Substrate recognition and catalysis by GH47 α-mannosidases involved in Asn-linked glycan maturation in the mammalian secretory pathway

Abstract

Maturation of Asn-linked oligosaccharides in the eukaryotic secretory pathway requires the trimming of nascent glycan chains to remove all glucose and several mannose residues before extension into complex-type structures on the cell surface and secreted glycoproteins. Multiple glycoside hydrolase family 47 (GH47) α-mannosidases, including endoplasmic reticulum (ER) α-mannosidase I (ERManI) and Golgi α-mannosidase IA (GMIA), are responsible for cleavage of terminal α1,2-linked mannose residues to produce uniquely trimmed oligomannose isomers that are necessary for ER glycoprotein quality control and glycan maturation. ERManI and GMIA have similar catalytic domain structures, but each enzyme cleaves distinct residues from tribranched oligomannose glycan substrates. The structural basis for branch-specific cleavage by ERManI and GMIA was explored by replacing an essential enzyme-bound Ca 2+ ion with a lanthanum (La 3+) ion. This ion swap led to enzyme inactivation while retaining high-affinity substrate interactions. Cocrystallization of La 3+ -bound enzymes with Man 9 GlcNAc 2 substrate analogs revealed enzyme-substrate complexes with distinct modes of glycan branch insertion into the respective enzyme active-site clefts. Both enzymes had glycan interactions that extended across the entire glycan structure, but each enzyme engaged a different glycan branch and used different sets of glycan interactions. Additional mutagenesis and time-course studies of glycan cleavage probed the structural basis of enzyme specificity. The results provide insights into the enzyme catalytic mechanisms and reveal structural snapshots of the sequential glycan cleavage events. The data also indicate that full steric access to glycan substrates determines the efficiency of mannose-trimming reactions that control the conversion to complex-type structures in mammalian cells.Asn-linked glycan processing | α-mannosidase | glycosidase mechanism | glycoside hydrolase | bioinorganic chemistry

Structural principles controlling HIV envelope glycosylation

“…With the advent of recent structural data, this apparent conflict can now be resolved [35,52]. Overlaying the crystal structure of the enzyme-substrate complex of the ER α1,2-mannosidase I [52] with the model of the fully glycosylated Env trimer derived from cryo-EM [53] and mass spectrometry analysis [29] shows how the mannosidase enzyme wraps around target glycans. In contrast, bNAbs are able to recognise glycans while also penetrating the glycan shield [8,53].…”

“…(A) ER α1,2-mannosidase I (PDB ID 5KIJ) [52] (cyan) was modelled to bind the Man 9 GlcNAc 2 N332 glycan (green) on a fully glycosylated model of a previously described model of BG505 SOSIP.664 [27]. The surrounding glycans that sterically clash with enzyme recognition of N332 are highlighted in red.…”

Genetic disruption of multiple α1,2-mannosidases generates mammalian cells producing recombinant proteins with high-mannose–type N-glycans

“…In mammals, there are five members (MAN1C1, MAN1B1, EDEM1, EDEM2, and EDEM3) other than MAN1A1 and MAN1A2 in the GH47 family ( Fig. 1C) (40). MAN1C1 and MAN1B1 are another Golgi-␣1,2-mannosidase I and ER-mannosidase I, respectively (34,41).…”

Use of Endoglycosidase H as a diagnostic tool for MAN1B1‐CDG patients

“…Different Golgi mannosidases exist. The Golgi mannosidases IA and IC are known to preferentially cleave the terminal mannoses of the A branch, while Golgi mannosidase IB preferably cleaves the C branch . In some extent, Golgi mannosidase IC can also remove the mannose of the B branch .…”

“…MAN1B1 encodes an α1,2‐mannosidase that generates the Man 8 GlcNAc 2 isomer B by catalyzing the removal of the mannose residue from the middle branch of Man 9 GlcNAc 2 structures (Fig. ) . The structural analysis of N‐linked glycans on serum glycoproteins in Man1B1‐deficient patients showed the presence of hybrid type glycan structures .…”