Chitotriosidase Activity Is Counterproductive in a Mouse Model of Systemic Candidiasis.

Mammalian cells do not produce chitin, an insoluble polymer of N-acetyl-D-glucosamine (GlcNAc), although chitin is a structural component of the cell wall of pathogenic microorganisms such as Candida albicans. Mammalian cells, including cells of the innate immune system elaborate chitinases, including chitotriosidase (Chit1), which may play a role in the anti-fungal immune response. In the current study, using knockout mice, we determined the role of Chit1 against systemic candidiasis. Chit1-deficient mice showed significant decrease in kidney fungal burden compared to mice expressing the functional enzyme. Using in vitro anti-candidal neutrophil functional assays, the introduction of the Chit1:chitin digestion end-product, chitobiose (N-acetyl-D-glucosamine dimer, GlcNAc2), decreased fungal-induced neutrophil swarming and Candida killing in vitro. Also, a role for the lectin-like binding site on the neutrophil integrin CR3 (Mac-1, CD11b/CD18) was found through physiological competitive interference by chitobiose. Furthermore, chitobiose treatment of wild type mice during systemic candidiasis resulted in the significant increase in fungal burden in the kidney. These data suggest a counterproductive role of Chit1 in mounting an efficient anti-fungal defense against systemic candidiasis.

Chitin Organizing and Modifying Enzymes and Proteins Involved In Remodeling of the Insect Cuticle.

Chitin, the extracellular matrix polysaccharide of insects and arthropods is widely distributed in nature in all kingdoms of life and serves a variety of functions. After synthesis by membrane-bound chitin synthases, it is extensively remodeled before incorporation into divergent matrices with wide-ranging physical and biological properties. This chapter discusses the properties of a variety of insect enzymes and proteins involved in this process. Chitin remodeling involves chitin synthases, which make the nascent chitin chains, and chitin deacetylases that partially deacetylate some of the N-acetylglucosamine residues either randomly or sequentially to yield local chitosan-like regions. Other proteins secreted into the procuticle or the midgut help in the assembly of single chitin chains into larger crystalline aggregates that measure in a few 100 nanometers. They are further embedded in a complex matrix of cuticular proteins or become associated with proteins containing chitin-binding domains to constitute the laminar procuticle or the lattice-like peritrophic matrix. During molting, previously formed laminar cuticle or PM are decrystallized/depolymerized to unmask the chitin chains, which then are degraded by a mixture of chitinolytic enzymes consisting of chitinases and N-acetylglucosaminidases present in molting fluid or in gut secretions. Some of the degradation products may be recycled for the synthesis of new matrices. We present a model of chitin synthesis, assembly, and degradation and the roles of these chitin-remodeling enzymes in this overall process.

Role of Vibrio cholerae exochitinase ChiA2 in horizontal gene transfer.

Vibrio cholerae exochitinase ChiA2 plays a key role in acquisition of nutrients by chitin hydrolysis in the natural environment as well as in pathogenesis in the intestinal milieu. In this study we demonstrate the importance of ChiA2 in horizontal gene transfer in the natural environment. We found that the expression of ChiA2 and TfoX, the central regulator of V. cholerae horizontal gene transfer, varied with changes in environmental conditions. The activity of ChiA2 was also dependent on these conditions. In 3 different environmental conditions tested here, we observed that the supporting environmental condition for maximum expression and activity of ChiA2 was 20 °C, pH 5.5, and 100 mmol/L salinity in the presence of chitin. The same condition also induced TfoX expression and was favorable for horizontal gene transfer in V. cholerae. High-performance liquid chromatography analysis showed that ChiA2 released a significant amount of (GlcNAc)2 from chitin hydrolysis under the favorable condition. We hypothesized that under the favorable environmental condition, ChiA2 was upregulated and maximally active to produce a significant amount of (GlcNAc)2 from chitin. The same environmental condition also induced tfoX expression, followed by its translational activation by the (GlcNAc)2 produced, leading to efficient horizontal gene transfer.

[Protein O-GlcNAcylation and regulation of cell signalling: involvement in pathophysiology]. / O-GlcNAc glycosylation des protéines et régulation de la signalisation cellulaire: implication en physiopathologie.

O-GlcNAcylation corresponds to the addition of N-acetyl glucosamine (GlcNAc) on serine or threonine residues of cytosolic and nuclear proteins. This reversible post-translational modification regulates protein phosphorylation, sub-cellular localisation, stability and activity. Only two enzymes, OGT (O-linked N-acetyl-glucosaminyltransferase) and OGA (O-linked N-acetyl-ß-D glucosaminidase), control the addition and removal of GlcNAc from more than a thousand of proteins. Alternative splicing generates different isoforms of OGT and OGA, and address these enzymes to different sub-cellular compartments (mitochondria, cytosol...), restraining their action to specific subsets of substrates. Moreover, interaction with adaptor proteins may also help address these enzymes to specific substrates. Alterations in protein O-GlcNAcylation have been observed in a number of important human diseases, such as Alzheimer, cancer and diabetes. A reciprocal relationship between Tau protein phosphorylation and O-GlcNAcylation has been observed, and decreased O-GlcNAcylation in the brain of patients with Alzheimer diseases may favour Tau aggregation, destabilisation of microtubules and neuronal alterations. Alterations in OGT/OGA expression levels, and in protein O-GlcNAcylation, have been described in different types of cancer, and much evidence indicates that O-GlcNAcylation may participate in abnormal proliferation and migration of cancer cells. O-GlcNAcylation of transcription factors and signalling effectors may also participate in defects observed in diabetes. Indeed, in situation of chronic hyperglycaemia, abnormal O-GlcNAcylation may have deleterious effect on insulin secretion and action, resulting in further impairment of glucose homeostasis. Therefore, O-GlcNAcylation appears to be a major regulator of cellular activities and may play an important part in different human diseases. However, because of the large spectrum of OGT and OGA substrates, targeting O-GlcNAc for treatment of these diseases will be a highly challenging task.

Chitinase 1 is a biomarker for and therapeutic target in scleroderma-associated interstitial lung disease that augments TGF-ß1 signaling.

Interstitial lung disease (ILD) with pulmonary fibrosis is an important manifestation in systemic sclerosis (SSc, scleroderma) where it portends a poor prognosis. However, biomarkers that predict the development and or severity of SSc-ILD have not been validated, and the pathogenetic mechanisms that engender this pulmonary response are poorly understood. In this study, we demonstrate in two different patient cohorts that the levels of chitotriosidase (Chit1) bioactivity and protein are significantly increased in the circulation and lungs of SSc patients compared with demographically matched controls. We also demonstrate that, compared with patients without lung involvement, patients with ILD show high levels of circulating Chit1 activity that correlate with disease severity. Murine modeling shows that in comparison with wild-type mice, bleomycin-induced pulmonary fibrosis was significantly reduced in Chit1⁻/⁻ mice and significantly enhanced in lungs from Chit1 overexpressing transgenic animals. In vitro studies also demonstrated that Chit1 interacts with TGF-ß1 to augment fibroblast TGF-ß receptors 1 and 2 expression and TGF-ß-induced Smad and MAPK/ERK activation. These studies indicate that Chit1 is potential biomarker for ILD in SSc and a therapeutic target in SSc-associated lung fibrosis and demonstrate that Chit1 augments TGF-ß1 effects by increasing receptor expression and canonical and noncanonical TGF-ß1 signaling.

Lung chitinolytic activity and chitotriosidase are elevated in chronic obstructive pulmonary disease and contribute to lung inflammation.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation and emphysematous alveolar destruction. In this study, we have investigated whether chitotriosidase (ChTRase) and acidic mammalian chitinase, two chitinases with chitinolytic activity, are selectively augmented in COPD and contribute to its pathogenesis. We found that smokers with COPD, but not asthmatics, had higher chitinolytic activity and increased levels of ChTRase in bronchoalveolar lavage, more ChTRase-positive cells in bronchial biopsies, and an elevated proportion of alveolar macrophages expressing ChTRase than smokers without COPD or never-smokers. ChTRase accounted for approximately 80% of bronchoalveolar lavage chitinolytic activity, while acidic mammalian chitinase was undetectable. Bronchoalveolar lavage chitinolytic activity and ChTRase were associated with airflow obstruction and emphysema and with the levels of interleukin (IL)-1beta, IL-8, tumor-necrosis factor (TNF)-alpha, and its type II soluble receptor. Tumor necrosis factor-alpha stimulated ChTRase release only from alveolar macrophages from smokers with COPD, and exposure of these cells to ChTRase promoted the release of IL-8, monocyte-chemoattractant protein-1, and metalloproteinase-9. Finally, ChTRase overexpression in the lung of normal mice promoted macrophage recruitment and the synthesis of the murine homologue of IL-8, keratinocyte-derived cytokine, and of monocyte-chemoattractant protein-1. We conclude that pulmonary ChTRase overexpression may represent a novel important mechanism involved in COPD onset and progression.

Characterisation of the biosynthesis and processing of the neutrophil granule membrane protein CD63 in myeloid cells.

The biosynthesis and processing of the neutrophil granule membrane protein CD63, present in azurophil granules, was investigated in four myeloid cell lines. The amount of CD63 synthesised differed, so did the amount of protein processed to high molecular weight forms, with the demonstration of a more prominent synthesis of CD63 in K562 cells. Newly synthesised CD63 was initially detected as two precursor forms with molecular weight of 32 and 35 kDa, respectively. These two initial forms were processed further to yield high molecular weight forms of CD63 with a mean molecular weight of 50 kDa. Treatment with endoglycosidase H or N-glycosidase F revealed a protein core, free from asparagine-linked carbohydrates, with a molecular weight of 23 kDa. Newly synthesised CD63 was susceptible to digestion with endoglycosidase H, and the protein was not completely resistant to endoglycosidase H until after 4 h of chase, indicating that transport through the medial and trans-Golgi complex with conversion of high-mannose carbohydrates to complex oligosaccharide side chains had occurred. This finding indicates a relatively long processing time for CD63 compared to that of soluble azurophil granule proteins. By digestion with O-glycanase, the existence of O-linked oligosaccharides on CD63 could not be demonstrated. Biosynthetic labelling of cells in the presence of brefeldin A showed the importance of a functional Golgi apparatus for processing of the protein to its high molecular weight forms.

LytG of Bacillus subtilis is a novel peptidoglycan hydrolase: the major active glucosaminidase.

LytG (YubE) of Bacillus subtilis is a novel 32 kDa autolysin produced during vegetative growth under the control of Esigma(A) RNA polymerase. Muropeptide analysis of vegetative cells of B. subtilis revealed LytG to be the major glucosaminidase responsible for peptidoglycan structural determination during vegetative growth. Overexpression and purification of LytG allowed its biochemical characterization. Despite sequence homology suggesting muramidase activity, LytG is a novel glucosaminidase with exoenzyme activity and may form part of a novel family of autolysins. It is involved in cell division, lysis, and motility on swarm plates.

Changes in the specificity of blood groups induced by enzymes from soil fungi.

Three strains of Cunninghamella elegance (C. elegance), Penicillium simplicissimum (P. simpl.), and Aspergillus niger (A. niger) were isolated from soil that produced the enzymes acting on blood groups A and B red cells. Culture filtrates from these fungi contained alpha-N-acetyl-D-galactosaminidase as an A-decomposing enzyme, which resulted in an almost complete loss of A specificity and an enhancement of blood group H activity as measured by elution tests using monoclonal antibodies. They also contained an alpha-D-galactosidase and an alpha-L-fucosidase, which partially destroyed the blood group B specific activity, but did not influence the blood group H specific activity.

Effect of hyaluronidase, beta-glucuronidase and beta-N-acetylglucosaminidase inhibitors on sperm penetration of the mouse oocyte.

The role of hyaluronidase, beta-glucuronidase and beta-N-acetylglucosaminidase in the penetration by mouse spermatozoa through the layers surrounding the oocyte was investigated by in vitro techniques. Myocrisin, fenoprofen, phosphorulated hesperidin and PS53 (a hydroquinone-sulfonic acid-formaldehyde polymer) inhibited fertilization when incubated with capacitated spermatozoa before the treated spermatozoa were mixed with intact oocytes but not when the inhibitor-treated, capacitated spermatozoa were added to oocytes free of follicle cells. The antifertility activity did not appear to be due to an effect on sperm motility or on the oocytes. These 4 compounds are known hyaluronidase inhibitors and, of the acrosomal enzymes tested, only share inhibition of hyaluronidase. Kinetic studies indicated that myocrisin is a reversible inhibitor of mouse sperm hyaluronidase whereas the other three are irreversible inhibitors. Adding saccharolactone, a beta-glucuronidase inhibitor, or N-acetylglucosaminolactone and N-acetylgalactosaminolactone, beta-N-acetylglucosaminidase inhibitors, to capacitated spermatozoa under the same conditions as the hyaluronidase inhibitors did not decrease fertilization. This was the case even though the beta-glucuronidase or beta-N-acetylglucosaminidase activities of the spermatozoa were completely inhibited, at least at the time that the inhibitor-treated, capacitated spermatozoa were mixed with the oocytes. The hyaluronidase activity of mouse spermatozoa remained unaltered during the incubation period required for capacitation; however, prolonged incubation caused a significant decrease in hyaluronidase. Untreated mouse spermatozoa caused hydrolysis of hyaluronic acid more effectively than did sperm extracts obtained by detergent extraction. These results are consistent with the theory of an essential role of hyaluronidase in mouse fertilization. At least in this species, the enzyme appears to be specifically involved in sperm penetration through the follicle cell layer. The data do not support an essential role for beta-glucuronidase and beta-N-acetylglucosaminidase in the penetration by mouse spermatozoa through the oocyte's investments. In contrast to some other species, sperm capacitation in mice does not result in a loss of hyaluronidase although part of the enzyme activity is lost on prolonged incubation. Mouse spermatozoa appear to be able to digest substrate (hyaluronic acid) even though hyaluronidase is not released.

Beta-N-acetyl-glucosaminidase: possible role in ocular neovascularization.

The possibility that lysosomal enzymes might be involved as angiogenic factors in ocular neovascularization (NV) was investigated. Beta-N-acetyl-glucosaminidase (NAGase) activity, and that of two other glycosidases, were present in the retinal derived protein fraction (RDPF) reported by others to be angiogenic. NAGase, but not the other glycosidases, was inhibited by vitreous. NAGase exhibited the same stability characteristics as RDPF. In diabetic rats there was a significant rise in vitreous but a fall in retinal NAGase activity. The sera of these animals, however, showed elevation in the activities of all five glycosidases. Preliminary experiments indicate that only the intermediate isoenzyme of NAGase, putatively insulin dependent, is elevated in the eyes of these diabetic rats. NAGase was also specifically elevated in the intraocular fluid from monkey eyes with retinal vein occlusion (RVO), and markedly so if NV was present. These results suggest the involvement of NAGase in the neovascular process in the eye.

Interaction between a Bacillus cereus spore hexosaminidase and specific germinants.

A purified coat-associated hexosaminidase from spores of Bacillus cereus was studied to determine whether it could promote germination of dormant spores. Spores of a coat-deficient mutant as well as chemically extracted spores were used as substrate. Both of these spore preparations responded poorly to most germinants. However, absorbance loss was accelerated when the hexosaminidase was added in the presence of L-alanine. Enzyme alone was not effective. The addition of D-alanine inhibited completely the absorbance loss caused by hexosaminidase and L-alanine. Calcium dipicolinate and L-alpha-aminobutyric acid activated the hexosaminidase to some extent, but these chemicals were much less effective than L-alanine. In addition to the absorbance loss, the spores treated with enzyme and germinants released hexosamine and lost heat resistance and phase whiteness. The results suggest that this particular enzyme might have a role in germination.

Digestion of asparagine-linked oligosaccharides by endo-beta-N-acetylglucosaminidase in the human skin fibroblasts obtained from fucosidosis patients.

The substrate specificity of human endo-beta-N-acetylglucosaminidase was studied by using the homogenate of cultured skin fibroblasts of fucosidosis patients as an enzyme source. The results indicate that biantennary complex type asparagine-linked sugar chains as well as high mannose type sugar chains are cleaved by the enzyme action. None of the sugar chains with a fucosyl residue on the proximal N-acetylglucosamine of their N,N'-diacetylchitobiose moieties was cleaved. These results proved enzymatically the mechanism of production of oligosaccharides detected in the urine of various exoglycosidase deficiencies.