Tráfego de vesículas na biossíntese do heparam sulfato: enfoque na rota retículo endoplasmático-golgi
Data
2019-07-25
Autores
Meneghetti, Maria Cecilia Zorel 
Orientadores
Lima, Marcelo Andrade de
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Desde a descrição do processo de biossíntese do heparam sulfato (HS), tem- se assumido que as modificações encontradas nesses polissacarídeos – polimerização, sulfatação e epimerização - ocorrem por meio de uma sequência hierárquica de eventos enzimáticos, sendo a última enzima desse processo a 3-O- sulfotransferase (HS3ST). Contudo, estudos recentes do nosso laboratório mostraram que a HS3ST poderia agir em diferentes estágios da biossíntese do HS. Assim, os diferentes padrões de substituição encontrados nesses glicosaminoglicanos poderiam resultar de distintos padrões de localização e/ou de transporte das enzimas de biossíntese na via RER-Golgi. Logo, o objetivo deste trabalho foi avaliar a influência do tráfego vesicular mediado pelas vesículas COPI e COPII na biossíntese do HS em células endoteliais de cordão umbilical transfectadas para a HS3ST5. As células transfectadas apresentaram uma pequena diminuição de expressão de sindecam-1 e sindecam-3, proteoglicanos de HS, quando comparado às células selvagens, contudo o perfil de glicosaminoglicanos sulfatados não foi alterado. Em relação ao tráfego vesicular, a enzima HS3ST5 apresentou uma distribuição semelhante entre as vesículas e as regiões do aparelho de golgi, contudo essa distribuição foi alterada na presença de agentes farmacológicos capazes de hiperestimular a síntese de HS, heparina, e inibir o tráfego vesicular, brefeldin A. Durante as primeiras horas de estímulo com heparina, a HS3ST5 localizou preferencialmente no trans golgi e essa localização coincidiu com uma mudança na composição dissacarídica do HS no extrato celular e no meio, indicando que o tráfego vesicular influencia a distribuição das enzimas na rota secretória e, consequentemente, no acesso ao substrato. Além disso, não houve aumento de expressão proteica das demais enzimas de biossíntese do HS e nem aumento de expressão gênica das PAPS sintases. Sob influência da heparina, a expressão gênica das subunidades de COPI e das isoformas de Sec24 da vesícula COPII, responsáveis pela seleção de carga, e das proteínas Rab1A, Rab1B e Rab2A, responsáveis pela especificidade do tráfego, foram alteradas, evidenciando o papel da regulação do tráfego na biossíntese do HS. Já na presença de brefeldin A, a HS3ST5 localizou preferencialmente em vesículas COPII e no trans golgi. Além disso, colocalizou com as demais enzimas modificadoras de HS de maneira semelhante. Portanto, nossos resultados mostraram que o tráfego vesicular xx influencia regula a biossíntese do HS por interferir na seleção de carga e na localização das enzimas nas cisternas do golgi, resultando em diferentes padrões de substituição encontrados no HS.
Since the byosynthesis description of the (HS) heparan sulfate, it has been assumed that the modifications found in these polysaccharides - sulfation and epimerization - occur through a hierarchical sequence of enzymatic events, being the last enzyme 3-O-sulfotransferase (HS3ST). However, recent studies from our laboratory have shown that HS3ST could act at different stages of the biosynthesis and on N-acetylated substrates. These different substitution patterns found in these glycosaminoglycans could result from different localization and transport patterns of the biosynthesis enzymes in the RER-Golgi pathway. Therefore, the aim of this work was to evaluate the influence of vesicular traffic mediated by COPI and COPII vesicles on the biosynthesis of HS in umbilical cord endothelial cells transfected for HS3ST5. Transfected cells showed a small decrease in expression of sindecam-1 and sindecam-3, HS proteoglycans, when compared to wild type cells, yet the sulfated glycosaminoglycan profile was not altered. In relation to vesicular traffic, the enzyme HS3ST5 showed a similar distribution between vesicles and golgi regions, but this distribution was altered in the presence of heparin and brefeldin A (BFA). Among the first hours of stimulation with heparin, HS3ST5 located preferentially in the trans golgi apparatus and this displacement coincided with a change in the disaccharide composition of the HS in the cell extract and in the medium, indicating that the vesicular traffic influences the distribution of the enzymes in the secretory route and, consequently, the access to the substrate. In addition, there were no increase in protein expression of the other HS biosynthesis enzymes and no increase in the gene expression of PAPS synthases. Under the influence of heparin, the genic expression of COPI subunits and the Sec24 isoforms of the COPII vesicle responsible for selection of cargo, and the Rab1A, Rab1B and Rab2A proteins, responsible for the specificity of the traffic, were altered, evidencing a role of heparin in traffic regulation. In the presence of BFA, HS3ST5 localized preferentially in COPII vesicles and trans golgi apparatus. In addition, it colocalized with the other HS modifying enzymes in a similar manner. Therefore, our results showed that vesicular traffic influences the regulation of HS biosynthesis by interfering with charge selection and the location of enzymes in golgi cisterns, which could result in different substitution patterns found in HS.
Since the byosynthesis description of the (HS) heparan sulfate, it has been assumed that the modifications found in these polysaccharides - sulfation and epimerization - occur through a hierarchical sequence of enzymatic events, being the last enzyme 3-O-sulfotransferase (HS3ST). However, recent studies from our laboratory have shown that HS3ST could act at different stages of the biosynthesis and on N-acetylated substrates. These different substitution patterns found in these glycosaminoglycans could result from different localization and transport patterns of the biosynthesis enzymes in the RER-Golgi pathway. Therefore, the aim of this work was to evaluate the influence of vesicular traffic mediated by COPI and COPII vesicles on the biosynthesis of HS in umbilical cord endothelial cells transfected for HS3ST5. Transfected cells showed a small decrease in expression of sindecam-1 and sindecam-3, HS proteoglycans, when compared to wild type cells, yet the sulfated glycosaminoglycan profile was not altered. In relation to vesicular traffic, the enzyme HS3ST5 showed a similar distribution between vesicles and golgi regions, but this distribution was altered in the presence of heparin and brefeldin A (BFA). Among the first hours of stimulation with heparin, HS3ST5 located preferentially in the trans golgi apparatus and this displacement coincided with a change in the disaccharide composition of the HS in the cell extract and in the medium, indicating that the vesicular traffic influences the distribution of the enzymes in the secretory route and, consequently, the access to the substrate. In addition, there were no increase in protein expression of the other HS biosynthesis enzymes and no increase in the gene expression of PAPS synthases. Under the influence of heparin, the genic expression of COPI subunits and the Sec24 isoforms of the COPII vesicle responsible for selection of cargo, and the Rab1A, Rab1B and Rab2A proteins, responsible for the specificity of the traffic, were altered, evidencing a role of heparin in traffic regulation. In the presence of BFA, HS3ST5 localized preferentially in COPII vesicles and trans golgi apparatus. In addition, it colocalized with the other HS modifying enzymes in a similar manner. Therefore, our results showed that vesicular traffic influences the regulation of HS biosynthesis by interfering with charge selection and the location of enzymes in golgi cisterns, which could result in different substitution patterns found in HS.
Descrição
Citação
MENEGHETTI, Maria Cecilia Zorél. Tráfego de vesículas na biossíntese do heparam sulfato: enfoque na rota retículo endoplasmático-golgi. 2019. 146f. Tese (Doutorado em Biologia Molecular) – Escola Paulista de Medicina, Universidade Federal de São Paulo. São Paulo, 2019.