Biochem 82:1090-1100, 2018) isolated a unique chitinase gene, comprising two catalytic domains almost homologous to each other (Cat1 and Cat2) and two chitin-binding domains (CBD1 and CBD2), from E. gracilis. We herein examined the mode of action and the specificity of the recombinant Cat2 by size exclusion chromatography and NMR spectroscopy. Both Cat1 and Cat2 appeared to act toward chitin substrate with non-processive/endo-splitting mode, recognizing two contiguous N-acetylglucosamine units at subsites - 2 and - 1. This is the first report on a chitinase having two endo-splitting catalytic domains. A cooperative action of two different endo-splitting domains may be advantageous for defensive action of the E. gracilis chitinase. The unicellular alga, E. gracilis, produces a chitinase consisting of two GH18 catalytic domains (Cat1 and Cat2) and two CBM18 chitin-binding domains (CBD1 and CBD2). Here, we produced a recombinant protein of the Cat2 domain to examine its mode of action as well as specificity. Cat2 hydrolyzed N-acetylglucosamine (A) oligomers (An, n = 4, 5, and 6) and partially N-acetylated chitosans with a non-processive/endo-splitting mode of action. NMR analysis of the product mixture from the enzymatic digestion of chitosan revealed that the reducing ends were exclusively A-unit, and the nearest neighbors of the reducing ends were mostly A-unit but not exclusively. Both A-unit and D-unit were found at the non-reducing ends and the nearest neighbors. These results indicated strong and absolute specificities for subsites - 2 and - 1, respectively, and no preference for A-unit at subsites + 1 and + 2. The same results were obtained from sugar sequence analysis of the individual enzymatic products from the chitosans. The subsite specificities of Cat2 are similar to those of GH18 human chitotriosidase, but differ from those of plant GH18 chitinases. Since the structures of Cat1 and Cat2 resemble to each other (99% similarity in amino acid sequences), Cat1 may hydrolyze the substrate with the same mode of action. Thus, the E. gracilis chitinase appears to act toward chitin polysaccharide chain through a cooperative action of the two endo-splitting catalytic domains, recognizing two contiguous A-units at subsites - 2 and - 1.

Identification of a maize (Zea mays) chitinase allele sequence suitable for a role in ear rot fungal resistance

Chitinases are thought to play a role in plant resistance to pathogens, but the extent of this role is unknown. The gene for a maize (Zea mays) chitinase, “chitinase 2”, previously reported to be induced by two ear rot pathogens in one maize inbred, was cloned from mRNA isolated from milk stage kernels of several different inbreds reported to be susceptible or resistant to ear rot pathogens. The chitinase gene sequence of some of the susceptible inbreds had frame shifts which would result in a nonfunctional protein. Other susceptible inbreds, and a few resistant inbreds, were missing regions that would contribute to chitin binding. A putative functional clone from a resistant inbred was expressed in yeast, and produced a protein with chitinase activity against different oligomers of N-acetyl glucosamine and modified chitin. The yeast-produced chitinase was also resistant to degradation by proteases from maize ear rot fungi, and enhanced antifungal activity of miconazole towards Fusarium graminearum. When introduced in maize callus transgenically, the callus expressing the chitinase 2 gene had significantly less growth of the ear rot pathogen, F. graminearium, than callus which did not express the gene. This information suggests susceptibility and resistance to ear rot pathogens is influenced not only by expression levels, but also by the sequence of putative resistance genes. Previous reports of increased expression of putative resistance genes need to be tempered with the realization that they may play no role in resistance if the genes code for nonfunctional or reduced function proteins.

Abstract W P106: Pericyte Protection by Edaravone After Tpa Treatment in Rat Cerebral Ischemia

i) Background & Objectives: Pericytes play a pivotal role in contraction, mediating inflammation, and regulation of blood flow in the brain. In the present study, changes of pericytes in the neurovascular unit (NVU) were examined in relation to the effects of exogenous tissue plasminogen activator (tPA) and a free radical scavenger, edaravone. ii) Methods: Immunohistochemistry and Western blot analyses showed that the overlap between PDGFRβ-positive pericytes and N-acetylglucosamine oligomers (NAGO)-positive endothelial cells increased significantly at 4 days after 90 min of transient middle cerebral artery occlusion (tMCAO). iii) Results: The number of pericytes and the overlap with NAGO decreased with tPA, but recovered with edaravone 4 days after tMCAO with proliferation. Thus, tPA treatment damaged pericytes resulting in the detachment from astrocytes and a decrease in GDNF secretion. However, treatment with edaravone greatly improved tPA-induced damage to pericytes. iv) Conclusion: The present study demonstrates that exogenous tPA strongly damages pericytes and destroys the integrity of the NVU, but edaravone treatment can extremely ameliorate such damage after acute cerebral ischemia in rats.

In vitro antitumor activity of heterochitooligosaccharides (a review)

This review presents an analysis of the literature data over the last decade in order to reveal the relationship between the structure and composition of chitin/chitosan oligomers and their antitumor activity. Oligosaccharides consisting of N-acetylglucosamine and/or glucosamine units connected to each other by β-1,4-O-glycoside bond are generally called chitooligosaccharides. Homochitooligosaccharides are the oligomers of N-acetylglucosamine (AGA) or glucosamine (GA). Heterooligosaccharides are a mixture of various oligomers differing in the degree of polymerization (DP), acetylation (DA), or deacetylation (DD), as well as in the location of N-acetyl residues in the oligomer chain. Heterochitooligomers with a polymerization degree of no more than 10 are usually water soluble. The solubility of chitooligosaccharides with a DP of more than 10 depends on the DA and pH of a solution. The pharmaceutical and food industries, as well as scientists engaged in basic research, are interested in the use of heterochitooligosaccharides. This is explained by their unique properties, such as good water solubility; minimal toxicity; biocompatibility; the ability to penetrate cell membranes, resulting in a high degree of absorption (unlike chitin and chitosan); and their biological activity. Therefore, in the last decade, researchers have focused their attention on studying the relationship of the structure of oligosaccharides and their specific activity, such as antitumor, antimicrobial, antioxidant, immunemodulatory, and other activities. This is shown by the number of publications, which has doubled compared to the number in 2001.

Microbial responses to chitin and chitosan in oxic and anoxic agricultural soil slurries

Abstract. Microbial degradation of chitin in soil substantially contributes to carbon cycling in terrestrial ecosystems. Chitin is globally the second most abundant biopolymer after cellulose and can be deacetylated to chitosan or can be hydrolyzed to N,N′-diacetylchitobiose and oligomers of N-acetylglucosamine by aerobic and anaerobic microorganisms. Which pathway of chitin hydrolysis is preferred by soil microbial communities is unknown. Supplementation of chitin stimulated microbial activity under oxic and anoxic conditions in agricultural soil slurries, whereas chitosan had no effect. Thus, the soil microbial community likely was more adapted to chitin as a substrate. In addition, this finding suggested that direct hydrolysis of chitin was preferred to the pathway that starts with deacetylation. Chitin was apparently degraded by aerobic respiration, ammonification, and nitrification to carbon dioxide and nitrate under oxic conditions. When oxygen was absent, fermentation products (acetate, butyrate, propionate, hydrogen, and carbon dioxide) and ammonia were detected, suggesting that butyric and propionic acid fermentation, along with ammonification, were likely responsible for anaerobic chitin degradation. In total, 42 different chiA genotypes were detected of which twenty were novel at an amino acid sequence dissimilarity of less than 50%. Various chiA genotypes responded to chitin supplementation and affiliated with a novel deep-branching bacterial chiA genotype (anoxic conditions), genotypes of Beta- and Gammaproteobacteria (oxic and anoxic conditions), and Planctomycetes (oxic conditions). Thus, this study provides evidence that detected chitinolytic bacteria were catabolically diverse and occupied different ecological niches with regard to oxygen availability enabling chitin degradation under various redox conditions on community level.

Pericyte protection by edaravone after tissue plasminogen activator treatment in rat cerebral ischemia.

Pericytes play a pivotal role in contraction, mediating inflammation and regulation of blood flow in the brain. In this study, changes of pericytes in the neurovascular unit (NVU) were examined in relation to the effects of exogenous tissue plasminogen activator (tPA) and a free radical scavenger, edaravone. Immunohistochemistry and Western blot analyses showed that the overlap between platelet-derived growth factor receptor β-positive pericytes and N-acetylglucosamine oligomers (NAGO)-positive endothelial cells increased significantly at 4 days after 90 min of transient middle cerebral artery occlusion (tMCAO). The number of pericytes and the overlap with NAGO decreased with tPA but recovered with edaravone 4 days after tMCAO with proliferation. Thus, tPA treatment damaged pericytes, resulting in the detachment from astrocytes and a decrease in glial cell line-derived neurotrophic factor secretion. However, treatment with edaravone greatly improved tPA-induced damage to pericytes. The present study demonstrates that exogenous tPA strongly damages pericytes and destroys the integrity of the NVU, but edaravone treatment can greatly ameliorate such damage after acute cerebral ischemia in rats. © 2014 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Characterization of a grape class IV chitinase.

A chitinase was purified from Vitis vinifera Manzoni Bianco grape juice and characterized. On the basis of proteomic analysis of tryptic peptides, a significant match identified the enzyme as a type IV grape chitinase previously found in juices of other V. vinifera varieties. The optimal pH and temperature for activity toward colloidal chitin were found to be 6 and 30 °C, respectively. The enzyme was found to hydrolyze chitin and oligomers of N-acetylglucosamine, generating N,N′-diacetylchitobiose and N-acetylglucosamine as products, but was inactive toward N,N′-diacetylchitobiose. The enzyme exhibited both endo- and exochitinase activities. Because yeast contains a small amount of chitin in the cell wall, the possibility of growth inhibition was tested. At a concentration and pH expected in ripe grapes, no inhibition of wine yeast growth by the chitinase was observed.

Functional Characterization of Staphylococcus epidermidis IcaB, a De-N-acetylase Important for Biofilm Formation

A polymer of partially de-N-acetylated β-1,6-linked N-acetylglucosamine (dPNAG), also known as the polysaccharide intercellular adhesin (PIA), is an important component of many bacterial biofilm matrices. In Staphyloccocus epidermidis, the poly-N-acetylglucosamine polymer is partially de-N-acetylated by the extracellular protein IcaB. To understand the mechanism of action of IcaB, the enzyme was overexpressed and purified. IcaB demonstrates metal-dependent de-N-acetylase activity on β-1,6-linked N-acetylglucosamine oligomers with a broad preference for divalent metals. Steady-state kinetic analysis reveals the low catalytic efficiency (pentasaccharide kcat/KM 0.03 M(-1) s(-1)) of the enzyme toward the oligomeric substrates. While IcaB displays similar rates of de-N-acetylation with tri- through hexasaccharide PNAG oligomers, position specific de-N-acetylation was only observed with penta- and hexasaccharides. The enzyme preferentially de-N-acetylates the second residue from the reducing terminus in the pentasaccharide and second and third residues from the reducing terminus in the hexasaccharide. The data described here represent an important step toward a detailed understanding of dPNAG biosynthesis in S. epidermidis, an important nosocomial pathogen, as well as in other Gram-positive bacteria. The low catalytic activity of IcaB is consistent with reports of other enzymes which act on biofilm-related polysaccharides, and this emerging trend may indicate a common feature among this group of polysaccharide processing enzymes.

Partially acetylated chitooligosaccharides bind to YKL-40 and stimulate growth of human osteoarthritic chondrocytes

Recent evidences indicating that cellular kinase signaling cascades are triggered by oligomers of N-acetylglucosamine (ChOS) and that condrocytes of human osteoarthritic cartilage secrete the inflammation associated chitolectin YKL-40, prompted us to study the binding affinity of partially acetylated ChOS to YKL-40 and their effect on primary chondrocytes in culture. Extensive chitinase digestion and filtration of partially deacetylated chitin yielded a mixture of ChOS (Oligomin™) and further ultrafiltration produced T-ChOS™, with substantially smaller fraction of the smallest sugars. YKL-40 binding affinity was determined for the different sized homologues, revealing micromolar affinities of the larger homologues to YKL-40. The response of osteoarthritic chondrocytes to Oligomin™ and T-ChOS™ was determined, revealing 2- to 3-fold increases in cell number. About 500μg/ml was needed for Oligomin™ and around five times lower concentration for T-ChOS™, higher concentrations abolished this effect for both products. Addition of chitotriose inhibited cellular responses mediated by larger oligosaccharides. These results, and the fact that the partially acetylated T-ChOS™ homologues should resist hydrolysis, point towards a new therapeutic concept for treating inflammatory joint diseases.

Purification, cDNA cloning and recombinant protein expression of a phloem lectin-like anti-insect defense protein BPLP from the phloem exudate of the wax gourd, Benincasa hispida

Latex and other exudates in plants contain various proteins that are thought to play important defensive roles against herbivorous insects and pathogens. Herein, the defensive effects of phloem exudates against the Eri silkworm, Samia ricini (Saturniidae, Lepidoptera) in several cucurbitaceous plants were investigated. It was found that phloem exudates are responsible for the defensive activities of cucurbitaceous plants, such as the wax gourd Benincasa hispida and Cucumis melo, especially in B. hispida, whose leaves showed the strongest growth-inhibitory activity of all the cucurbitaceous plants tested. A 35 kDa proteinaceous growth-inhibitory factor against insects designated BPLP (B. hispida Phloem Lectin-like Protein) was next isolated and purified from the B. hispida exudate, using anion exchange and gel filtration chromatography. A very low concentration (70 μg/g) of BPLP significantly inhibited growth of S. ricini larvae. The full-length cDNA (1076 bp) encoding BPLP was cloned and its nucleotide sequence was determined. The deduced amino acid sequence of BPLP had 51% identity with a cucurbitaceous phloem lectin (phloem protein 2, PP2), and showed binding specificity to oligomers of N-acetylglucosamine. Some features of BPLP indicated that it does not have a cysteine residue and it is composed of two repeats of similar sequences, suggesting that BPLP is distinct from PP2. Recombinant BPLP, obtained by expressing the cDNA in Escherichia coli, showed both chitin-binding lectin activity and growth-inhibitory activity against S. ricini larvae. The present study thus provides experimental evidence that phloem exudates of Cucurbitaceae plants, analogous to plant latex, play defensive roles against insect herbivores, especially against chewing insects, and contain defensive substances toxic to them.

The binding affinity and molecular basis of the structure-binding relationship between urinary Tamm-Horsfall glycoprotein and tumor necrosis factor-α.

In a previous study we noted significant THP binding to TNF-α, but did not explore the molecular basis of the structure-binding relationship. In this study, we used lectin-binding ELISA to assess the carbohydrate compositions of THP, BSA, IgG, TNF-α, and IFN-γ. We identified β(1,4)-N-acetylglucosamine oligomers (GlcNAc) and GlcNAc/branched mannose in BSA, IgG, TNF-α, and THP, but not in IFN-γ. These carbohydrate moieties mediated binding with THP. Small amounts of Siaα(2,3)Gal/ GalNAc, Sia(2,6)Gal/GalNAc, and mannose residues were also present in THP and TNF-α. Binding affinity (Kd) between THP and TNF-α by Scatchard plot analysis was 1.4–1.7 × 10−6 M, lower than antigen-antibody or ligand-receptor binding affinities. To elucidate the structure-binding relationship of THP-TNF-α, THP was digested with neuraminidase, β-galactosidase, O-sialoglycoprotein endopeptidase, carboxypeptidase Y, or proteinase K. β-galactosidase increased binding capacity of THP for TNF-α. Monosaccharide inhibition suggested that α-methyl-D-mannoside, GlcNAc, and GalNAc, but not sialic acid, suppress THP-TNF-α binding as detected by ELISA. We conclude that sugar-lectin and sugar-protein interactions between cognate sites in THP and TNF-α mediate their binding.

Human YKL-39 is a pseudo-chitinase with retained chitooligosaccharide-binding properties.

The chitinase-like proteins YKL-39 (chitinase 3-like-2) and YKL-40 (chitinase 3-like-1) are highly expressed in a number of human cells independent of their origin (mesenchymal, epithelial or haemapoietic). Elevated serum levels of YKL-40 have been associated with a negative outcome in a number of diseases ranging from cancer to inflammation and asthma. YKL-39 expression has been associated with osteoarthritis. However, despite the reported association with disease, the physiological or pathological role of these proteins is still very poorly understood. Although YKL-39 is homologous to the two family 18 chitinases in the human genome, it has been reported to lack any chitinase activity. In the present study, we show that human YKL-39 possesses a chitinase-like fold, but lacks key active-site residues required for catalysis. A glycan screen identified oligomers of N-acetylglucosamine as preferred binding partners. YKL-39 binds chitooligosaccharides and a newly synthesized derivative of the bisdionin chitinase-inhibitor class with micromolar affinity, through a number of conserved tryptophan residues. Strikingly, the chitinase activity of YKL-39 was recovered by reverting two non-conservative substitutions in the active site to those found in the active enzymes, suggesting that YKL-39 is a pseudo-chitinase with retention of chitinase-like ligand-binding properties.

Identification and Characterization of a Novel Polysaccharide Deacetylase C (PdaC) from Bacillus subtilis

Cell wall metabolism and cell wall modification are very important processes that bacteria use to adjust to various environmental conditions. One of the main modifications is deacetylation of peptidoglycan. The polysaccharide deacetylase homologue, Bacillus subtilis YjeA (renamed PdaC), was characterized and found to be a unique deacetylase. The pdaC deletion mutant was sensitive to lysozyme treatment, indicating that PdaC acts as a deacetylase. The purified recombinant and truncated PdaC from Escherichia coli deacetylated B. subtilis peptidoglycan and its polymer, (-GlcNAc-MurNAc[-L-Ala-D-Glu]-)(n). Surprisingly, RP-HPLC and ESI-MS/MS analyses showed that the enzyme deacetylates N-acetylmuramic acid (MurNAc) not GlcNAc from the polymer. Contrary to Streptococcus pneumoniae PgdA, which shows high amino acid sequence similarity with PdaC and is a zinc-dependent GlcNAc deacetylase toward peptidoglycan, there was less dependence on zinc ion for deacetylation of peptidoglycan by PdaC than other metal ions (Mn(2+), Mg(2+), Ca(2+)). The kinetic values of the activity toward B. subtilis peptidoglycan were K(m) = 4.8 mM and k(cat) = 0.32 s(-1). PdaC also deacetylated N-acetylglucosamine (GlcNAc) oligomers with a K(m) = 12.3 mM and k(cat) = 0.24 s(-1) toward GlcNAc(4). Therefore, PdaC has GlcNAc deacetylase activity toward GlcNAc oligomers and MurNAc deacetylase activity toward B. subtilis peptidoglycan.

A class V chitinase from Arabidopsis thaliana: gene responses, enzymatic properties, and crystallographic analysis

Expression of a class V chitinase gene (At4g19810, AtChiC) in Arabidopsis thaliana was examined by quantitative real-time PCR and by analyzing microarray data available at Genevestigator. The gene expression was induced by the plant stress-related hormones abscisic acid (ABA) and jasmonic acid (JA) and by the stress resulting from the elicitor flagellin, NaCl, and osmosis. The recombinant AtChiC protein was produced in E. coli, purified, and characterized with respect to the structure and function. The recombinant AtChiC hydrolyzed N-acetylglucosamine oligomers producing dimers from the non-reducing end of the substrates. The crystal structure of AtChiC was determined by the molecular replacement method at 2.0Å resolution. AtChiC was found to adopt an (β/α)(8) fold with a small insertion domain composed of an α-helix and a five-stranded β-sheet. From docking simulation of AtChiC with pentameric substrate, the amino acid residues responsible for substrate binding were found to be well conserved when compared with those of the class V chitinase from Nicotiana tabacum (NtChiV). All of the structural and functional properties of AtChiC are quite similar to those obtained for NtChiV, and seem to be common to class V chitinases from higher plants.