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Mechanism of glycosylation orchestrated by C1GalT1 and ST6GalNAc-I enzymes unveiled by NMR
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A O-glicosilação do tipo mucina é um mecanismo complexo altamente regulado pela atividade coordenada de enzimas específicas denominadas glicosiltransferases (GTs). No cancro, a desregulação da sua expressão e/ou atividade é comum, e conduz a uma glicosilação aberrante e à formação de antigénios de hidratos de carbono associados a tumores (TACAs). Os antigénios TF- (Galβ1-3GalNAcα1-O-Ser/Thr) e STn- (Neu5Acα2-6GalNAcα1-O-Ser/Thr) são dois dos TACAs mais encontrados em células cancerígenas. O antigénio TF é catalisado pela C1GalT1, uma GT com mecanismo de inversão. Esta enzima transfere uma molécula de galactose (Gal) do dador UDP-α-Gal para glicopéptidos que contêm o antigénio Tn (GalNAcα1-O-Ser/Thr). Este antigénio existe normalmente em células saudáveis mascarado por outros O-glicanos complexos. Contudo, no cancro, fica exposto. Já o antigénio STn é exclusivo das células cancerígenas. Através de um mecanismo de inversão, a enzima ST6GalNAc-I transfere um resíduo de ácido siálico (Neu5Ac) do dador CMP-β-Neu5Ac para substratos que contenham GalNAc. Contudo, apesar da relevância biológica das enzimas C1GalT1 e ST6GalNAc-I tanto na saúde como na doença, o seu mecanismo de ação a nível molecular ainda permanece relativamente desconhecido.
Tendo em conta a falta de informação estrutural, conformacional e mecanística destas enzimas, o foco desta tese é compreender o seu mecanismo molecular de O-glicosilação usando a mucina 1 (MUC1) como modelo. Desta forma, utilizou-se uma estratégia multidisciplinar que combina técnicas de RMN com simulações de dinâmica molecular, protocolos de biologia molecular, cristalografia de raio-X e outras técnicas biofísicas para o fazer. A tese está organizada em dois capítulos científicos onde se pretende determinar como a C1GalT1 reconhece os seus substratos a nível atómico, através de uma combinação de cristalografia de raio-X, métodos calorimétricos, simulações de dinâmica molecular e métodos de RMN baseados na deteção do ligando assim como elucidar o mecanismo de glicosilação da MUC1 pela C1GalT1 e a ST6GalNAc-I através de técnicas de RMN.
O capítulo 3 determina o processo de reconhecimento molecular de glicopéptidos contendo GalNAc pela C1GalT1. Neste capítulo foi demonstrado que a C1GalT1 é uma metaloenzima com um mecanismo de inversão do tipo SN2 que necessita de UDP-Gal e MnCl2 para se ligar especificamente a péptidos contendo GalNAc. A C1GalT1 reconhece os seus substratos através da unidade de GalNAc, porém a sequência peptídica estabelece interações adicionais que permitem estabilizar o complexo. Curiosamente, a C1GalT1 parece necessitar de uma conformação alternada para reconhecer os seus substratos, o que pode explicar a capacidade desta enzima para galactosilar ambos os resíduos Tn-Thr e Tn-Ser.
No capítulo 4, as preferências de glicosilação das enzimas C1GalT1 e ST6GalNAc-I foram determinadas para diferentes glicodomínios da MUC1, usando como modelo um glicodomínio com quatro repetições em tandem ((GVT*S*APDT*RPAPGS*T*APPAH)4, MUC14TR, * representa os possíveis sítios de glicosilação) desta mucina. Para a galactosilação observou-se que a atividade da C1GalT1 é influenciada por substratos com sítios de glicosilação adjacentes em substratos complexos, enquanto para sítios de glicosilação isolados a conformação e/ou sequência peptídica parece governar a sua atividade. O mesmo não foi observado para a ST6GalNAc-I. Enquanto a ST6GalNAc-I prefere glicosilar Tn-Thr, a C1GalT1 glicosila Tn-Thr e Tn-Ser identicamente. Em termos de conformação, a adição do segundo açúcar apenas afeta a conformação local do sítio de glicosilação, sem impactar a conformação global da MUC1. A alteração conformacional global ocorre após a adição de GalNAc pelas GalNAc-Ts. A informação estrutural obtida permitirá no futuro desenvolver de forma racional novos glicodomínios MUC1 com diferentes densidades de antigénios TF e STn.
Mucin-type O-glycosylation is a complex mechanism regulated by the coordinated activity of specific glycosyltransferases (GTs). Altered regulation of several GTs is a common feature of cancer which yields tumour-associated carbohydrate antigens (TACAs). Two of the most popular TACAs are the TF- (Galβ1-3GalNAcα1-O-Ser/Thr) and STn- (Neu5Acα2-6GalNAcα1-O-Ser/Thr) antigens. The TF-antigen is formed by the inverting C1GalT1 enzyme, which transfers a Gal residue from the sugar donor UDP-α-Gal to Tn- (GalNAcα1-O-Ser/Thr) peptides. This antigen is naturally present as a hidden part of more complex O-glycans, however, truncation of O-glycans is a common characteristic of cancer cells, which exposes TF-epitopes in malignancy. The STn-antigen, exclusively found in cancer cells, is generated by the inverting ST6GalNAc-I enzyme, which adds a Neu5Ac residue from CMP-β-Neu5Ac sugar donor to GalNAc-containing substrates. Despite the significance of C1GalT1 and ST6GalNAc-I in health and disease, the molecular mechanisms underlying the recognition and catalysis of these enzymes remained elusive during the initial stages of this thesis. On this basis, this thesis is focused on deciphering the molecular mechanism of mucin-1 (MUC1) O-glycosylation by C1GalT1 and ST6GalNAc-I, through the concerted integration of NMR with molecular dynamics (MD) simulations, and assisted by molecular biology protocols, X-ray crystallography and other biophysical techniques. This thesis is organized in two main scientific chapters: 1) the substrate molecular recognition mechanism of C1GalT1 through the combination of ligand-based NMR methods, X-ray crystallography, MD simulations and calorimetric measurements and 2) the elucidation of MUC1 glycosylation mechanism by C1GalT1 and ST6GalNAc-I unveiled by NMR. Chapter 3 describes the structural insights for the substrate recognition and the synthesis of TF-antigen by C1GalT1. It is demonstrated that C1GalT1 is a metal-dependent inverting GT that follows an SN2 mechanism and needs UDP-Gal and MnCl2 for specific binding to Tn-peptides. The binding event is mainly driven by the GalNAc unit while the peptide sequence provides optimal kinetic and binding parameters. Key highlight is the evidence that C1GalT1 recognizes both Tn-Thr and Tn-Ser in a similar staggered conformation, which might explain the activity of C1GalT1 targeting both types of substrates in mucins. In chapter 4, the glycosylation preferences of C1GalT1 and ST6GalNAc-I, targeting a four tandem repeat (TR) of MUC1 Tn-glycodomain ((GVT*S*APDT*RPAPGS*T*APPAH)4, MUC14TR, * Tn possible glycosylation sites), were scrutinized. Neighbouring glycosylation governs galactosylation by C1GalT1 in clusters of Tn-glycodomains, while in isolated glycosylation sites the peptide sequence or/and peptide conformation seems to play a role. For ST6GalNAc-I, this effect is not observed. However, ST6GalNAc-I prefers to glycosylate Tn-Thr over Tn-Ser, while C1GalT1 identically glycosylates Tn-Ser and Tn-Thr sites. Lastly, the introduction of the second sugar moiety affects the local conformation surrounding the glycosylation site, however, does not have a major impact on the peptide backbone conformation of MUC1. The main conformational change on the MUC1 peptide backbone is dictated by the first GalNAc residue introduced by GalNAc-Ts. All the structural information uncovered in chapter 4 can now be used to precise engineer TF- and STn-MUC1 glycodomains with different densities.
Mucin-type O-glycosylation is a complex mechanism regulated by the coordinated activity of specific glycosyltransferases (GTs). Altered regulation of several GTs is a common feature of cancer which yields tumour-associated carbohydrate antigens (TACAs). Two of the most popular TACAs are the TF- (Galβ1-3GalNAcα1-O-Ser/Thr) and STn- (Neu5Acα2-6GalNAcα1-O-Ser/Thr) antigens. The TF-antigen is formed by the inverting C1GalT1 enzyme, which transfers a Gal residue from the sugar donor UDP-α-Gal to Tn- (GalNAcα1-O-Ser/Thr) peptides. This antigen is naturally present as a hidden part of more complex O-glycans, however, truncation of O-glycans is a common characteristic of cancer cells, which exposes TF-epitopes in malignancy. The STn-antigen, exclusively found in cancer cells, is generated by the inverting ST6GalNAc-I enzyme, which adds a Neu5Ac residue from CMP-β-Neu5Ac sugar donor to GalNAc-containing substrates. Despite the significance of C1GalT1 and ST6GalNAc-I in health and disease, the molecular mechanisms underlying the recognition and catalysis of these enzymes remained elusive during the initial stages of this thesis. On this basis, this thesis is focused on deciphering the molecular mechanism of mucin-1 (MUC1) O-glycosylation by C1GalT1 and ST6GalNAc-I, through the concerted integration of NMR with molecular dynamics (MD) simulations, and assisted by molecular biology protocols, X-ray crystallography and other biophysical techniques. This thesis is organized in two main scientific chapters: 1) the substrate molecular recognition mechanism of C1GalT1 through the combination of ligand-based NMR methods, X-ray crystallography, MD simulations and calorimetric measurements and 2) the elucidation of MUC1 glycosylation mechanism by C1GalT1 and ST6GalNAc-I unveiled by NMR. Chapter 3 describes the structural insights for the substrate recognition and the synthesis of TF-antigen by C1GalT1. It is demonstrated that C1GalT1 is a metal-dependent inverting GT that follows an SN2 mechanism and needs UDP-Gal and MnCl2 for specific binding to Tn-peptides. The binding event is mainly driven by the GalNAc unit while the peptide sequence provides optimal kinetic and binding parameters. Key highlight is the evidence that C1GalT1 recognizes both Tn-Thr and Tn-Ser in a similar staggered conformation, which might explain the activity of C1GalT1 targeting both types of substrates in mucins. In chapter 4, the glycosylation preferences of C1GalT1 and ST6GalNAc-I, targeting a four tandem repeat (TR) of MUC1 Tn-glycodomain ((GVT*S*APDT*RPAPGS*T*APPAH)4, MUC14TR, * Tn possible glycosylation sites), were scrutinized. Neighbouring glycosylation governs galactosylation by C1GalT1 in clusters of Tn-glycodomains, while in isolated glycosylation sites the peptide sequence or/and peptide conformation seems to play a role. For ST6GalNAc-I, this effect is not observed. However, ST6GalNAc-I prefers to glycosylate Tn-Thr over Tn-Ser, while C1GalT1 identically glycosylates Tn-Ser and Tn-Thr sites. Lastly, the introduction of the second sugar moiety affects the local conformation surrounding the glycosylation site, however, does not have a major impact on the peptide backbone conformation of MUC1. The main conformational change on the MUC1 peptide backbone is dictated by the first GalNAc residue introduced by GalNAc-Ts. All the structural information uncovered in chapter 4 can now be used to precise engineer TF- and STn-MUC1 glycodomains with different densities.
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Palavras-chave
Glycosylation C1GalT1 ST6GalNAc-I MUC1 NMR Spectroscopy Molecular Dynamics simulations