Interaction study between carob galactomannans and proteins using carbohydrate microarray tecnology
Autor(a) principal: | |
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Data de Publicação: | 2021 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10773/32832 |
Resumo: | Carbohydrate microarrays are a powerful, sensitive and high-throughput method to study carbohydrate-protein interactions, enabling the elucidation of several physiological functions and pathological mechanisms. Galactomannans (GMs) are a polysaccharide family which specific structure can have several particularities upon the source and/or undergo several changes upon the application of different extraction/purification methods. In this study, 2 lots of GMs derived from locust bean gum (LBG), were purified to ≈100% of carbohydrates, lot 1 and 2, presenting distinctive mannose/galactose (M/G) ratios, 2.4 and 3.3, respectively. Due to a closer structural simillarity reported for LBG with lot 2, this sample was selected for further treatment: dissolved and purified by diafiltration achieving reproductive mass yields of 63-67%. The purified samples were submitted to a partial acid hydrolysis treatment assisted by microwaves (MW), yielding 45% of mass and allowing a viscosity reduction, and depolymerisation effect occuring in minutes time-length. The aplication of an ultrafiltration separation process allowed a large portfolio of GMs with different molecular weights (Mw) fractions (6-21% of mass in each range of cut-off). Structural analysis, both neutral sugars and methylation, were used to characterise the structure of the carbohydrates obtained, and the main glycosidic bonds identified were 4-Manp (43-49%), 4,6-Manp (23-31%) and T-Gal (19-24%) in accordance with GMs composition. The purified LBG lot 1 and 2, and higher Mw retentates were non-convalently immobilized onto nitrocellulose-coated glass slides, with other well characterized saccharides as controls, constructing a microarray. The array was probed for recognition with different carbohydrate specific proteins, such as monoclonal antibodies (mAbs), and plant and mammalian lectins. Microarray analysis showed that, on one hand, the mannan recognizing mAbs 400-4 and LM21, confirmed the presence of β1,4-mannose backbone in the LBG-derived fractions. On the other hand, anti-galactomannan CCRC-M70 demonstrated binding correlation, associating recognition to the M/G ratio differences instead of the source, and beeing less tolerant to lower Mw. Microarray analysis of the two plant lectins used, RCA120 and ConA revelead that RCA120 binding to the saccharides presented, was unable to demonstrate a different identification between GMs and arabinogalactans, while ConA non significant binding confirms that only β-Man is present in the samples. Microarray analysis of the immune related lectins, DC-SIGN and SIGNR1, showed no to negligible binding, showing a potencial application of LBG fractions obtained during this study to clinical applications not involving an immune recognition. |
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Interaction study between carob galactomannans and proteins using carbohydrate microarray tecnologyCarbohydrate-protein interactionGalactomannansLocust bean (carob) gumCarbohydrate microarraysCarbohydrate-binding proteinsStructure-function relationshipCarbohydrate microarrays are a powerful, sensitive and high-throughput method to study carbohydrate-protein interactions, enabling the elucidation of several physiological functions and pathological mechanisms. Galactomannans (GMs) are a polysaccharide family which specific structure can have several particularities upon the source and/or undergo several changes upon the application of different extraction/purification methods. In this study, 2 lots of GMs derived from locust bean gum (LBG), were purified to ≈100% of carbohydrates, lot 1 and 2, presenting distinctive mannose/galactose (M/G) ratios, 2.4 and 3.3, respectively. Due to a closer structural simillarity reported for LBG with lot 2, this sample was selected for further treatment: dissolved and purified by diafiltration achieving reproductive mass yields of 63-67%. The purified samples were submitted to a partial acid hydrolysis treatment assisted by microwaves (MW), yielding 45% of mass and allowing a viscosity reduction, and depolymerisation effect occuring in minutes time-length. The aplication of an ultrafiltration separation process allowed a large portfolio of GMs with different molecular weights (Mw) fractions (6-21% of mass in each range of cut-off). Structural analysis, both neutral sugars and methylation, were used to characterise the structure of the carbohydrates obtained, and the main glycosidic bonds identified were 4-Manp (43-49%), 4,6-Manp (23-31%) and T-Gal (19-24%) in accordance with GMs composition. The purified LBG lot 1 and 2, and higher Mw retentates were non-convalently immobilized onto nitrocellulose-coated glass slides, with other well characterized saccharides as controls, constructing a microarray. The array was probed for recognition with different carbohydrate specific proteins, such as monoclonal antibodies (mAbs), and plant and mammalian lectins. Microarray analysis showed that, on one hand, the mannan recognizing mAbs 400-4 and LM21, confirmed the presence of β1,4-mannose backbone in the LBG-derived fractions. On the other hand, anti-galactomannan CCRC-M70 demonstrated binding correlation, associating recognition to the M/G ratio differences instead of the source, and beeing less tolerant to lower Mw. Microarray analysis of the two plant lectins used, RCA120 and ConA revelead that RCA120 binding to the saccharides presented, was unable to demonstrate a different identification between GMs and arabinogalactans, while ConA non significant binding confirms that only β-Man is present in the samples. Microarray analysis of the immune related lectins, DC-SIGN and SIGNR1, showed no to negligible binding, showing a potencial application of LBG fractions obtained during this study to clinical applications not involving an immune recognition.Microarrays de hidratos de carbono são um método poderoso, sensível e de alto rendimento, capacitado para a elucidação de diferentes funções fisiológicas e mecanismos patológicos. As galactomananas (GMs) são uma família de polissacarídeos cuja estrutura específica pode ter várias particularidades dependendo da origem e/ou sofrer diferentes mudanças baseada na aplicação de diferentes métodos de extração/purificação. Neste estudo, 2 lotes de GMs obtidos de goma de alfarroba (LBG) foram purificados a ≈100% de hidratos de carbono, lote 1 e 2 apresentando rácios manose/galactose (M/G) distintos, 2.4 e 3.3, respetivamente. Devido à proximidade estrutural do lote 2 às GMs de LBG reportadas, esta amostra foi escolhida para os tratamentos consequentes: dissolução e purificação por diafiltração, atingindo um rendimento em massa reprodutível de 63-67%. As amostras purificadas foram submetidas a um tratamento de hidrólise ácida parcial assistida por microondas (MW), com um rendimento em massa de 45% que permitiu uma redução de viscosidade, e um efeito despolimerizador em minutos. A aplicação de um processo de separação por ultrafiltração permitiu a criação de várias frações de diferentes pesos moleculares (Mw) criando um portefólio de GMs (6-21% de massa em cada intervalo de fração). As análises estruturais, nomeadamente açúcares neutros e metilação, foram usadas para caraterizar a estrutura dos hidratos de carbono obtidos e as principais ligações identificadas foram a 4-Manp (43-49%), 4,6-Manp (23-31%) e T-Gal (19-24%), concordantes com a composições em GMs. Os lotes 1 e 2 purificados, assim como os retentatos de alto Mw foram não-covalentemente imobilizados em suportes de nitrocelulose, em conjunto com outros sacarídeos bem caraterizados como controlos, construindo um microarray. O array foi avaliado com proteínas de especificidade conhecida para diferentes hidratos de carbono, como anticorpos monoclonais (mAbs), e lectinas de plantas e mamíferos. A análise dos microarrays mostrou que, por um lado, os mAbs que reconhecem mananas, 400-4 e LM21, confirmaram a presença de uma cadeia principal de β1,4-manose nas amostras obtidas da LBG. Por outro lado, o anti-galactomananas CCRC-M70 demonstrou uma correlação da sua ligação a diferentes rácio M/G ao invés da fonte de origem, mostrando ainda ser menos tolerante a menores Mws. A análise do microarray às duas lectinas de plantas usadas RCA120 e ConA revelaram que a ligação da RCA120 aos sacarídeos presentes, foi incapaz de demonstrar a distinção entre GMs e arabinogalactanas, enquanto as ligações não significativas de ConA demonstrou apenas a presença de β-Manose nas amostras. A análise do array às lectinas relacionadas com o sistema imune, DC-SIGN e SIGNR1, mostrou ligações inexistentes a negligenciáveis, mostrando o potencial em aplicações clínicas destas frações de LBG quando não se pretender um reconhecimento imune.2022-12-14T00:00:00Z2021-12-06T00:00:00Z2021-12-06info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10773/32832engFerreira, Bernardo Almeida de Castroinfo:eu-repo/semantics/embargoedAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-02-22T12:03:16Zoai:ria.ua.pt:10773/32832Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:04:24.083048Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Interaction study between carob galactomannans and proteins using carbohydrate microarray tecnology |
title |
Interaction study between carob galactomannans and proteins using carbohydrate microarray tecnology |
spellingShingle |
Interaction study between carob galactomannans and proteins using carbohydrate microarray tecnology Ferreira, Bernardo Almeida de Castro Carbohydrate-protein interaction Galactomannans Locust bean (carob) gum Carbohydrate microarrays Carbohydrate-binding proteins Structure-function relationship |
title_short |
Interaction study between carob galactomannans and proteins using carbohydrate microarray tecnology |
title_full |
Interaction study between carob galactomannans and proteins using carbohydrate microarray tecnology |
title_fullStr |
Interaction study between carob galactomannans and proteins using carbohydrate microarray tecnology |
title_full_unstemmed |
Interaction study between carob galactomannans and proteins using carbohydrate microarray tecnology |
title_sort |
Interaction study between carob galactomannans and proteins using carbohydrate microarray tecnology |
author |
Ferreira, Bernardo Almeida de Castro |
author_facet |
Ferreira, Bernardo Almeida de Castro |
author_role |
author |
dc.contributor.author.fl_str_mv |
Ferreira, Bernardo Almeida de Castro |
dc.subject.por.fl_str_mv |
Carbohydrate-protein interaction Galactomannans Locust bean (carob) gum Carbohydrate microarrays Carbohydrate-binding proteins Structure-function relationship |
topic |
Carbohydrate-protein interaction Galactomannans Locust bean (carob) gum Carbohydrate microarrays Carbohydrate-binding proteins Structure-function relationship |
description |
Carbohydrate microarrays are a powerful, sensitive and high-throughput method to study carbohydrate-protein interactions, enabling the elucidation of several physiological functions and pathological mechanisms. Galactomannans (GMs) are a polysaccharide family which specific structure can have several particularities upon the source and/or undergo several changes upon the application of different extraction/purification methods. In this study, 2 lots of GMs derived from locust bean gum (LBG), were purified to ≈100% of carbohydrates, lot 1 and 2, presenting distinctive mannose/galactose (M/G) ratios, 2.4 and 3.3, respectively. Due to a closer structural simillarity reported for LBG with lot 2, this sample was selected for further treatment: dissolved and purified by diafiltration achieving reproductive mass yields of 63-67%. The purified samples were submitted to a partial acid hydrolysis treatment assisted by microwaves (MW), yielding 45% of mass and allowing a viscosity reduction, and depolymerisation effect occuring in minutes time-length. The aplication of an ultrafiltration separation process allowed a large portfolio of GMs with different molecular weights (Mw) fractions (6-21% of mass in each range of cut-off). Structural analysis, both neutral sugars and methylation, were used to characterise the structure of the carbohydrates obtained, and the main glycosidic bonds identified were 4-Manp (43-49%), 4,6-Manp (23-31%) and T-Gal (19-24%) in accordance with GMs composition. The purified LBG lot 1 and 2, and higher Mw retentates were non-convalently immobilized onto nitrocellulose-coated glass slides, with other well characterized saccharides as controls, constructing a microarray. The array was probed for recognition with different carbohydrate specific proteins, such as monoclonal antibodies (mAbs), and plant and mammalian lectins. Microarray analysis showed that, on one hand, the mannan recognizing mAbs 400-4 and LM21, confirmed the presence of β1,4-mannose backbone in the LBG-derived fractions. On the other hand, anti-galactomannan CCRC-M70 demonstrated binding correlation, associating recognition to the M/G ratio differences instead of the source, and beeing less tolerant to lower Mw. Microarray analysis of the two plant lectins used, RCA120 and ConA revelead that RCA120 binding to the saccharides presented, was unable to demonstrate a different identification between GMs and arabinogalactans, while ConA non significant binding confirms that only β-Man is present in the samples. Microarray analysis of the immune related lectins, DC-SIGN and SIGNR1, showed no to negligible binding, showing a potencial application of LBG fractions obtained during this study to clinical applications not involving an immune recognition. |
publishDate |
2021 |
dc.date.none.fl_str_mv |
2021-12-06T00:00:00Z 2021-12-06 2022-12-14T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10773/32832 |
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http://hdl.handle.net/10773/32832 |
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eng |
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eng |
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info:eu-repo/semantics/embargoedAccess |
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embargoedAccess |
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application/pdf |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
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RCAAP |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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