GH18 endo-β-N-acetylglucosaminidases use distinct mechanisms to process hybrid-type N-linked glycans

Beatriz Trastoy,Jonathan J Du,Chao Li,Mikel García-Alija,Erik H Klontz,Blaine R Roberts,Thomas C Donahue,Lai-Xi Wang,Eric J Sundberg,Marcelo E Guerin

doi:10.1016/j.jbc.2021.101011

Abstract

N-glycosylation is one of the most abundant posttranslational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology but also for the development of new drugs for a wide range of human diseases. Chemoenzymatic synthesis using engineered endo-β-N-acetylglucosaminidases (ENGases) has been used extensively to modulate the chemistry of N-glycosylated proteins. However, defining the molecular mechanisms by which ENGases specifically recognize and process N-glycans remains a major challenge. Here we present the X-ray crystal structure of the ENGase EndoBT-3987 from Bacteroides thetaiotaomicron in complex with a hybrid-type glycan product. In combination with alanine scanning mutagenesis, molecular docking calculations and enzymatic activity measurements conducted on a chemically engineered monoclonal antibody substrate unveil two mechanisms for hybrid-type recognition and processing by paradigmatic ENGases. Altogether, the experimental data provide pivotal insight into the molecular mechanism of substrate recognition and specificity for GH18 ENGases and further advance our understanding of chemoenzymatic synthesis and remodeling of homogeneous N-glycan glycoproteins.

Highlights

Pyrophosphate derivative to specific Asn residues in nascent polypeptide chains [1]
We focus on two GH18 enzymes, EndoS2 from S. pyogenes and EndoBT-3987 from Bacteroides thetaiotaomicron, a key enzyme that catalyzes the first step in the degradation and processing of mammalian highmannose type (HM-type) N-glycans in the human gut [36,37,38]
We observed that EndoBT-3987 hydrolyzes hybrid type (Hy-type) N-glycans less efficiently than HM-type N-glycans (see Experimental procedures and ref. [31])

Summary

Introduction

Pyrophosphate derivative to specific Asn residues in nascent polypeptide chains [1]. The presence of the glycosylation consensus sequence (Asn-X-Ser/Thr, where X may be any amino acid except for Pro) is necessary but not sufficient for glycosylation. A glutamic acid acts as an acid in this first step protonating the anomeric carbon and as a base in a second step deprotonating a water molecule that produces the second nucleophilic attack These enzymes, when in the presence of the two products of hydrolysis, the hydrolyzed Nglycan and the protein bearing a GlcNAc residue, are capable of catalyzing the reverse reaction toward the glycosidic bond formation. The efficiency of this reaction is increased using a glycosynthase ENGase mutant [17] and a N-glycan oxazoline as an activated glycosyl donor substrate (Fig. 1) [18,19,20,21]. No enzyme has been described to exclusively hydrolyze only HM-type or Hy-type N-glycans

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Conclusion