Estudio d las preferencias de glicosilación de las pp-GalNAc-Ts: implicación en el desarrollo de inhibidores

Esta tesis se enmarca en el estudio de las GalNAc transferasas polipeptídicas (ppGalNAc-Ts), proteínas encargadas de transferir un residuo de GalNAc desde UDP-GalNAc a cadenas laterales de Ser/Thr, iniciando así la glicosilación encontrada en un gran número de proteínas como es el caso de las mucinas. Las 20 isoformas de ppGalNAc-Ts encontradas en mamíferos se han clasificado en dos grandes clases, en función de sus diferentes especificidades por sustratos aceptores no modificados o previamente glicosilados. Todas ellas contienen un dominio catalítico en el N-terminal unido por un linker flexible a un dominio de tipo lectina en el extremo C-terminal; este último es el que las diferencia de las glicosiltransferasas encontradas en la naturaleza. La importancia de esta familia de enzimas radica en que la glicosilación tipo “O- GalNAc” llevada a cabo por las mismas es la más abundante, a la par que la más compleja y regulada, de todas las modificaciones postraduccionales. Son, además, responsables de glicosilar a más del 80% de todas las proteínas que pasan a través de la vía secretora. Estas isoformas de ppGalNAc-Ts, si bien no parecen ser esenciales en mamíferos, desempeñan papeles importantes en fisiología. Un ejemplo claro de su importancia es la presencia de calcinosis tumoral en personas que presentan una actividad parcial o nula de la isoforma GalNAc-T3, causada por mutaciones presentes en la misma, que impide la correcta glicosilación del factor de crecimiento de fibroblasto 23 (FGF23). Otro ejemplo es el de la presencia homozigótica del mutante F104S en la isoforma GalNAc-T2, que conlleva niveles bajos de HDL en humanos, monos y roedores (Khetarpal, S. A. et al., Cell Metab, 2016, 24(2), 234-245). Sin embargo, y pese a que se conocen las estructuras de varias de las GalNAc-Ts, todavía no están bien definidos los requerimientos necesarios para llevar a cabo la glicosilación de proteínas, ni los aspectos sobre la dinámica global de su interacción con diferentes ligandos. Es, así, en este contexto en el que se ha elaborado esta tesis doctoral, estructurada según la modalidad de compendio de artículos. En ella se recogen cinco publicaciones, todas estrechamente relacionadas entre sí y centradas en el estudio y caracterización bioquímica y estructural de diversas isoformas de estas enzimas ppGalNAc-Ts, así como de diversos mutantes de las mismas. Con ellas se pretende: a) Ahondar en la justificación molecular del selectivo y regulado comportamiento de algunas de las isoformas más representativas de las ppGalNAc-Ts (Lira-Navarrete, de las Rivas et al., 2015; de las Rivas et al., 2017). b) Llevar a cabo estudios de orientación más aplicada en los que, desde la comprensión de las bases moleculares subyacentes, se intentan desarrollar moduladores selectivos de la actividad enzimática (Ghirardello, de las Rivas et al., 2016; Liu, Xu, Xu, de las Rivas et al., 2017) y se propone una explicación para la inactivación de una variante patogénica de la HsGalNAc-T2 asociada con problemas en el metabolismo del colesterol y cardiovasculares (de las Rivas et al., 2018). Referencia completa de las publicaciones que constituyen el compendio de artículos recogido en la tesis: -Lira-Navarrete, E., DE LAS RIVAS, M., Compañón, I., Carmen Pallarés, M. C., Kong, Y., Iglesias-Fernández, J., Bernades, G. J. L., Peregrina, J. M., Rovira, C., Bernadó, P., Bruscolini, P., Clausen, H., Lostao, A., Corzana, F. & Hurtado-Guerrero, R. (2015). Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation. Nature Communications, 6(6937), DOI: 10.1038/ncomms7937.-DE LAS RIVAS, M., Lira-Navarrete, E., James Paul Daniel, E., Compañón, I., Coelho, H., Diniz, A., Jiménez-Barbero, J., Peregrina, J. M., Clausen, H., Corzana, F., Marcelo, F., Jiménez-Osés, F., Gerken, . A. & Hurtado-Guerrero, R. (2017). The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences. Nature Communications, 8(1959), DOI: 10.1038/s41467-017-02006-0. -Ghirardello, M., DE LAS RIVAS, M., Lacetera, A., Delso, I., Lira-Navarrete, E,. Tejero, T., Martín-Santamaría, S., Hurtado-Guerrero, R., & Merino, P. (2016). Glycomimetics Targeting Glycosyltransferases: Synthetic, Computational and Structural Studies of Less-Polar Conjugates, Chemistry - A European Journal, 22, 7215-7224.-Liu, F., Xu, K., Xu, Z., DE LAS RIVAS, M., Wang, C., Li, X., Lu, J., Zhou, Y., Delso, I., Merino, P., Hurtado-Guerrero, R., Zhang, Y. & Wu, F. (2017). The small molecule luteolin inhibits N-acetyl-alpha-galactosaminyltransferases and reduces mucin-type O-glycosylation of amyloid precursor protein. Journal of Biological Chemistry, 292(52), pp. 21304-21319. -DE LAS RIVAS, M., Coelho, H., Diniz, A., Lira-Navarrete, E., Compañón, I., Jiménez-Barberó, J., Schjoldager, K., T., Bennett, E. P., Vakhrushev, S. Y., Clausen, H., Corzana, F., Marcelo, F. & Hurtado-Guerrero, R. (2018). Structural analysis of a GalNAc-T2 mutant reveals an induced-fit catalytic mechanism for GalNAc-Ts. Chemistry - A European Journal. DOI: 10.1002/chem.201800701.<br /

Plasmodium falciparum Apicomplexan-Specific Glucosamine-6-Phosphate <i>N</i>-Acetyltransferase Is Key for Amino Sugar Metabolism and Asexual Blood Stage Development.

--- - i: - N - N - O - N - Plasmodium falciparum - Cryptosporidium parvum - P. falciparum - N - N - P. falciparum - C. parvum b: - IMPORTANCE content: - UDP- - "-acetylglucosamine (UDP-GlcNAc), the main product of the hexosamine biosynthetic pathway, is an important metabolite in protozoan parasites since its sugar moiety is incorporated into glycosylphosphatidylinositol (GPI) glycolipids and " - "- and " - "-linked glycans. Apicomplexan parasites have a hexosamine pathway comparable to other eukaryotic organisms, with the exception of the glucosamine-phosphate " - "-acetyltransferase (GNA1) enzymatic step that has an independent evolutionary origin and significant differences from nonapicomplexan GNA1s. By using conditional genetic engineering, we demonstrate the requirement of GNA1 for the generation of a pool of UDP-GlcNAc and for the development of intraerythrocytic asexual " - " parasites. Furthermore, we present the 1.95\xE2\x80\x89\xC3\x85 resolution structure of the GNA1 ortholog from " - ", an apicomplexan parasite which is a leading cause of diarrhea in developing countries, as a surrogate for " - " GNA1. The in-depth analysis of the crystal shows the presence of specific residues relevant for GNA1 enzymatic activity that are further investigated by the creation of site-specific mutants. The experiments reveal distinct features in apicomplexan GNA1 enzymes that could be exploitable for the generation of selective inhibitors against these parasites, by targeting the hexosamine pathway. This work underscores the potential of apicomplexan GNA1 as a drug target against malaria." - " Apicomplexan parasites cause a major burden on global health and economy. The absence of treatments, the emergence of resistances against available therapies, and the parasite's ability to manipulate host cells and evade immune systems highlight the urgent need to characterize new drug targets to treat infections caused by these parasites. We demonstrate that glucosamine-6-phosphate " - -acetyltransferase (GNA1), required for the biosynthesis of UDP- - "-acetylglucosamine (UDP-GlcNAc), is essential for " - " asexual blood stage development and that the disruption of the gene encoding this enzyme quickly causes the death of the parasite within a life cycle. The high-resolution crystal structure of the GNA1 ortholog from the apicomplexan parasite " - ", used here as a surrogate, highlights significant differences from human GNA1. These divergences can be exploited for the design of specific inhibitors against the malaria parasite.

The small molecule luteolin inhibits N-acetyl-α-galactosaminyltransferases and reduces mucin-type O-glycosylation of amyloid precursor protein

Mucin-type O-glycosylation is the most abundant type of O-glycosylation. It is initiated by the members of the polypeptide N-acetyl-α-galactosaminyltransferase (ppGalNAc-T) family and closely associated with both physiological and pathological conditions, such as coronary artery disease or Alzheimer's disease. The lack of direct and selective inhibitors of ppGalNAc-Ts has largely impeded research progress in understanding the molecular events in mucin-type O-glycosylation. Here, we report that a small molecule, the plant flavonoid luteolin, selectively inhibits ppGalNAc-Ts in vitro and in cells. We found that luteolin inhibits ppGalNAc-T2 in a peptide/protein-competitive manner but not promiscuously (e.g. via aggregation-based activity). X-ray structural analysis revealed that luteolin binds to the PXP motif-binding site found in most protein substrates, which was further validated by comparing the interactions of luteolin with wild-type enzyme and with mutants using 1H NMR-based binding experiments. Functional studies disclosed that luteolin at least partially reduced production of β-amyloid protein by selectively inhibiting the activity of ppGalNAc-T isoforms. In conclusion, our study provides key structural and functional details on luteolin inhibiting ppGalNAc-T activity, opening up the way for further optimization of more potent and specific ppGalNAc-T inhibitors. Moreover, our findings may inform future investigations into site-specific O-GalNAc glycosylation and into the molecular mechanism of luteolin-mediated ppGalNAc-T inhibition.This work was supported by the National Basic Research Program of China Grants 2012CB822103 and 2011CB910603 (to Y. Z.); National High Technology Research and Development Program of China Grant 2012AA020203 (to Y. Z.); National Natural Science Foundation Grants 31170771 (to Y. Z.), 31370806 (to Y. Z.), and 31570796 (to Y. Z.); National Basic Research Program of China Grant 2012CB822103 (to F. W.); National Natural Science Foundation Grants 31270853 and 81102377 (to F. W.); Agencia Aragonesa para la Investigación y Desarrollo (ARAID), Ministerio de Economía y Competitividad, Grants CTQ2013-44367-C2-2-P and BFU2016-75633-P (to R. H.-G.); Diputación General de Aragón (DGA) Grant B89 (to R. H.-G.); and the EU Seventh Framework Programme (2007–2013) under BioStruct-X (Grant Agreement 283570 and BIOSTRUCTX 5186) (to R. H.-G.).Peer Reviewe

Structural and mechanistic insights into the catalytic-domain-mediated short-range glycosylation preferences of GalNAc-T4

17 pags, 4 figs, 2 tabsMucin-type O-glycosylation is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) which are type-II transmembrane proteins that contain Golgi luminal catalytic and lectin domains that are connected by a flexible linker. Several GalNAc-Ts, including GalNAc-T4, show both long-range and short-range prior glycosylation specificity, governed by their lectin and catalytic domains, respectively. While the mechanism of the lectin-domain-dependent glycosylation is well-known, the molecular basis for the catalytic-domain-dependent glycosylation of glycopeptides is unclear. Herein, we report the crystal structure of GalNAc-T4 bound to the diglycopeptide GAT GAGAGAGT TPGPG (containing two α-GalNAc glycosylated Thr (T ), the PXP motif and a "naked" Thr acceptor site) that describes its catalytic domain glycopeptide GalNAc binding site. Kinetic studies of wild-type and GalNAc binding site mutant enzymes show the lectin domain GalNAc binding activity dominates over the catalytic domain GalNAc binding activity and that these activities can be independently eliminated. Surprisingly, a flexible loop protruding from the lectin domain was found essential for the optimal activity of the catalytic domain. This work provides the first structural basis for the short-range glycosylation preferences of a GalNAc-T.We thank synchrotron radiation sources DLS (Oxford) and in particular beamline I03 (experiment number MX10121-15). We thank ARAID, MEC (CTQ2013-44367-C2-2-P, BFU2016-75633-P, CTQ2015-67727-R, CTQ2015-70524-R, and CTQ2017-85496-P), AGAUR (SGR2017-1189), the National Institutes of Health (R01-GM113534, and instrument Grant GM113534-01S to T. A. Gerken), the Danish National Research Foundation (DNRF107), the FCT-Portugal [UID/Multi/04378/2013 cofinanced by the FEDER (POCI- 01-0145-FEDER-007728)], and the DGA (E34_R17) for financial support. I. Compañón thanks Universidad de La Rioja for the FPI grant. F. Marcelo thanks FCT-Portugal for IF Investigator grant (IF/00780/2015) and PTNMR supported by Project 022161. E. Lira-Navarrete acknowledges her postdoctoral EMBO fellowship ALTF 1553-2015 cofunded by the European Commission (LTFCOFUND2013, GA-2013-609409) and Marie Curie Actions. H. Coelho and J. Jiménez-Barbero thank EU for the TOLLerant project. The research leading to these results has also received funding from the FP7 (2007−2013) under BioStruct-X (Grant agreement 283570 and BIOSTRUCTX_5186). We would also like to acknowledge the assistance of Juwan Lee in obtaining the GalNAc-T4 random peptide motif

The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences

11 pags, 3 figs, 2 tabsThe polypeptide GalNAc-transferases (GalNAc-Ts), that initiate mucin-type O-glycosylation, consist of a catalytic and a lectin domain connected by a flexible linker. In addition to recognizing polypeptide sequence, the GalNAc-Ts exhibit unique long-range N- A nd/or C-terminal prior glycosylation (GalNAc-O-Ser/Thr) preferences modulated by the lectin domain. Here we report studies on GalNAc-T4 that reveal the origins of its unique N-terminal long-range glycopeptide specificity, which is the opposite of GalNAc-T2. The GalNAc-T4 structure bound to a monoglycopeptide shows that the GalNAc-binding site of its lectin domain is rotated relative to the homologous GalNAc-T2 structure, explaining their different long-range preferences. Kinetics and molecular dynamics simulations on several GalNAc-T2 flexible linker constructs show altered remote prior glycosylation preferences, confirming that the flexible linker dictates the rotation of the lectin domain, thus modulating the GalNAc-Ts' long-range preferences. This work for the first time provides the structural basis for the different remote prior glycosylation preferences of the GalNAc-Ts.We thank synchrotron radiation sources DLS (Oxford) and in particular beamline I03 (experiment number MX10121-7). We thank ARAID, MEC (CTQ2013-44367-C2-2-P, BFU2016-75633-P, CTQ2015-67727-R, CTQ2015-70524-R, and RYC-2013-14706), the National Institutes of Health (GM113534, and instrument grant GM113534-01S), the Danish National Research Foundation (DNRF107), the FCT-Portugal (UID/Multi/04378/2013 and PTNMR Project No 022161), and the DGA (B89) for the financial support. I.C. thanks Universidad de La Rioja for the FPI grant. F.M. thanks FCT-Portugal for IF Investigator. E.L.-N. acknowledges her postdoctoral EMBO fellowship ALTF 1553-2015 co-funded by the European Commission (LTFCOFUND2013, GA-2013-609409) and Marie Curie Actions. H.C. and J.J.-B. thank EU for the TOLLerant project. The research leading to these results has also received funding from the FP7 (2007-2013) under BioStruct-X (grant agreement No. 283570 and BIOSTRUCTX_5186). We also thank BIFI (Memento cluster) and CESGA for computer support.Peer reviewe

Atomic and Specificity Details of Mucin 1 O-Glycosylation Process by Multiple Polypeptide GalNAc-Transferase Isoforms Unveiled by NMR and Molecular Modeling

IF/00780/2015 PTDC/BIA-MIB/31028/2017 UIDP/04378/2020 UIDB/04378/2020 LA/P/0140/2020 SFRH/BD/140394/2018 PD/BD/142847/2018 PD00065/2013 DL 57/2016 ROTEIRO/0031/2013-PINFRA/22161/2016 BFU2016-75633-P PID2019-105451GB-I00 E34_R17 LMP58_18 to R.H-G RTI2018-099592-B-C21 ITN, GA-642157 COST Action GLYCONanoProbes (CA18132) ERC-2017-AdG, project number 788143-RECGLYCANMR RTI218-094751-B-C21) DNRF107The large family of polypeptide GalNAc-transferases (GalNAc-Ts) controls with precision how GalNAc O-glycans are added in the tandem repeat regions of mucins (e.g., MUC1). However, the structural features behind the creation of well-defined and clustered patterns of O-glycans in mucins are poorly understood. In this context, herein, we disclose the full process of MUC1 O-glycosylation by GalNAc-T2/T3/T4 isoforms by NMR spectroscopy assisted by molecular modeling protocols. By using MUC1, with four tandem repeat domains as a substrate, we confirmed the glycosylation preferences of different GalNAc-Ts isoforms and highlighted the importance of the lectin domain in the glycosylation site selection after the addition of the first GalNAc residue. In a glycosylated substrate, with yet multiple acceptor sites, the lectin domain contributes to orientate acceptor sites to the catalytic domain. Our experiments suggest that during this process, neighboring tandem repeats are critical for further glycosylation of acceptor sites by GalNAc-T2/T4 in a lectin-assisted manner. Our studies also show local conformational changes in the peptide backbone during incorporation of GalNAc residues, which might explain GalNAc-T2/T3/T4 fine specificities toward the MUC1 substrate. Interestingly, we postulate that a specific salt-bridge and the inverse γ-turn conformation of the PDTRP sequence in MUC1 are the main structural motifs behind the GalNAc-T4 specificity toward this region. In addition, in-cell analysis shows that the GalNAc-T4 isoform is the only isoform glycosylating the Thr of the immunogenic epitope PDTRP in vivo, which highlights the relevance of GalNAc-T4 in the glycosylation of this epitope. Finally, the NMR methodology established herein can be extended to other glycosyltransferases, such as C1GalT1 and ST6GalNAc-I, to determine the specificity toward complex mucin acceptor substrates.publishersversionepub_ahead_of_prin

Realización del congreso Relevance of Physical and Chemical Sciences and Scientists from a Socio-Historical Perspective por parte de los alumnos de la asignatura de Física y Química de 3º ESO del Colegio Privado Bilingüe Juan de Lanuza, en Zaragoza.

This Master's degree project aims at reducing Secondary students' demotivation and lack of interest regarding scientific subjects, with our main focus placed on Physics and Chemistry. Aiming to understand the origin and underlying causes of this attitudinal estrangement, a bibliographic revision has been carried out, revealing that the main trigger of such a estrangement is thought to be the excessively propedeutic and conceptual teaching to which the students are subjected to, and which doesn't allow them to establish enough links between Science, Technology and Society (STS). Among these, the History of Physics and Chemistry, the contextualized role and input of male scientists - as well as that of the, usually forgotten, women scientists -, and the cultural relevance of socio-scientific issues, have been singled out as effective strategies to stimulate intrinsic motivation among Secondary School (SS) students. Taking this background into context and working within this framework, a series of objectives have been outlined, with the ultimate aim of increasing the interest in Physics and Chemistry of the 3rd grade Secondary School students from "Colegio Juan de Lanuza" in Zaragoza. In order to achieve that, a practical intervention proposal has been defined, by which said students would have to carry out a research project around some famous female scientist or relevant socio-scientific issue and proceed to its subsequent presentation in a sort of formal scientificconference manner. Methodology, sources and evaluation tools are also specified. Thus, we can say that, despite the limiting lack of empiric validation, the designed didactic proposal manages to achieve the desired effect and accomplish the established objectives in a more than satisfactory manner. And thus, one could speculate about how its proper implementation should be able to diminish the above-mentioned difficulties.Este Trabajo de Fin de Máster (TFM) nace con objeto de paliar la desmotivación y falta de interés de los estudiantes de Secundaria por el estudio de las Ciencias y, más concretamente, la Física y la Química. Con el fin de comprender las causas de este alejamiento actitudinal, se ha realizado una revisión bibliográfica que ha evidenciado cómo la principal causante de esta desconexión es una enseñanza excesivamente conceptual y propedéutica, que no brinda a los alumnos suficientes relaciones entre Ciencia, Tecnología y Sociedad (CTS). Dentro de las mismas, el papel de la Historia de la Física y la Química, la participación contextualizada de científicos y, aunque a veces no lo parezca, científicas, así como la relevancia histórica y cultural de numerosos dilemas socio-científicos, ha sido reseñada como un eficaz recurso didáctico para despertar la motivación intrínseca entre el alumnado de la ESO. En este contexto, se plantean una serie de objetivos gracias a los cuales se persigue el fin último de incrementar el interés de los alumnos de la asignatura de Física y Química del curso de 3º de ESO del Colegio Juan de Lanuza, en Zaragoza, a través de la puesta en práctica de una propuesta de intervención didáctica en la que dichos alumnos tendrían que realizar una investigación sobre una científica de la Historia de la Física y la Química o una cuestión socio-científica relevante, que posteriormente presentarían en el contexto de un congreso científico formal. Se especifican la metodología y recursos y se diseñan también procedimientos para la evaluación de resultados. Finalmente, se llega a la conclusión de que, pese a la limitante falta de comprobación empírica, la propuesta de intervención diseñada cumplimenta sobradamente los objetivos iniciales, y se prevé cómo su puesta en práctica habría de ser capaz de atenuar la problemática descrita

Polypeptide GalNAc-Ts:from redundancy to specificity

Mucin-type O-glycosylation is a post-translational modification (PTM) that is predicted to occur in more than the 80% of the proteins that pass through the Golgi apparatus. This PTM is initiated by a family of polypeptide GalNAc-transferases (GalNAc-Ts) that modify Ser and Thr residues of proteins through the addition of a GalNAc moiety. These enzymes are type II membrane proteins that consist of a Golgi luminal catalytic domain connected by a flexible linker to a ricin type lectin domain. Together, both domains account for the different glycosylation preferences observed among isoenzymes. Although it is well accepted that most of the family members share some degree of redundancy toward their protein and glycoprotein substrates, it has been recently found that several GalNAc-Ts also possess activity toward specific targets. Despite the high similarity between isoenzymes, structural differences have recently been reported that are key to understanding the molecular basis of both their redundancy and specificity. The present review focuses on the molecular aspects of the protein substrate recognition and the different glycosylation preferences of these enzymes, which in turn will serve as a roadmap to the rational design of specific modulators of mucin-type O-glycosylation

Interruptores moleculares biomiméticos

Durante las últimas décadas, los científicos han tratado de construir dispositivos moleculares eficientes pero, hasta ahora, todos los intentos de diseñar sistemas artificiales palidecen frente a las máquinas moleculares naturales en funcionamiento. Aprendiendo de la naturaleza podríamos ser capaces de hacer uso de las excelentes propiedades que han sido refinadas por una lenta evolución. A continuación revisaremos brevemente algunas de las máquinas moleculares naturales más conocidas y las tomaremos como fuente de inspiración para el diseño de interruptores artificiales biomiméticos. Tomando el trabajo de la naturaleza como punto de partida, señalaremos algunos de los requisitos para construir máquinas moleculares eficientes.For the last decades, scientists have tried to build efficient molecular devices but, until now, every attempt to design artificial systems pales beside the natures molecular machines at work. By learning from nature, we could be able to make use of the excellent properties refined by slow evolution. Here we will briefly review some of the better-known natural molecular machines as an inspiration for the design of biomimetic artificial switches. Taking the natures work as a starting point, we will specify some of the requirements to build efficient molecular machines

Structural analysis of a GalNAc-T2 mutant reveals an induced-fit catalytic mechanism for GalNAc-Ts

The family of polypeptide N-acetylgalactosamine (GalNAc) transferases (GalNAc-Ts) orchestrates the initiating step of mucin-type protein O-glycosylation by transfer of GalNAc moieties to serine and threonine residues in proteins. Deficiencies and dysregulation of GalNAc-T isoenzymes are related to different diseases. Recently, it has been demonstrated that an inactive GalNAc-T2 mutant (F104S), which is not located at the active site, induces low levels of high-density lipoprotein cholesterol (HDL-C) in humans. Herein, the molecular basis for F104S mutant inactivation has been deciphered. Saturation transfer difference NMR spectroscopy experiments demonstrate that the mutation induces loss of binding to peptide substrates. Analysis of the crystal structure of the F104S mutant bound to UDP-GalNAc (UDP=uridine diphosphate), combined with molecular dynamics (MD) simulations, has revealed that the flexible loop is disordered and displays larger conformational changes in the mutant enzyme than that in the wild-type (WT) enzyme. 19F NMR spectroscopy experiments reveal that the WT enzyme only reaches the active state in the presence of UDP-GalNAc, which provides compelling evidence that GalNAc-T2 adopts a UDP-GalNAc-dependent induced-fit mechanism. The F104S mutation precludes the enzyme from achieving the active conformation and concomitantly binding peptide substrates. This study provides new insights into the catalytic mechanism of the large family of GalNAc-Ts and how these enzymes orchestrate protein O-glycosylation