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).
