Structural and mechanistic insight into N-glycan processing by endo-α-mannosidase

Abstract

N-linked glycans play key roles in protein folding, stability, and function. Biosynthetic modification of N-linked glycans, within the endoplasmic reticulum, features sequential trimming and readornment steps. One unusual enzyme, endo-α-mannosidase, cleaves mannoside linkages internally within an N-linked glycan chain, short circuiting the classical N-glycan biosynthetic pathway. Here, using two bacterial orthologs, we present the first structural and mechanistic dissection of endo-α-mannosidase. Structures solved at resolutions 1.7–2.1 Å reveal a ( β / α ) 8 barrel fold in which the catalytic center is present in a long substrate-binding groove, consistent with cleavage within the N-glycan chain. Enzymatic cleavage of authentic Glc 1/3 Man 9 GlcNAc 2 yields Glc 1/3 -Man. Using the bespoke substrate α-Glc-1,3-α-Man fluoride, the enzyme was shown to act with retention of anomeric configuration. Complexes with the established endo-α-mannosidase inhibitor α-Glc-1,3-deoxymannonojirimycin and a newly developed inhibitor, α-Glc-1,3-isofagomine, and with the reducing-end product α-1,2-mannobiose structurally define the -2 to +2 subsites of the enzyme. These structural and mechanistic data provide a foundation upon which to develop new enzyme inhibitors targeting the hijacking of N-glycan synthesis in viral disease and cancer.

Spiro‐epoxyglycosides as Activity‐Based Probes for Glycoside Hydrolase Family 99 Endomannosidase/Endomannanase

“…We report here the first activity‐based probes for detection of GH99 enzymes, which were designed based on the proposed mechanism of this enzyme. In this proposed mechanism a 1,2‐anhydro‐epoxide intermediate is formed by general base assisted deprotonation of O2 by a carboxylate residue 23. Our design strategy includes a reactive C2 spiro‐epoxide that can potentially covalently label the general base (acting as a nucleophile), and includes a fluorescent label for visualization.…”

Measurement of endo-α-mannosidase activity using a fluorescently labeled oligosaccharide derivative

“…based on the mechanism of endo-α-mannosidase activity. 6) To construct this tetrasaccharide, we prepared 2 mannosyl donors (compounds 4 and 7 as shown in Scheme 1). Preparation of mannosyl chloride donor 4 proceeded in six steps starting from D-mannose as previously described.…”

“…4,5) Recently, crystal structures of GH99 from two types of enteric bacteria have been determined. 6) The kinetics and catalytic mechanisms of mammalian endo-α-mannosidase remain incompletely understood.…”

Characterization of the - and -Mannosidases of Porphyromonas gingivalis

“…There are only a few reports in the literature on the occurrence of ␣-mannosidases in Gram-negative bacteria. Thompson et al (38) described the 3D structures and mechanism of N-glycan processing by endo-␣-mannosidases from B. thetaiotaomicron and Bacteroides xylanisolvens. These endomannosidases are classified in the Carbohydrate-Active Enzymes Database family GH99 (www.cazy.org) and hydrolyze the ␣-1,2-mannosidic bond between the glucose-substituted mannose and the remainder of the N-glycan and act on structures of Glc 1-3 Man 9 GlcNAc 2 , as well as structures that have been trimmed by ER-mannosidases ERM1 and ERM2 in the 6=-pentamannosyl branch, releasing Glc 1-3-1,3-␣-Man oligosaccharides (38).…”