Periodic Reporting for period 1 - GlycoMabs (Chemoenzymatic glyco-engineering of therapeutic monoclonal antibodies)
Reporting period: 2019-04-16 to 2021-04-15
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).
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.