Periodic Reporting for period 1 - GlycoMabs (Chemoenzymatic glyco-engineering of therapeutic monoclonal antibodies)
Periodo di rendicontazione: 2019-04-16 al 2021-04-15
Monoclonal antibodies (mAbs) are a prominent and rapidly expanding class of drugs used to treat various diseases, e.g. cancer, autoimmunity, neurodegenerative and infectious diseases, among others. Most of the therapeutically available mAbs are based on immunoglobulin G (IgG). IgGs have two conserved N-glycosylation sites on each heavy chain of the fragment crystallizable (Fc) region. The effector functions (e.g. Antibody-dependent cell-mediated cytotoxicity, complement activation) mediated by the Fc region of the antibodies are determined not only by the presence of this N-glycan of the Fc region but also by its carbohydrate composition. N-glycosylation is a highly complex and heterogenous post-translational modification that generates enormous diversity of glycan structures attached to an asparagine (Asn) residue of a glycoprotein. Controlling the composition of the N-glycan to generate a homogenous glycosylation profile has become a major challenge in recent years to improve the pharmacokinetic and functional properties of these drugs. Chemoenzymatic synthesis, using endo--acetylglucosaminidases (ENGases) mutants and N-glycans oxazoline derivatives, is a successful in vitro approach to obtain defined and homogenous N-glycans chemistry in mAbs. ENGases are endoglycosidases that hydrolyze the (1-4) linkage between the first two GlcNAc residues of N-linked glycans on proteins. They are classified in two glycosyl hydrolase families (GH18 and GH20) according to the CAZy database. Although they share a common catalytic mechanism, the substrate specificity is distinct for each enzyme. ENGases in general do not show protein specificity, except for EndoS and EndoS2 from Streptococcus pyogenes which are the only enzymes with a known specific protein substrate, the Fc region of IgG antibodies. But they also show a selectivity for N-glycan that are capable of hydrolyzing. N-glycans in eukaryotes can be classified into three main groups: complex-type (CT), high-mannose (HM) type and hybrid (Hy) type. EndoS is highly restrictive, only capable of hydrolyzing biantennary CT N-glycans, while EndoS2 is able to hydrolyze the three major N-glycan in the Fc of IgGs. In contrast, EndoF1 from Elizabethkingia meningoseptica and EndoH from Streptococcus plicatus hydrolyze HM and Hy-type N-glycans but they cannot process CT N-glycans. Therefore, the objective of this project is to understand the glycan specificity of these glycosyl hydrolases that modify the N-glycan of mAbs in order to rationally design enzymes with new and/or improved glycan selectivity.
IIn the past, we have determined the structural basis of N-glycan specificity by EndoS and EndoS2 that hydrolyze specifically CT- and the three main types of N-glycans on mAbs, respectively. However, the mechanism by which ENGases recognize specifically HM- and not CT remained unknown. In this project, we were focused on the study of the structural basis of HM-N-glycan processing by EndoBT-3987 from Bacteroides thetaiotaomicron. EndoBT-3987, enzyme that belongs to the same glycosyl hydrolase family than EndoS or EndoS2 (GH18), initiates the HM N-glycan degradation pathway in the human gut. Furthermore, EndoBT-3987 shows a high structural homology with EndoH from Streptomyces plicatus, the ENGase most used as enzymatic reagent in glycoprotein research. The molecular mechanism by which EndoBT-3987 or other GH18 ENGase, including EndoH, specifically hydrolyze HM N-glycans has yet to be defined. In order to understand this N-glycan recognition mechanism:
1. We determined the X-ray crystal structure of the catalytic inactive EndoBT-3987 in complex with the glycan substrate, AsnGlcNAc2Man9. This structure represents the first example of an enzyme-substrate complex in the GH18 ENGase family. We were able to visualize how the enzyme guides the N-glycan into the catalytic site where the reaction takes place. In addition, we observed that the Asn attached to the N-glycan do not interact with the protein, supporting the idea that this enzyme only shows specificity for the glycan but not for the protein.
2. We elucidated the X-ray crystal structure of the wild type EndoBT-3987 in complex with two glycan products, Man9GlcNAc and Man5GlcNAc. We revealed the mechanism of product released and complete the structural snapshots of the catalytic cycle of the EndoBT-3987.
3. We performed alanine scanning mutagenesis of the residues that are part of the loops that interact with the N-glycans in the crystal structures. Furthermore, we measured the hydrolytic activity of these alanine mutant against the monoclonal antibody, Rituximab, to further investigate the HM-glycan recognition mechanism of this enzyme. We observed that the mutations that most affected the activity of the enzyme were in the loops interacting with the antenna (1,6) of the N-glycan.
4. We conducted structural comparison studies with other GH18 ENGases, including EndoS, EndoS2 and EndoH, which suggested that HM-hydrolyzing enzymes (EndoBT-3987, EndoH) specifically recognize the antenna (1,6), whereas enzymes capable of hydrolyzing CT-glycans recognize the antenna (1,3).
This work is described in the publication: B. Trastoy, J. J. Du, E. H. Klontz, C. Li, J. O. Cifuente, L. X. Wang, E. J. Sundberg, M. E. Guerin, Structural basis of mammalian high-mannose N-glycan processing by human gut Bacteroides. Nat. Commun. 11, 899 (2020).
1. We determined the X-ray crystal structure of the catalytic inactive EndoBT-3987 in complex with the glycan substrate, AsnGlcNAc2Man9. This structure represents the first example of an enzyme-substrate complex in the GH18 ENGase family. We were able to visualize how the enzyme guides the N-glycan into the catalytic site where the reaction takes place. In addition, we observed that the Asn attached to the N-glycan do not interact with the protein, supporting the idea that this enzyme only shows specificity for the glycan but not for the protein.
2. We elucidated the X-ray crystal structure of the wild type EndoBT-3987 in complex with two glycan products, Man9GlcNAc and Man5GlcNAc. We revealed the mechanism of product released and complete the structural snapshots of the catalytic cycle of the EndoBT-3987.
3. We performed alanine scanning mutagenesis of the residues that are part of the loops that interact with the N-glycans in the crystal structures. Furthermore, we measured the hydrolytic activity of these alanine mutant against the monoclonal antibody, Rituximab, to further investigate the HM-glycan recognition mechanism of this enzyme. We observed that the mutations that most affected the activity of the enzyme were in the loops interacting with the antenna (1,6) of the N-glycan.
4. We conducted structural comparison studies with other GH18 ENGases, including EndoS, EndoS2 and EndoH, which suggested that HM-hydrolyzing enzymes (EndoBT-3987, EndoH) specifically recognize the antenna (1,6), whereas enzymes capable of hydrolyzing CT-glycans recognize the antenna (1,3).
This work is described in the publication: B. Trastoy, J. J. Du, E. H. Klontz, C. Li, J. O. Cifuente, L. X. Wang, E. J. Sundberg, M. E. Guerin, Structural basis of mammalian high-mannose N-glycan processing by human gut Bacteroides. Nat. Commun. 11, 899 (2020).
Although the COVID-19 outbreak negatively affected ongoing or planned activities of this proposal, we were still able to successfully redirect this research project. We determined the molecular mechanism by which key enzymes for in vitro glyco-remodeling, specifically recognize high-mannose and complex-type N-glycans on therapeutic monoclonal antibodies. This structure-function studies provide the basis for rationally manipulating endoglycosidase functionalities in order to enhace the glycoengineering capabilities of therapeutic monoclonal antibodies, an emerging class of drugs that are used to treat a wide variety of diseases.
Regarding the impact on my career prospects, the training received during the implementation of GlycoMabs project has increased my competency in fundamental scientific areas: technical and scientific training in state-of-the art techniques, project management, dissemination of results in high-impact journals, meetings and lectures in the Master program of Molecular Biology and Biomedicine of the University of Cantabria.
Regarding the impact on my career prospects, the training received during the implementation of GlycoMabs project has increased my competency in fundamental scientific areas: technical and scientific training in state-of-the art techniques, project management, dissemination of results in high-impact journals, meetings and lectures in the Master program of Molecular Biology and Biomedicine of the University of Cantabria.