Letteratura scientifica selezionata sul tema "Human Chitinase"

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.

Chitinases are hydrolytic enzymes widely distributed in nature. Despite their physiologic and pathophysiologic roles are not well understood, chitinases are emerging as biomarkers in a broad range of neurologic disorders, where in many cases, protein levels measured in the CSF have been shown to correlate with disease activity and progression. In this review, we will summarize the structural features of human chitinases and chitinase-like proteins and their potential physiologic and pathologic functions in the CNS. We will also review existing evidence for the role of chitinases and chitinase-like proteins as diagnostic and prognostic biomarkers in inflammatory, neurodegenerative diseases, and psychiatric disorders. Finally, we will comment on future perspectives of chitinase studies in neurologic conditions.

Background: Chitinases are the evolutionary conserved glycosidic enzymes that are characterized by their ability to cleave the naturally abundant polysaccharide chitin. The potential role of chitinases has been identified in the manifestation of various allergies and inflammatory diseases. In recent years, chitinases inhibitors are emerging as an alluring area of interest for the researchers and scientists and there is a dire need for the development of potential and safe chitinase antagonists for the prophylaxis and treatment of several diseases. Objective: The present review expedites the role of chitinases and their inhibitors in inflammation and related disorders. Methods: At first, an exhaustive survey of literature and various patents available related to chitinases were carried out. Useful information on chitinases and their inhibitor was gathered from the authentic scientific databases namely SCOPUS, EMBASE, PUBMED, GOOGLE SCHOLAR, MEDLINE, EMBASE, EBSCO, WEB OF SCIENCE, etc. This information was further analyzed and compiled up to prepare the framework of the review article. The search strategy was conducted by using queries with key terms “ chitin”, “chitinase”, “chitotrisidase”, “acidic mammalian chitinase”, “chitinase inhibitors”, “asthma” and “chitinases associated inflammatory disorders”, etc. The patents were searched using the key terms “chitinases and uses thereof”, “chitinase inhibitors”, “chitin-chitinase associated pathological disorders” etc. from www.google.com/patents, www.freepatentsonline.com, and www.scopus.com. Results: The present review provides a vision for apprehending human chitinases and their participation in several diseases. The patents available also signify the extended role and effectiveness of chitinase inhibitors in the prevention and treatment of various diseases viz. asthma, acute and chronic inflammatory diseases, autoimmune diseases, dental diseases, neurologic diseases, metabolic diseases, liver diseases, polycystic ovary syndrome, endometriosis, and cancer. In this regard, extensive pre-clinical and clinical investigations are required to develop some novel, potent and selective drug molecules for the treatment of various inflammatory diseases, allergies and cancers in the foreseeable future. Conclusion: In conclusion, chitinases can be used as potential biomarkers in prognosis and diagnosis of several inflammatory diseases and allergies and the design of novel chitinase inhibitors may act as key and rational scaffolds in designing some novel therapeutic agents in the treatment of variety of inflammatory diseases.

Abstract Immunosuppressed patients are highly susceptible to invasive fungal infections (IFI) such as invasive pulmonary aspergillosis, which is predominantly caused by the fungus Aspergillus fumigatus. An important component of the fungal cell wall is chitin, a polymer of N-acetyl-D-glucosamine (GlcNAc). Chitin is not produced by humans, however, the chitin degrading enzymes (chitinases) chitotriosidase (Chit-1) and acidic mammalian chitinase (AMCase) are. Chitinase is predominantly produced by activated macrophages, and may possibly aid in the defense against chitin-containing pathogens. We show that serum and bronchoalveolar lavage (BAL) chitinase levels are increased in patients with IFI, and in mice after pulmonary exposure to A. fumigatus conidia. Several different stimuli, including stimulation with chitin can lead to chitinase responses. In vitro stimulation of U937 human monocytes and RAW mouse macrophages with either chitin-particles (7-12 µm) or GlcNAc increased secreted and intracellular chitinase activity, the accumulation of intracellular Chit-1, and significantly increased the mRNA levels of Chit-1 and AMCase. Accordingly, we hypothesize that Chit-1 expression is regulated through a positive feedback mechanism involving the degradation of chitin to GlcNAc by host chitinases and GlcNAc recognition that in turn upregulates chitinase expression. This potential feedback mechanism of chitinase regulation may have utility in the diagnosis of IFIs.

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.

ABSTRACT During mosquito transmission, malaria ookinetes must cross a chitin-containing structure known as the peritrophic matrix (PM), which surrounds the infected blood meal in the mosquito midgut. In turn, ookinetes produce multiple chitinase activities presumably aimed at disrupting this physical barrier to allow ookinete invasion of the midgut epithelium. Plasmodium chitinase activities are demonstrated targets for human and avian malaria transmission blockade with the chitinase inhibitor allosamidin. Here, we identify and characterize the first chitinase gene of a rodent malaria parasite,Plasmodium berghei. We show that the gene, namedPbCHT1, is a structural ortholog ofPgCHT1 of the avian malaria parasite Plasmodium gallinaceum and a paralog of PfCHT1 of the human malaria parasite Plasmodium falciparum. Targeted disruption of PbCHT1 reduced parasite infectivity inAnopheles stephensi mosquitoes by up to 90%. Reductions in infectivity were also observed in ookinete feeds—an artificial situation where midgut invasion occurs before PM formation—suggesting that PbCHT1 plays a role other than PM disruption. PbCHT1 null mutants had no residual ookinete-derived chitinase activity in vitro, suggesting that P. berghei ookinetes express only one chitinase gene. Moreover, PbCHT1 activity appeared insensitive to allosamidin inhibition, an observation that raises questions about the use of allosamidin and components like it as potential malaria transmission-blocking drugs. Taken together, these findings suggest a fundamental divergence among rodent, avian, and human malaria parasite chitinases, with implications for the evolution ofPlasmodium-mosquito interactions.

Recently, the second mammalian chitinase, designated acidic mammalian chitinase (AMCase), has been identified in human, mouse, and cow. In contrast to the earlier identified macrophage-derived chitinase (chitotriosidase), this chitinase is richly expressed in the gastrointestinal (GI) tract, suggesting its role in digestion of chitin-containing foods as well as defense against chitin-coated microorganisms and parasites. This in situ hybridization study first revealed cellular localization of the gut-type chitinase in the mouse and chicken. In adult mice, the parotid gland, von Ebner's gland, and gastric chief cells, all of which are exocrine cells of the serous type, expressed the gut chitinase mRNA. In the chicken, oxyntico-peptic cells in glandular stomach (proventriculus) and hepatocytes expressed the chitinase mRNA. Because cattle produce the gut chitinase (chitin-binding protein b04) only in the liver, the gut chitinases in mammals and birds have three major sources of production, i.e., the salivary gland, stomach, and liver. During ontogenetic development, the expression level in the parotid gland and stomach of mice increased to the adult level before weaning, whereas in the stomach of chickens intense signals were detectable in embryos from incubation day 7.

ABSTRACT Chitinases of pathogens have been proposed as potential targets of vaccines or specific inhibitors. We studied the genomic organization, transcript levels, developmental expression, and biological function of chitinases in the rodent filarial nematode Acanthocheilonema viteae, a model organism for human-pathogenic filarial worms. Characterization of nine genomic clones from an A. viteae phage library and Southern blot experiments revealed the existence of three different chitinase genes, two of which could theoretically yield functional transcripts. The deduced proteins of these genes had the common modular organization of family 18 chitinases. Northern blot experiments and rapid amplification of cDNA ends-PCR with adult worms and larval stages showed that only one gene is expressed, with high variation in transcript levels, as determined by real-time PCR. Chitinase transcript levels were lowest in the late male stage 4 larva (L4) and peaked in the stage 3 larva (L3), which was corroborated by Western blotting. RNA interference (RNAi) experiments showed that treatment of L3 and adult female worms with double-stranded RNA of chitinase inhibited molting of L3 worms and hatching of microfilariae. RNAi also led to the death of 50% of female worms, revealing the essential role of chitinase in the life cycle of filarial nematodes.

SUMMARYHaemoglobin or blood in the growth medium ofLeishmania majorinhibited the formation of infective promastigotes and the secretion of chitinases. Inoculation of mice with stationary-phase parasites from control medium caused infections in 20/29 mice, compared to 3/20 mice injected with parasites grown with 10% rabbit blood, or 1/30 mice that received parasites grown with rabbit haemoglobin. The concentration of peanut lectin (PNA) required to agglutinate promastigotes was used as an index of their infectivity, ranging from a high concentration for infective populations to a low concentration for relatively non-infective populations. Agglutination of 50% of the parasites from control medium or from medium containing rabbit haemoglobin required 4·1 μg PNA/ml and 0·1 μg PNA/ml, respectively. Chitinase activities/107parasites decreased from 4·8 units chitinase and 12·5 unitsN-acetylglucosaminidase (NAGase) in the control to 2·0 units chitinase and 8·5 units NAGase in cultures containing rabbit haemoglobin. Rabbit, human, bovine and pigeon haemoglobins had various inhibitory effects on the activity of chitinases and not on the virulence, as expressed by PNA agglutination. The relevance of the results to the cycle ofLeishmaniais discussed.

Chitinases are enzymes that hydrolyze chitin, a glucosamine polymer synthesized by lower organisms for structural purposes [1]. While humans do not synthetize chitin, they express two active chitinases, Chitotriosidase (hCHIT1) and Acidic Mammalian Chitinase (hAMCase). Both enzymes attracted attention due to their upregulation in immune system disorders [2,3]. They consist of a catalytic domain of 39 kDa and a chitin binding domain, joined by a hinge. The active site shows a cluster of three conserved acidic residues, E140, D138 and D136, linked by H-bonds, where D138 and E140 are involved in the hydrolysis reaction [1,3]. To increase our knowledge on the catalytic mechanism of human chitinases, we conducted a detailed structural analysis on hCHIT1. For this, we have improved the X-ray resolution of the apo hCHIT1 catalytic domain to 1Å. We investigated the protonation state on the catalytic site and detected a double conformation of D138, one in contact with D136 and a second one in contact with E140. Our analysis revealed for the first time different protonation states for each conformation of D138 (fig1). Interestingly, our X-ray data suggest that the catalytic E140, supposed to donate a proton in the catalytic reaction, is deprotonated in the apo form. To gain insight on the proton transition pathway during the hydrolysis, we have solved the X-ray structure of hCHIT1 complexed with the substrate at 1.05 Å. In comparison with the apo form, this structure shows a rearrangement of the protonation states of the catalytic triad triggered by the binding of the substrate. Our results led us to suggest a new hydrolysis model involving changes in the hydrogen bond network of the catalytic triad accompanied by a flip of D138 towards D136. This contributes to protonate E140, which then donates the proton to the substrate. To confirm the role of the active site's hydrogen network, we are currently studying CHIT1 by neutron crystallography and quantum mechanics.

La chitotriosidase (CHIT1) est une chitinase humaine appartenant à la famille glycosyl hydrolase 18 (GH18) qui hydrolyse la chitine. CHIT1 présente plusieurs caractéristiques enzymatiques conservées dans la famille GH18 qui ne sont pas complètement comprises. Pour renforcer nos connaissances sur le mécanisme catalytique de CHIT1 et de la famille GH18, j'ai amélioré la résolution des structures obtenues par diffraction de rayon-X du domaine catalytique de CHIT1. Ces structures correspondent à la forme apo de CHIT1, pseudo-apo ainsi qu’en complexe avec la chitobiose ont été obtenues à des résolutions comprises entre 0.95Å et 1.10Å. Mes résultats m’ont permis de proposer un nouveau mécanisme d’hydrolyse des chaines chito-oligosaccharidiques. En outre, grâce à une nouvelle stratégie de cristallisation, la première structure cristalline de CHIT1 complète a pu être obtenue à une résolution de 1.95Å. Mon étude donne de nouvelles perspectives sur le mode d'action de CHIT1 et les caractéristiques enzymatiques conservées dans la famille GH18
Chitotriosidase (CHIT1) is a human chitinase belonging to the glycosyl hydrolase family 18 (GH18), a highly conserved enzyme family. GH18 enzymes hydrolyze chitin, a N-acetyl glucosamine polymer. CHIT1 is characterized by many enzymatic features that are conserved in GH18 and not completely understood. To increase our knowledge on the catalytic mechanism in CHIT1 and GH18 family, I improved the X-ray resolution crystal structure of CHIT1 catalytic domain in apo and pseudo apo forms as well as in complex with a synthetic substrate to a resolution range between 0.95Å and at 1.10Å. My results allow me to suggest a new mechanism for chito-oligosaccharide chains hydrolysis. Moreover, thanks to a new a crystallogenesis strategy, I obtained the first crystal structure of full length CHIT1 at 1.95Å resolution. My study presents many structural and mechanistic aspects of CHIT1 which gives new insights onto its mode of action and shed light into the conserved enzymatic features in GH18 chitinase family

Systemic fungal infections represent a threat to public health, and annually more than 150 million people suffer from fungal diseases. This worrisome data reflects the growing group of patients with immunocompromised conditions: due to cancer chemotherapy, organ transplanting or affected by AIDS, and the outbreaks of azoles resistant strains. Moreover, the global emergency caused by SARS-CoV-2 led to long term hospitalizations, and intubation increased the susceptibility to developing fungal infections. Therefore, now more than ever, the challenge of developing new antifungal drugs is dramatically urgent. Our research group has been interested in the great potential of guanylated compounds as new antifungal agents since 2007. During these 15 years, three series of derivatives, characterized by an amidinoureas scaffold, have been developed. The structure of these compounds is new and not shared with other antifungal drugs present on the market. Consequently, they show remarkably antifungal activity, especially among Candida strains resistant to azole drugs. The first chapter of my thesis deals with synthesizing new antifungal compounds with a macrocyclic amidinourea scaffold. Firstly, a novel compound, BM37, was synthesized through a convergent approach using the ring-closing metathesis (RCM)as a key step. Secondly, we decided to conduct advanced biological investigations of our lead compound, BM1. Consequently, we face the need to prepare this compound on a gram scale. To achieve this result, we changed the synthetic route and took inspiration from Fukuyama’s work designing a new strategy to obtain 1 gram of BM1. The second chapter of my thesis explores the design and synthesis of novel inhibitors targeting human chitinases. This project started when we investigated a putative target for the amidinoureas compounds endowed with antifungal activity. This research led us to the Chitinase family. In particular, our interest fell on human chitinases due to their involvement in chronic inflammatory lung diseases. The development of new human chitinase inhibitors, characterized by two different chemical scaffolds, is the aim of this second chapter. The former was the macrocyclic amidinoureas scaffold. Here three derivatives: BM56, BM57 and BM58, were synthesized and evaluated on human chitinases. The latter explored the chemical space related to the 6-piperazine-1-ylpyrazine-2-carboxamide, a new scaffold that emerged from a structure-based virtual screening. In this case, we synthesized a small, focused library of derivatives. The third chapter of my thesis describes my work as visiting PhD student at Uppsala University. During this period, I have been involved in the alkylation of the N position of 3-methyl indole with several cyclic ketones using a green and efficient amide coupling reagent, the TP3®. Finally, the last chapter contains chemical and biological data of all the compounds presented in the thesis.

Chitinase proteins are expressed in most of reigns, from fungi to mammals. This kind of proteins can cleave the chitin, the second most abundant polysaccharide in nature. Mammals do not synthesize nor are able to use it, but they express Chitinase proteins. It has been proved that this kind of proteins are involved in several pathologies. Human Acidic Mammalian Chitinase is involved in the development of pathologies related to the Th2 inflammation. It has been discovered that the inhibition of this protein allowed the reduction of the inflammation. This effect makes this protein a very interesting target for the treatment of this kind of pathologies. In this PhD thesis, the first subject is the target fishing procedure applied to a series of macrocyclic compounds endowed with antifungal activity. The result of the target fishing has been the chitinase protein, and it has been rationally designed a new derivative able to inhibit the chitinase protein with higher potency. The second theme of this work has been the computational study the complex between the rationally designed chitinase inhibitor and the human Acidic Mammalian Chitinase, to identify the most reliable binding mode of the compound inside the binding pocket. The identification of the binding mode has been followed by the design of a library of derivatives, using the information acquired after the study, to improve the activity of the series of compounds towards the Acidic Mammalian Chitinase. The last topic has been the Structure Based Virtual Screening on the Acidic Mammalian Chitinase to find novel scaffolds active as AMCase inhibitor. A first screening based on pharmacophoric filtration and docking calculations has been done, identifying a preliminary hit, on which it has been performed a substructure search, that allowed the discovery of a more active derivative. This molecule has been examined with Molecular Dynamics simulations observing modification in the binding mode during each replica. It has been done a cluster analysis used for the generation of a new pharmacophoric model. The selected cluster representatives have been used as receptors for an ensemble docking calculation that allowed the identification of a third molecule biologically active as AMCase inhibitor.

Espécies do gênero Paracoccidioides spp são fungos patogênicos, termodimórficos, agentes etiológicos de doença endêmica em diversas regiões da América Latina. O indivíduo infectado desenvolve uma resposta específica que, quando associada à alta produção de TNF-? e IFN-?, favorece a resistência ao fungo. Componentes de alguns fungos patogênicos foram caracterizados, por técnicas de knockdown gênico, como importantes para a virulência fúngica. Nosso grupo identificou paracoccina (PCN) como um componente de leveduras de P. brasiliensis; trata-se de uma proteína com um domínio enzimático, dotado de atividade quitinase e um domínio lectínico, ligante de GlcNAc. PCN é dotada das seguintes propriedades: (a) contribui para o crescimento do fungo; (b) promove a adesão da levedura à matriz extracelular, por ligar-se à laminina; (c) interage com N-glicanas de TLR2 e TLR4 e promove ativação celular; (d) estimula macrófagos a produzirem mediadores pró-inflamatórios como IL- 12, TNF-? e NO; (e) promove a polarização M1 de macrófagos; (f) induz atividade fungicida em neutrófilos, bem como formação de NETs e supressão da apoptose, eventos que se mostraram dependentes da síntese de novo de proteínas pelos neutrófilos estimulados. Dada a relevância das atividades biológicas de PCN, promovemos recentemente o silenciamento do gene que codifica essa proteína, através de metodologia que usa RNA anti-sense e transformação mediada por Agrobacterium tumefaciens (ATMT). Uma vez PCN silenciada, a levedura perdeu a capacidade de fazer a transição para micélio e diminuiu a resistência à atividade fungicida de macrófagos. A infecção de camundongos com as cepas silenciadas, em comparação com as WT, causou doença de menor gravidade, com carga fúngica reduzida e baixa taxa de mortalidade. Essas observações sugerem de que PCN funcione como um fator de virulência em P. brasiliensis, que afeta a patogênese da infecção. Neste trabalho, ampliamos as ferramentas moleculares de manipulação do fungo e viabilizamos a superexpressão de PCN em leveduras de P. brasiliensis, tendo como objetivos estudar seu papel na virulência e na patogênese da infecção, bem como determinar os mecanismos responsáveis por tais atividades. A inoculação de leveduras que superexpressam PCN (ov-PCN) em camundongos causou doença pulmonar muito grave, em comparação à doença leve e moderada causada por leveduras silenciadas em PCN e leveduras WT, respectivamente. Nesse sentido, nossos esforços se dedicaram à busca dos mecanismos dos mecanismos através dos quais PCN influencia o curso da infecção experimental. Na tentativa de identificar o papel exercido pelo domínio quitinase da PCN, coletamos o sobrenadante de culturas de leveduras ov-PCN e WT. Partículas de quitina presentes nesses sobrenadantes foram purificadas por afinidade à lectina WGA (wheat germ agglutinin). Através de medida da área das partículas capturadas, através de microscopia eletrônica e aplicação do programa ImageJ, verificamos que a superexpressão de PCN resultou em clivagem mais eficiente da quitina da parede de leveduras, uma vez que apenas partículas muito pequenas (mediana das medidas = 2 nm2) foram detectadas, enquanto as áreas das partículas de quitina obtidas de leveduras selvagens (WT) forneceram mediana 3 vezes maior (6 nm2). As partículas de quitina foram então utilizadas para estimular macrófagos a produzirem citocinas. As obtidas de ov-PCN estimularam preponderantemente a secreção da citocina antiinflamatória IL-10, enquanto os macrófagos estimulados com partículas de leveduras WT produziram mais TNF-? e IL-1?, ambas de efeito pró-inflamatório. Esses resultados permitiram a identificação de um mecanismo importante para que a superexpressão de PCN se associe à ocorrência de doença pulmonar muito grave: o microambiente anti-inflamatório criado pelo estímulo de macrófagos por PCN leva ao desenvolvimento de resposta imune não protetora do tipo Th2 e lesões mais graves. Um segundo mecanismo foi identificado ao compararmos a resistência de leveduras ov-PCN e WT às respostas efetoras de macrófagos. A superexpressão de PCN associou-se à maior internalização das leveduras e maior resistência à atividade fungicida exercida por macrófagos. O estudo demonstra que diferentes níveis da expressão de uma quitinase (como PCN) levam à resistência a atividades antifúngicas de macrófagos e a diferentes graus de clivagem de quitina. A clivagem, por sua vez, pode alterar a estrutura da parede celular fúngica e a geração de fragmentos de quitina, cujos tamanhos e concentrações influenciam a produção de citocinas pelos macrófagos. Sob a ação de citocinas pró- ou antiinflamatórias liberadas pelos macrófagos e, consequentemente, a montagem de respostas adaptativas pode ser decisiva para haver suscetibilidade ou resistência à infecção por P. brasiliensis. Este trabalho proporciona um importante avanço no conhecimento do papel de quitinases na resposta anti-fúngica do hospedeiro.
Species of the genus Paracoccidioides spp are thermodymorphic fungi that cause a systemic disease, which is endemic in several regions of the Latin America. The infected individual develops a specific response that, when associated with the high production of TNF-? and IFN- ?, favors resistance to the fungus. Components of some pathogenic fungi were characterized by gene knockdown techniques as important the for fungal virulence. Our group has identified a component of P. brasiliensis, named Paracoccin (PCN); it is a bifunctional protein with an enzymatic domain, endowed with chitinase activity and a lectin domain, which binds GlcNAc and chitin, a GlcNAc polymers. PCN has the following properties: (a) contributes to the fungus growth; (b) promotes the yeast adhesion to the extracellular matrix, by binding to laminina glycans; (c) interacts with TLR2 and TLR4 N-glycans, which triggers cell activation; (d) stimulates macrophages to produce proinflammatory mediators, such as IL-12, TNF-? and NO; (e) promotes the M1 polarization of macrophages; (f) induces the neutrophils fungicidal activity, NETs formation, and suppression of neutrophils apoptosis, which are depending events on the de novo protein synthesis by neutrophils. Given the relevant biological activities exerted by PCN, we have performed recently the silencing of the gene that codes for this protein through a system that uses RNA anti-sense and Agrobacterium tumefaciens mediated transformation (ATMT). Once having the PCN gene silenced, yeast lost the ability of doing the transition to mycelium and decreased its resistance to macrophages fungicidal activities. Mice infection with PCN-silenced yeasts, compared to the infected with WT yeasts, exhibited a milder pulmonary disease with reduced fungal burden and low mortality rate. These observations suggest that PCN acts as a P. brasiliensis virulence factor that affects the pathogenesis of the fungal infection. In the present study, we expanded the molecular tools for the fungus manipulation and enabled the overexpression of PCN in P. brasiliensis yeasts, aiming to elucidate the PCN role in the fungus virulence and the infection pathogenesis, as well as determining the responsible mechanisms for the PCN activities. Inoculation of the PCN overexpressing yeasts (ov-PCN) into mice caused a very severe lung disease, compared to the mild and moderate diseases caused by PCN-silenced and WT yeasts, respectively. Then our efforts became dedicated to the search of mechanisms through which PCN influences the course of the experimental fungal disease. In an attempt to identify the role of the PCN chitinase domain, we harvested the supernatant of the ov-PCN and WT yeasts cultures. Chitin particles contained in the supernatants have been captured by affinity to the immobilized WGA (wheat germ agglutinin) lectin. By measuring through electron microscopy and application of the ImageJ program the area of the isolated chitin particles, we verified that the overexpression of PCN resulted in a more efficient cleavage of whole chitin molecules contained in the yeast cell wall, since only very small particles (median of the measurements = 2 nm2) were detected, while the the chitin particles areas obtained from WT-yeasts provided a median 3 fold higher (6 nm2). Then, the preparations of chitin particles were taken to stimulate macrophages to produce cytokines. The particles obtained from ov-PCN have stimulated preponderantly the secretion of the anti-inflammatory cytokine IL-10, whereas the macrophages stimulated with WT yeast particles have produced higher concentrations of TNF-? and IL-1?, which are known proinflammatory cytokines. These results allowed the identification of an important mechanism for the association of PCN overexpression to the occurrence of very severe pulmonary disease: the anti-inflammatory microenvironment created by the macrophages stimulation with PCN leads to the development of a non-protective Th2-type immune response and the more severe pulmonary injury. A second mechanism was identified as implicated in the severity of the lung disease associated to PCN overexpression. We compared the sensitivity of ov-PCN and WT yeasts to macrophages effector functions. PCN overexpressing yeasts were better internalized by macrophages and more resistant to the fungicidal activity of these cells, events that contributes for the high pulmonary fungal load verified in mice infected with ov-PCN yeasts. The study demonstrates that different levels of a chitinase (PCN) expression and enzymatic activity lead yeasts to change their sensitivity to macrophages antifungal activities as well as to different grades of chitin cleavage. The cleavage, in its turn, leads to changes in the structure of the fungal cell wall and generation of chitin fragments, whose sizes and concentrations influence the cytokines production by macrophages. Under the influence of pro-inflammatory or anti-inflammatory cytokines released by macrophages, the mounted adaptative responses can be decisive in conferring susceptibility or resistance to the P. brasiliensis infection. This study provides an important advance in the knowledge on the role of a chitinase in the host antifungal response.

碩士
大同大學
生物工程研究所碩士班
95
Abstract Chitotriosidase activity in human body has been proposed as a biochemical defense marker of macrophage activation and related to various infectious disease, inflammatory reaction, and immunological response. It is obviously in serum of patients suffering from Gaucher’s disease. In order to evaluate the correlation among chitotriosidase activity, pathological blood value and biological function in human serum, 186 sample including 60 healthy person, 30 patients with hyperlipedemia, and 96 patients with uremia were collect for the analyses of biochemical and hematology values. The protein quantitative assay and assay of reducing sugar releasing by enzyme hydrolysis were also applied to analyze the activity of chitinase in human serum. It was found that the average values of chitinase activities in patients with hyperlipedemia, in patients with uremia, and in healthy group were 583.3 U/mg, 546.4 U/mg, and 495.5 U/mg, respectively. Both of the former two patient groups were higher than the last one. The chitotriosidase activities with the fluorescence substrate, 4-mmethylumbelliferyl-β-D-N,N′,N′′-triacetyl-chitotrioside, as well as biochemical and hematology values were measured in 90 individual serums, which were separated into six groups including healthy persons and patients with thalassemia, uremia, inflammation (CRP>10 mg), cancer, and gout. The chitotriosidase activities were insignificant statistically, but the activities of chitotriosidase in serum for the patients with ferritin over 1000 ng/ml were 370 ± 159 nmol/ml.h (mean ± SD) that were higher than the 54.8 ± 29 nmol/ml.h (mean ± SD) in healthy group. Age and sex for the chitotriosidase activity were also no particularly positive correlation (P= 0.086). The increase of plasma chitotriosidase activity in thalassemia patients with high ferritin overload could be related to an iron mediated damage to the lysosomal apparatus similarly to Gaucher’s disease. It is proved that some disease causing the abnormal in human blood values positive correlation with the chitotriosidase liberation from activated macrophage. Hence, the measurement of chitotriosidase could not only speculate the cause of disease but also observe the timing of therapeutic intervention.

碩士
中臺科技大學
醫學檢驗生物技術系碩士班
99
Chitinase 3-like 1 (CHI3L1) is one kind of mammalian chitinase family proteins, as well as the 18th glycohydrolase family. Increased expression of CHI3L1 has been correlated with the presence of many kinds of different diseases, including cancers, autoimmune diseases, and chronic inflammatory conditions. According to further genetic analysis, some European researchers found that CHI3L1 gene was the susceptible gene of allergy. There was an indication that single nucleotide polymorphysiums (SNPs) in CHI3L1 might be associated with some clinical allergy diseases. Genetic variations in CHI3L1 were identified and genotyped in 143 allergy adults and 36 non-allergy adults. The variations of CHI3L1 SNPs (-131C/G and -247C/T) were examined by three different PCR methods (1) DNA sequence analysis, (2) TaqMan analysis and (3) High Resolution Melt analysis. We recorded and compared these data by statistical analysis, and we investigated the relationship between adult allergy and CHI3L1 gene. Our preliminary studies have shown that, by comparing DNA sequence analysis with TaqMan analysis and HRM analysis, the sensitivity are 65.85 and 89.74% and the specificity are 82.35 and 94.34% respectively. The variation of -131C/G promoter region had P values of <0.001, strongly supporting that the -131C/G polymorphism in the CHI3L1 is associated with high allergy risk. These results agreed well with those of the European researchers, the variation of -247C/T promoter region had a P values of >0.05, indicating that the SNP in the area CHI3L1 -247 is not associated with atopy. However, our results were different from those obtained by the Korean researchers. In follow-up studies we plan to collect more clinical specimens and do more clinical experiments to characterize the relationship between CHI3L1 gene and allergy.

碩士
大同大學
生物工程學系(所)
94
Chitinolytic enzymes exist in various species in nature. As a glycosyl hydrolases, they can catalyze the hydrolysis of chitin, the second most abundant polysaccharide on earth. Chitinolytic enzymes are necessary in the development of metabolism for arthropoda and fish. In human, there are many kinds of chitinolytic enzymes and chitinase-like proteins. Their physiological functions are yet appreciated. In this study, chitinase and related proteins from human plasma were identified using liquid chromatography. In one method, Blue Sepharose 6 affinity liquid chromatography and DEAE Sepharose ion exchange liquid chromatography were used to separate proteins from human plasma. A protein with molecular weight of 150 kDa, could be identified by western blot using ChBD antiserum. In other way, chitin affinity liquid chromatography and DEAE Sepharose ion exchange liquid chromatography were used to separate proteins from human plasma. A protein with molecular weight of 30 kDa was identified with ability to catalyze the hydrolysis of glycol chitin by in-situ activity staining on SDS-PAGE.

碩士
國立臺灣海洋大學
食品科學系
92
Abstract This research was conducted to isolate chitinolytic Clostridium perfringens from human feces, to develop a medium that rendered the bacterium to produce higher chitinase activity, and to purify and characterize the chitinase(s) produced by the bacterium. A medium mainly containing peptone and yeast extract was employed to cultivate the bacterium for production of higher chitinase activity after cultivation (37℃) for 25 h at anaerobic condition. When the proteins in crude enzyme solution of C. perfringens were precipitated by ammonium sulfate and then followed by dialysis, condensation and gel filtration, two chitinases (P-1 and P-2) were fractionated. Both chitinases could hydrolyse 4-methylumbellifery-N-acetylchitobiose [4-MU-(NAG)2] and 4-methylumbellifery-N-acetylchitotriose [4-MU-(NAG)3]. After the chromatography using Sephacryl S-300 and SDS-PAGE, chitinase P-1 revealed the molecular weight of 139.7 kDa composed of two subunits (87.9 and 52.4 kDa); P-2 showed the molecular weight of 196.8 kDa composed of two subunits (97 and 49.9 kDa). Due to the similar characteristics on the stabilities of reaction temperatures and pHs and the activation of metal ions, both enzymes were supposed to be the same protein. When the bacterium was incubated at the same condition for 96 h and the crude enzyme solution was dealt with the same treatments, there was only a chitinolytic peak revealed on DEAE or HIC chromatography, which was coded as chitinase A-4. The molecular weight of A-4 was 75 kDa and could hydrolyse both 4-MU-(NAG)2 and 4-MU-(NAG)3. Using 4-MU-(NAG)2 as a substrate, the optimum reaction temperature was 40℃ and the values of Km and Vmax were 0.14 mM and 416.67 nmole/ min/ mg, respectively; while using 4-MU-(NAG)3 as the substate, the optimum reaction temperature was 50℃, and Km and Vmax values were 0.34 mM and 384.62 nmole/ min/ mg, respectively. When using both substrates above-mentioned, the optimum reaction pH was the same (6.0) and the enzyme activity was enhanced by dithiothreitol or 2-iodoacetamide (1 mM), but inhibited by iodoacetic acid, phenyl methyl sulfonyl fluoride, Fe2+, Fe3+ or Hg2+. However, Ba2+, Ca2+, Cu2+, K+, Mg2+, Mg2+, Mn2+ or Na+ could only activate chitinase A-4 to hydrolyse 4-MU-(NAG)2. N-acetychitobiose [(NAG)2] was the main chitinolytic product when A-4 was employed to hydrolyse chitins from different sources. Among them, the highest quality of (NAG)2 was obtained from squid pen chitin after hydrolysation. Due to the existence of other enzymes in the crude enzyme solution, chitinase P-1 and P-2 could be modified to form A-4. Consequently, A-4 was the final stable chitinase of C. perfringens.