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

摘要

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 read-ornment steps. One unusual enzyme, endo-a-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-a-mannosidase. Structures solved at resolutions 1.7-2.1 A 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 GIc_(1/3)Man_9GlcNAc_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-deox-ymannonojirimycin 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.