Research

Glycans are not only one of the major components of the cell but also are essential molecules that modulate a variety of important biological processes in all living organisms. Glycans are used primarily as energy storage and metabolic intermediates as well as being main structural constituents in bacteria and plants. Moreover, as a consequence of protein and lipid glycosylation, glycans generate a significant amount of structural diversity in biological systems. This structural information is particularly apparent in molecular recognition events including cell-cell interactions during critical steps of development, the immune response and host-pathogen interactions.

We investigate the structural and chemical biology of proteins involved in the biosynthesis and modification of glycans. We use a multidisciplinary approach including protein biophysics, protein biochemistry, molecular biology and structural biology, in the form of X-ray crystallography, X-ray free electron laser (XFEL), cryo electron microscopy (Cryo-EM), and small angle X-ray scattering, to elucidate mechanistic aspects of these processes at the molecular level.

In that context, research is concentrated - but not limited - on:

1. Rationalizing glycoengineering strategies for immunotherapeutic antibodies.

Therapeutic immunoglobulin G (IgG) monoclonal antibodies are a prominent and expanding class of drugs used for the treatment of several human disorders including cancer, autoimmunity, and infectious diseases. The presence of a N-linked glycosylation site is critical for IgG function contributing both to Fc γ receptor binding and activation of the complement pathway. IgG antibodies including those produced for clinical use typically exist as mixtures of more than 20 glycoforms, which significantly impacts their efficacies, stabilities and the effector functions. To better control their therapeutic properties, the chemoenzymatic synthesis of homogeneously N-glycosylated antibodies has been developed. Experiments are designed to capitalize on our growing understanding of a family of antibody-modifying enzymes in order to use them more effectively to modify antibodies in novel ways, so as to unleash the full potential of antibody drugs.

Figure 1. Structural insights into the mechanisms and specificities of IgG-active endoglycosidases. a. Schematic representation of the chemoenzymatic synthesis of homogenous IgGs using EndoS(2) WT, EndoS(2) glycosynthase mutants and glycan oxazoline derivatives. b. Cartoon (left) and surface (right) representations showing the general fold and secondary structure organization of EndoSD233A/E235L. c. In the upper panel: surface representation of the residues studied by alanine scanning mutagenesis, coloured by loop number. Hydrolytic activity retained by alanine mutants is in parentheses, with the substrate glycan tested superimposed onto its crystal structure: EndoS with CT glycan (left), EndoS2 with CT glycan (center) and EndoS2 with HM glycan (right). In the lower panel: enzyme residues that form glycan contacts. Residues that mediate conserved interactions in the three crystal structures are labeled in black, while residues that mediate a unique contact are in orange (Trastoy et al. Nat. Commun. 9:1874, 2018; Klontz and Trastoy et al., ACS Cent Sci. 5:524-538, 2019; Trastoy et al., Nat. Commun. 11:899, 2020).

2. Unveiling the role of gut microbiota in health and disease.

The composition and physiology of the gut microbiota plays a pivotal role in human health and disease. Perturbations of such equilibrium have been associated with the occurrence of several pathologies, including metabolic disease, cardiovascular disease, type-2 diabetes, cancer and inflammatory bowel disease. One major factor that influence the balance of bacterial species in the gut is the influx of glycans into the intestine, mostly from diet as well as host mucosal secretions and secreted epithelial cells. The processing of a broad diversity of glycans present in the human gut requires of several glycosidic linkage-specific degradative enzymes. Human intestinal enzymes are capable of fully degrading a small set of glycans containing only one or two different sugar linkages. Gut symbiotic microorganisms provide the complementary enzymatic machinery necessary to depolymerize glycans into their sugar components that otherwise cannot be processed by the host. We investigate the strategies and the molecular mechanisms utilized by our resident gut bacteria to recognize, acquire and degrade complex glycans.

Figure 2. The overall structure of EndoBT-3987 and the substrate Man9GlcNAc2Asn glycan binding site. a Surface representation, with annotated domains and GH loops. b Cartoon representation of two views of the EndoBT-3987D312A/E314L-Man9GlcNAc2Asn crystal structure. EndoBT-3987 is a structural homologue of EndoH, an enzyme extensively used in biotechnology, and for which the mechanism of substrate recognition was largely unknown.c The electron density of Man9GlcNAc2Asn substrate shown at 1.0 σ r.m.s. deviation (Trastoy et al., Nat. Commun. 11:899, 2020).

3. Understanding and engineering substrate specificity of Carbohydrate Active enZymes.

CAZymes catalyze the biosynthesis, breakdown, modification and transport of glycan structures, generating a tremendous amount of structural diversity in biological systems. The long term-goal of this project is to understand the catalysis and structural determinants (the understanding), and the modulation (the engineering) of substrate specificity of prototype CAZyme targets, with a strong commitment to apply that knowledge in areas including biomedicine, bioengineering and biotechnology.

  • We envision to visualize the reaction catalyzed by selected glycoside hydrolase and glycosyltransferase enzymes in full, that means “recording” the reaction as it occurs, by using structural biology methods.

Figure 3. The catalytic cycle of the retaining glucosyltransferase GpgS. Structural snapshots of the reaction center of the retaining glucosyl-3-phosphoglycerate synthase GpgS, decipher multiple conformations of the enzyme/substrate/products intermediates in its native state, supporting an SNi catalytic mechanism. (Albesa-Jové et al., Angew. Chem. Int. Ed. Engl. 54:9898-902, 2015; Albesa-Jové and Guerin, Curr. Opin. Struct. Biol. 40:23-32, 2016; Albesa-Jové et al., Angew. Chem. Int. Ed. 56:14853-14857, 2017; Albesa-Jové et al., Structure 25:1034-1044, 2017; Albesa-Jové et al., Methods Enzymol. 621:261-279, 2019 - Review).

  • We study a family of enzymes responsible for the biosynthesis of nucleotide sugars, nucleotide-sugar pyrophosphorylases (NDP-sugar PPases). Nucleotide sugars act as sugar donors of glycosyltransferases in the biosynthesis of most of the natural disaccharides, oligosaccharides, polysaccharides and glycoconjugates in nature. In this context, we envision to understand the allosteric regulation mechanisms of bacterial glycogen and plant starch biosynthetic pathways.

Figure 4. Overall structures of the Escherichia coli AGPase in complex with the positive and negative allosteric regulators, FBP and AMP, respectively, as visualized by cryoEM. a,c The overall views of the electron density cryoEM maps of the EcAGPase-FBPD2 and EcAGPase-AMPD2 complexes, colored according to the four protomers (orange, yellow, blue and green) of the homotetramer, respectively. b,d A selected region corresponding to the neighborhood of the FBP and AMP binding sites are masked to reveal the atomic models built into the corresponding electron densities, with the protein shown as ribbons and the ligands FBP and AMP in stick representations, respectively (Cifuente et al., Structure 24:1613-1622, 2016; Cifuente et al., Curr. Res. Struct. Biol. 2:89-103, 2020; Cifuente et al., Biochem. J. 476:2059-2092, 2019 - Review).

  • We study a family of carbohydrate esterases, chitin de-N-acetylases (CDAs), that catalyze the hydrolysis of the acetamido group in GlcNAc residues of chitin, chitosan, and chitooligosaccharides (COSs). One of the most striking examples that highlight the importance of COSs is represented by the Nod factors, key signal molecules produced by rhizobia that initiate the development of root nodules in leguminous host plants. Most of the biological activities associated with COSs seem to be largely dependent on the degree of polymerization and the specific de-N-acetylation pattern, which define the charge density and the distribution of GlcNAc and GlcNH2 moieties in chitosan and COS. A major challenge is to understand how CDAs specifically define the distribution of GlcNAc and GlcNH2 moieties in the oligomeric chain.

Figure 6. Structural basis of the 'Subsite Capping Model'. We solved crystal structures of the Vibrio cholera CDA (VcCDA) in four relevant states of its catalytic cycle. We unraveled an induced-fit mechanism with a significant conformational change of a loop closing the active site. It is proposed that the deacetylation pattern exhibited by different CDAs is governed by critical loops that shape and differentially block accessible subsites in the binding cleft of CE4 enzymes (Andrés and Albesa-Jové et al., Angew. Chem. Int. Ed. Engl. 53:6882-6887, 2014).

4. The cell envelope of Mycobacterium tuberculosis

The cell envelope of M. tuberculosis contains glycans and lipids of exceptional structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell envelope is currently intensively studied in order to identify novel drug targets. The cell envelope of M. tuberculosis comprises four main layers: (i) the plasma membrane or inner membrane, (ii) the peptidoglycan–arabinogalactan complex (AGP), (iii) an assymetrical outer membrane or ‘mycomembrane’, that is covalently linked to AGP through the mycolic acids, and (iv) the external capsule. The mycolic acids form the inner leaflet of the asymmetrical outer membrane, with the outer layer consisting of a variety of non-covalently attached (glyco)lipids, lipoglycans, and (lipo)proteins some of which are glycosylated. The capsule is composed of polysaccharides and proteins with only minor amounts of lipids. Our long-term goals are (i) to investigate the mechanistic aspects of proteins involved in the biosynthesis and regulation of this complex structure, and (ii) to understand the how the cell envelope is organized in space and time, within the context of its interaction with the host.

  • Structural basis of PIM, LM and LAM biosynthetic pathways - Phosphatidylinositol mannosides (PIMs) and metabolically related lipoglycans comprising lipomannan (LM) and lipoarabinomannan (LAM) are non-covalently anchored through their PI moiety to the inner and outer membranes of the cell envelope. PIMs, LM and LAM are key structural elements and virulence factors of M. tuberculosis. PIMs can contain one to six mannose residues and up to four acyl chains, with tri- and tetra-acylated phosphatidylinositol dimannoside (PIM2) and phosphatidylinositol hexamannoside (PIM6) as the predominant species. Specifically, PIM2 and its acylated versions Ac1PIM2 and Ac2PIM2 are considered both metabolic end-products and intermediates in the biosynthesis of Ac1PIM6 and Ac2PIM6, LM, and LAM. We focus on the identification and characterization at the molecular level of proteins involved in the biosynthesis of PIMs, LM and LAM.

Figure 7. Early steps of PIM biosynthetic pathway. In vitro experimental evidence indicates that although two pathways might co-exist in mycobacteria, the sequence of events PI→PIM1→PIM2→Ac1PIM2 is favored (Guerin et al., J. Biol. Chem. 285:33577-33583, 2010 - Review).

Of particular relevance, we visualized the occurrence of a conformational switch during the catalytic cycle of the retaining glycosyltransferase PimA, the enzyme that initiate the pathway, involving both α-helix–to–β-strand and β-strand–to–α-helix transitions. These structural changes seem to be promoted by interactions of the protein with anionic phospholipids in the membrane surface, and to modulate catalysis. We reported crystal structures of PatA, a membrane associated acyltransferase that transfers a palmitoyl moiety from palmitoyl–CoA to the 6-position of the mannose ring added by PimA. The structures reveal an α/β architecture, with the acyl chain of palmitoyl-CoA deeply buried into a hydrophobic pocket that runs perpendicular to a long groove where the active site is located. Enzyme catalysis is mediated by an unprecedented charge relay system, which markedly diverges from the canonical HX4D motif. We found that PimA and PatA are essential enzymes for M. tuberculosis growth in vitro and in vivo. Altogether, the experimental data highlight the importance of the PIMs biosynthetic pathway for M. tuberculosis, providing exciting possibilities for inhibitor design/drug discovery programs.

Figure 8. Secondary structure reshuffling involves α-helix–to–β-strand and β-strand–to–α-helix transitions transitions. The dynamical N-terminal region (residues 110-170 shown in rainbow-colored cartoon representation) as seen in different crystal structures of PimA, including two unliganded structures (molecular surfaces in yellow and red), the PimA-GDP-Man complex (in blue) and the PimA-GDP-diC8PI complex (in orange; Giganti et al., Nat. Chem. Biol. 11:16-18, 2015, Highlighted in the News and Views section: Membrane enzymes: Transformers at the interface. Brodhun and Tittmann. Nat. Chem. Biol. 11:102-103, 2015).

Figure 9. The donor and acceptor binding mechanism of PatA. Cartoon representation showing the assembled active site and location of (i) Man-C16 and (ii) S-C16CoA into PatA. β-strands and α-helices are shown in orange and yellow, respectively. Two views showing the structural superposition of (i) Man-C16 and (ii) S−C16CoA (Albesa-Jové et al., Nat. Commun. 7:10906, 2016; Tersa et al., ACS Chem. Biol. 13:131-140, 2018).


  • Drug discovery program - M. tuberculosis is the second most deadly infectious agent in the world after HIV. Drug treatment is a laborious process, requiring daily dosage of a combination of four first-line drugs over 6 months: isoniazid (INH), rifampicin (RIF), ethambutol, and pyrazinamide. During recent years there has been an alarming rise of multidrug-resistant (MDR) and extensively drug-resistant (XDR) cases of TB. Under such circumstances, the discovery of novel anti-TB drugs with bactericidal mechanisms different from those of currently available agents has become a worldwide priority. From a structural biology perspective, our laboratory is committed to contribute to the discovery of new drugs against TB.