Chitinases and Chitinase-Like Proteins as Therapeutic Targets in Inflammatory Diseases,with a Special Focus on Inflammatory Bowel Diseases

Chitinases belong to the evolutionarily conserved glycosyl hydrolase family 18 (GH18). They catalyze degradation of chitin to N-acetylglucosamine by hydrolysis of the β-(1-4)-glycosidic bonds. Although mammals do not synthesize chitin, they possess two enzymatically active chitinases, i.e., chitotriosidase (CHIT1) and acidic mammalian chitinase (AMCase), as well as several chitinase-like proteins (YKL-40, YKL-39, oviductin, and stabilin-interacting protein). The latter lack enzymatic activity but still display oligosaccharides-binding ability. The physiologic functions of chitinases are still unclear, but they have been shown to be involved in the pathogenesis of various human fibrotic and inflammatory disorders, particularly those of the lung (idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, sarcoidosis, and asthma) and the gastrointestinal tract (inflammatory bowel diseases (IBDs) and colon cancer). In this review, we summarize the current knowledge about chitinases, particularly in IBDs, and demonstrate that chitinases can serve as prognostic biomarkers of disease progression. Moreover, we suggest that the inhibition of chitinase activity may be considered as a novel therapeutic strategy for the treatment of IBDs.     

Functional Properties of the Catalytic Domain of Mouse Acidic Mammalian Chitinase Expressed in Escherichia coli

Mouse acidic mammalian chitinase (AMCase) plays important physiological roles in defense and nutrition. AMCase is composed of an N-terminal catalytic domain (CatD) and a C-terminal chitin-binding domain (CBD). We expressed CatD of mouse AMCase as a recombinant fusion protein with Protein A and V5-His in Escherichia coli (Protein A-CatD-V5-His), evaluated its functional properties and compared them to the full-length AMCase (Protein A-AMCase-V5-His). Under our experimental conditions, the chitinolytic activity of both proteins against 4-nitrophenyl N,N''-diacetyl-β-d-chitobioside was equivalent with regard to their specific enzymatic activities, optimal pH and temperature as well as to the pH and temperature stability. CatD bound to chitin beads and cleaved the N-acetylglucosamine hexamer, colloidal and crystalline chitin as well as the shrimp shell, and released primarily N,N''-diacetylchitobiose fragments at pH 2.0. These results indicate that the primary structure of CatD is sufficient to form a proper tertiary structure required for chitinolytic activity, recognize chitin substrates and degrade them in the absence of a CBD. Our recombinant proteins can be used for further studies evaluating pathophysiological roles of AMCase in different diseases.     

Contribution of Chitinase A’s C-Terminal Vacuolar Sorting Determinant to the Study of Soluble Protein Compartmentation

Plant chitinases have been studied for their importance in the defense of crop plants from pathogen attacks and for their peculiar vacuolar sorting determinants. A peculiarity of the sequence of many family 19 chitinases is the presence of a C-terminal extension that seems to be important for their correct recognition by the vacuole sorting machinery. The 7 amino acids long C-terminal vacuolar sorting determinant (CtVSD) of tobacco chitinase A is necessary and sufficient for the transport to the vacuole. This VSD shares no homology with other CtVSDs such as the phaseolin’s tetrapeptide AFVY (AlaPheValTyr) and it is also sorted by different mechanisms. While a receptor for this signal has not yet been convincingly identified, the research using the chitinase CtVSD has been very informative, leading to the observation of phenomena otherwise difficult to observe such as the presence of separate vacuoles in differentiating cells and the existence of a Golgi-independent route to the vacuole. Thanks to these new insights in the endoplasmic reticulum (ER)-to-vacuole transport, GFPChi (Green Fluorescent Protein carrying the chitinase A CtVSD) and other markers based on chitinase signals will continue to help the investigation of vacuolar biogenesis in plants.     

Enzymatic Synthesis of Galactosylated Serine/Threonine Derivatives by β-Galactosidase from Escherichia coli

The transgalactosylations of serine/threonine derivatives were investigated using β-galactosidase from Escherichia coli as biocatalyst. Using ortho-nitrophenyl-β-d-galactoside as donor, the highest bioconversion yield of transgalactosylated N-carboxy benzyl l-serine benzyl ester (23.2%) was achieved in heptane:buffer medium (70:30), whereas with the lactose, the highest bioconversion yield (3.94%) was obtained in the buffer reaction system. The structures of most abundant galactosylated serine products were characterized by MS/MS. The molecular docking simulation revealed that the binding of serine/threonine derivatives to the enzyme’s active site was stronger (−4.6~−7.9 kcal/mol) than that of the natural acceptor, glucose, and mainly occurred through interactions with aromatic residues. For N-tert-butoxycarbonyl serine methyl ester (6.8%) and N-carboxybenzyl serine benzyl ester (3.4%), their binding affinities and the distances between their hydroxyl side chain and the 1′-OH group of galactose moiety were in good accordance with the quantified bioconversion yields. Despite its lower predicted bioconversion yield, the high experimental bioconversion yield obtained with N-carboxybenzyl serine methyl ester (23.2%) demonstrated the importance of the thermodynamically-driven nature of the transgalactosylation reaction.     

Green Conversion of Agroindustrial Wastes into Chitin and Chitosan by Rhizopus arrhizus and Cunninghamella elegans Strains

This article sets out a method for producing chitin and chitosan by Cunninghamella elegans and Rhizopus arrhizus strains using a green metabolic conversion of agroindustrial wastes (corn steep liquor and molasses). The physicochemical characteristics of the biopolymers and antimicrobial activity are described. Chitin and chitosan were extracted by alkali-acid treatment, and characterized by infrared spectroscopy, viscosity and X-ray diffraction. The effectiveness of chitosan from C. elegans and R. arrhizus in inhibiting the growth of Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica, Escherichia coli and Yersinia enterocolitica were evaluated by determining the minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC). The highest production of biomass (24.60 g/L), chitin (83.20 mg/g) and chitosan (49.31 mg/g) was obtained by R. arrhizus. Chitin and chitosan from both fungi showed a similar degree of deacetylation, respectively of 25% and 82%, crystallinity indices of 33.80% and 32.80% for chitin, and 20.30% and 17.80% for chitosan. Both chitin and chitosan presented similar viscosimetry of 3.79–3.40 cP and low molecular weight of 5.08 × 10³ and 4.68 × 10³ g/mol. They both showed identical MIC and MBC for all bacteria assayed. These results suggest that: agricultural wastes can be produced in an environmentally friendly way; chitin and chitosan can be produced economically; and that chitosan has antimicrobial potential against pathogenic bacteria.     

New Glycosylated Dihydrochalcones Obtained by Biotransformation of 2′-Hydroxy-2-methylchalcone in Cultures of Entomopathogenic Filamentous Fungi

Flavonoids, including chalcones, are more stable and bioavailable in the form of glycosylated and methylated derivatives. The combined chemical and biotechnological methods can be applied to obtain such compounds. In the present study, 2′-hydroxy-2-methylchalcone was synthesized and biotransformed in the cultures of entomopathogenic filamentous fungi Beauveria bassiana KCH J1.5, Isaria fumosorosea KCH J2 and Isaria farinosa KCH J2.6, which have been known for their extensive enzymatic system and ability to perform glycosylation of flavonoids. As a result, five new glycosylated dihydrochalcones were obtained. Biotransformation of 2′-hydroxy-2-methylchalcone by B. bassiana KCH J1.5 resulted in four glycosylated dihydrochalcones: 2′-hydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, 2′,3-dihydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, 2′-hydroxy-2-hydroxymethyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, and 2′,4-dihydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside. In the culture of I. fumosorosea KCH J2 only one product was formed—3-hydroxy-2-methyldihydrochalcone 2′-O-β-d-(4″-O-methyl)-glucopyranoside. Biotransformation performed by I. farinosa KCH J2.6 resulted in the formation of two products: 2′-hydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside and 2′,3-dihydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside. The structures of all obtained products were established based on the NMR spectroscopy. All products mentioned above may be used in further studies as potentially bioactive compounds with improved stability and bioavailability. These compounds can be considered as flavor enhancers and potential sweeteners.     

Changes in the microstructure and enzymatic hydrolysis performance of chitin treated by steam explosion,high-pressure homogenization,and γ radiation

This research intends to reduce the crystallinity of chitin with physical methods so as to improve the enzymatic hydrolysis efficiency of chitin. Scanning electron microscopy showed that both steam explosion and high-pressure homogenization significantly enlarged the pores in shrimp shells, which are made up of chitin, but γ radiation did not. X-ray diffraction showed that the crystallinity index of chitin decreased by 23.2% in the (110) plane and 24.7% in the (020) plane after three rounds of steam explosion and decreased by 12.4% in the (110) plane and 28.7% in the (020) plane after three rounds of high-pressure homogenization. After γ radiation, however, there was no significant change. The corresponding deacetylation degrees of the chitin treated with steam explosion and high-pressure homogenization increased by 13.2 and 5.7%, respectively. The natural, steam-exploded and high-pressure homogenized chitins respectively produced 0.251, 0.145, and 0.407 mg/ml reducing sugars with the hydrolysis of Streptomyces griseus chitinase. However, when hydrolyzed by Paenibacillus sp. A1 chitinase the steam-exploded and the high-pressure homogenized chitin respectively released 40 and 300% more reducing sugars than the natural chitin. The results found that steam explosion and high-pressure homogenization can reduce the crystallinity of chitin, and make it prone to enzymatic hydrolysis.     

Role of Long-Range Protein Dynamics in Different Thymidylate Synthase Catalyzed Reactions

Recent studies of Escherichia coli thymidylate synthase (ecTSase) showed that a highly conserved residue, Y209, that is located 8 Å away from the reaction site, plays a key role in the protein’s dynamics. Those crystallographic studies indicated that Y209W mutant is a structurally identical but dynamically altered relative to the wild type (WT) enzyme, and that its turnover catalytic rate governed by a slow hydride-transfer has been affected. The most challenging test of an examination of a fast chemical conversion that precedes the rate-limiting step has been achieved here. The physical nature of both fast and slow C-H bond activations have been compared between the WT and mutant by means of observed and intrinsic kinetic isotope effects (KIEs) and their temperature dependence. The findings indicate that the proton abstraction step has not been altered as much as the hydride transfer step. Additionally, the comparison indicated that other kinetic steps in the TSase catalyzed reaction were substantially affected, including the order of the substrate binding. Enigmatically, although Y209 is H-bonded to 3''-OH of 2''-deoxyuridine-5''-mono­phosphate (dUMP), its altered dynamics is more pronounced on the binding of the remote cofactor, (6R)-N⁵,N¹⁰-methylene-5,6,7,8-tetrahydrofolate (CH₂H₄folate), revealing the importance of long-range dynamics of the enzymatic complex and its catalytic function.     

Lysin Motif (LysM) Proteins: Interlinking Manipulation of Plant Immunity and Fungi

The proteins with lysin motif (LysM) are carbohydrate-binding protein modules that play a critical role in the host-pathogen interactions. The plant LysM proteins mostly function as pattern recognition receptors (PRRs) that sense chitin to induce the plant’s immunity. In contrast, fungal LysM blocks chitin sensing or signaling to inhibit chitin-induced host immunity. In this review, we provide historical perspectives on plant and fungal LysMs to demonstrate how these proteins are involved in the regulation of plant’s immune response by microbes. Plants employ LysM proteins to recognize fungal chitins that are then degraded by plant chitinases to induce immunity. In contrast, fungal pathogens recruit LysM proteins to protect their cell wall from hydrolysis by plant chitinase to prevent activation of chitin-induced immunity. Uncovering this coevolutionary arms race in which LysM plays a pivotal role in manipulating facilitates a greater understanding of the mechanisms governing plant-fungus interactions.     

Fungal Biotransformation of 2′-Methylflavanone and 2′-Methylflavone as a Method to Obtain Glycosylated Derivatives

Methylated flavonoids are promising pharmaceutical agents due to their improved metabolic stability and increased activity compared to unmethylated forms. The biotransformation in cultures of entomopathogenic filamentous fungi is a valuable method to obtain glycosylated flavones and flavanones with increased aqueous solubility and bioavailability. In the present study, we combined chemical synthesis and biotransformation to obtain methylated and glycosylated flavonoid derivatives. In the first step, we synthesized 2′-methylflavanone and 2′-methylflavone. Afterwards, both compounds were biotransformed in the cultures of two strains of entomopathogenic filamentous fungi Beauveria bassiana KCH J1.5 and Isaria fumosorosea KCH J2. We determined the structures of biotransformation products based on NMR spectroscopy. Biotransformations of 2′-methyflavanone in the culture of B. bassiana KCH J1.5 resulted in three glycosylated flavanones: 2′-methylflavanone 6-O-β-d-(4″-O-methyl)-glucopyranoside, 3′-hydroxy-2′-methylflavanone 6-O-β-d-(4″-O-methyl)-glucopyranoside, and 2-(2′-methylphenyl)-chromane 4-O-β-d-(4″-O-methyl)-glucopyranoside, whereas in the culture of I. fumosorosea KCH J2, two other products were obtained: 2′-methylflavanone 3′-O-β-d-(4″-O-methyl)-glucopyranoside and 2-methylbenzoic acid 4-O-β-d-(4′-O-methyl)-glucopyranoside. 2′-Methylflavone was effectively biotransformed only by I. fumosorosea KCH J2 into three derivatives: 2′-methylflavone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, 2′-methylflavone 4′-O-β-d-(4″-O-methyl)-glucopyranoside, and 2′-methylflavone 5′-O-β-d-(4″-O-methyl)-glucopyranoside. All obtained glycosylated flavonoids have not been described in the literature until now and need further research on their biological activity and pharmacological efficacy as potential drugs.     

The Chitinolytic Activities of Streptomyces sp. TH-11

Chitin is an abundant biopolymer composed of units of N-acetyl-D-glucosamine linked by β-1,4 glycosidic bonds. Chitin is the main component of the shells of mollusks, the cell wall of fungi and yeast and of the exoskeleton of crustaceans and insects. The degradation of chitin is catalyzed by chitinases that occur in a wide range of organisms. Among them, the chitinases from microorganisms are extremely important for the degradation and recycling of the carbon and nitrogen trapped in the large amount of insoluble chitin in nature. Streptomyces sp. TH-11 was isolated from the sediment of the Tou-Chien River, Taiwan. The chitinolytic enzyme activities were detected using a rapid in-gel detection method from the cell-free preparation of the culture medium of TH-11. The chitinolytic enzyme activity during prolonged liquid culturing was also analyzed by direct measurement of the chitin consumption. Decomposition of the exoskeleton of shrimps was demonstrated using electron microscopy and atomic force microscopy.     

Rational Design of Adenylate Kinase Thermostability through Coevolution and Sequence Divergence Analysis

Protein engineering is actively pursued in industrial and laboratory settings for high thermostability. Among the many protein engineering methods, rational design by bioinformatics provides theoretical guidance without time-consuming experimental screenings. However, most rational design methods either rely on protein tertiary structure information or have limited accuracies. We proposed a primary-sequence-based algorithm for increasing the heat resistance of a protein while maintaining its functions. Using adenylate kinase (ADK) family as a model system, this method identified a series of amino acid sites closely related to thermostability. Single- and double-point mutants constructed based on this method increase the thermal denaturation temperature of the mesophilic Escherichia coli (E. coli) ADK by 5.5 and 8.3 °C, respectively, while preserving most of the catalytic function at ambient temperatures. Additionally, the constructed mutants have improved enzymatic activity at higher temperature.     

Synthesis,DNA Binding,and Antiproliferative Activity of Novel Acridine-Thiosemicarbazone Derivatives

In this work, the acridine nucleus was used as a lead-compound for structural modification by adding different substituted thiosemicarbazide moieties. Eight new (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide derivatives (3a–h) were synthesized, their antiproliferative activities were evaluated, and DNA binding properties were performed with calf thymus DNA (ctDNA) by electronic absorption and fluorescence spectroscopies. Both hyperchromic and hypochromic effects, as well as red or blue shifts were demonstrated by addition of ctDNA to the derivatives. The calculated binding constants ranged from 1.74 × 10⁴ to 1.0 × 10⁶ M⁻¹ and quenching constants from −0.2 × 10⁴ to 2.18 × 10⁴ M⁻¹ indicating high affinity to ctDNA base pairs. The most efficient compound in binding to ctDNA in vitro was (Z)-2-(acridin-9-ylmethylene)-N-(4-chlorophenyl) hydrazinecarbothioamide (3f), while the most active compound in antiproliferative assay was (Z)-2-(acridin-9-ylmethylene)-N-phenylhydrazinecarbothioamide (3a). There was no correlation between DNA-binding and in vitro antiproliferative activity, but the results suggest that DNA binding can be involved in the biological activity mechanism. This study may guide the choice of the size and shape of the intercalating part of the ligand and the strategic selection of substituents that increase DNA-binding or antiproliferative properties.     

Hydrolysis of Oligosaccharides by a Thermostable α-Galactosidase from Termitomyces eurrhizus

The genus of Termitomyces purchased from the market has been identified as Termitomyces eurrhizus using the Internal Transcribed Spacer (ITS) method. An α-galactosidase from T. eurrhizus (TEG), a monomeric protein with a molecular mass of 72 kDa, was purified 146 fold by employing ion exchange chromatography and gel filtration. The optimum pH and temperature was 5.0 and 60 °C, respectively. TEG was stable over pH 2–6, and also exhibited good thermostablility, retaining 100% of the original activity after incubation at 60 °C for 2 h. Inhibition of the enzyme activity by N-bromosuccinimide (NBS) constituted evidence for an essential role of tryptophan in the catalytic action of the isolated enzyme. Besides 4-nitro-phenyl α-d-galactophyranoside (pNPGal), natural substrates could also be effectively hydrolyzed by TEG. Results of thin-layer chromatography (TLC) revealed complete enzymatic hydrolysis of raffinose and stachyose to galactose at 50 °C within 6 h. These properties of TEG advocate its utilization for elevating the nutritional value of soymilk.     

A new stilbazolium salt with perfectly aligned chromophores for second-order nonlinear optics: 4-N,N-Dimethylamino-4′-N′-methyl-stilbazolium 3-carboxy-4-hydroxybenzenesulfonate

A new organic nonlinear optical crystal 4-N,N-dimethylamino-4′-N′-methyl-stilbazolium 3-carboxy-4-hydroxybenzenesulfonate (DSCHS) has been developed with very promising properties for quadratic nonlinear optical applications. DSCHS single crystals with non-centrosymmetric structure have been obtained from aqueous methanol solution. X-ray crystallographic analysis revealed that the crystal structure of DSCHS is triclinic P1 with the chromophores aligned perfectly parallel, leading to the maximum possible order parameter <cos³θ> = 1 in the crystalline state, which is optimal for electro-optics, THz-wave generation and field detection applications. Kurtz powder test has shown that DSCHS exhibits a very large second-order optical nonlinearity, with a 30 percent higher second-harmonic signal than the well-known organic nonlinear optical crystal 4-N,N-dimethylamino-4′-N′-methyl-stilbazolium tosylate (DAST).     

Surface maleylation and naphthaloylation of chitin nanofibers for property enhancement

Surface N-maleylation and N-naphthaloylation of chitin nanofiber (NF) were achieved by reaction with maleic and naphthalic anhydrides in water, respectively. Maleyl and naphthaloyl groups were sufficiently introduced to an amino group on a surface-deacetylated chitin NF. The characteristic nanofiber morphology was maintained after maleylation and naphthaloylation. These NFs were homogeneously dispersed in several organic solvents. Especially, naphthaloyl chitin NF was dispersed even in aromatic solvents, owing to the high level of solvation interactions with the naphthaloyl group. The NF dispersions in those aromatic solvents exhibited a dispersive-to-precipitate transition response at approximately 29 °C. Moreover, the naphthaloyl chitin NF dispersion cut harmful ultraviolet light. The maleyl chitin NF formed a self-standing gel after cross-linking polymerization.     

An Engineered sgsh Mutant Zebrafish Recapitulates Molecular and Behavioural Pathobiology of Sanfilippo Syndrome A/MPS IIIA

Mucopolysaccharidosis IIIA (MPS IIIA, Sanfilippo syndrome type A), a paediatric neurological lysosomal storage disease, is caused by impaired function of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH) resulting in impaired catabolism of heparan sulfate glycosaminoglycan (HS GAG) and its accumulation in tissues. MPS IIIA represents a significant proportion of childhood dementias. This condition generally leads to patient death in the teenage years, yet no effective therapy exists for MPS IIIA and a complete understanding of the mechanisms of MPS IIIA pathogenesis is lacking. Here, we employ targeted CRISPR/Cas9 mutagenesis to generate a model of MPS IIIA in the zebrafish, a model organism with strong genetic tractability and amenity for high-throughput screening. The sgsh^Δex5−6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgsh^Δex5−6 zebrafish faithfully recapitulate diverse CNS-specific features of MPS IIIA, including neuronal lysosomal overabundance, complex behavioural phenotypes, and profound, lifelong neuroinflammation. We further demonstrate that neuroinflammation in sgsh^Δex5−6 zebrafish is largely dependent on interleukin-1β and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgsh^Δex5−6 mutant larvae in a context-dependent manner. We expect the sgsh^Δex5−6 zebrafish mutant to be a valuable resource in gaining a better understanding of MPS IIIA pathobiology towards the development of timely and effective therapeutic interventions.