Selective Manipulation of Discrete Mannosidase Activities in the Endoplasmic Reticulum by Using Reciprocally Selective Inhibitors

Abstract

Within the endoplasmic reticulum, immature glycoproteins are sorted into secretion and degradation pathways through the sequential trimming of mannose residues from Man GlcNAc to Man GlcNAc by the combined actions of assorted α-1,2-mannosidases. It has been speculated that specific glycoforms encode signals for secretion and degradation. However, it is unclear whether the specific signal glycoforms are produced by random mannosidase action or are produced regioselectively in a sequenced manner by specific α-1,2-mannosidases. Here, we report the identification of a set of selective mannosidase inhibitors and development of conditions for their use that enable production of distinct pools of Man GlcNAc isomers from a structurally defined synthetic Man GlcNAc substrate in an endoplasmic reticulum fraction. Glycan processing analysis with these inhibitors provides the first biochemical evidence for selective production of the signal glycoforms contributing to traffic control in glycoprotein quality control.

Mannosidase activity of EDEM1 and EDEM2 depends on an unfolded state of their glycoprotein substrates

“…Given the preference of ERManI for the quick removal of the terminal middle branch B mannose yielding M8B, EDEM1 likely removes then an α1,2 mannose residue from branches A or C, producing M7. However, we cannot exclude some contribution of an alternative pathway where the middle branch mannose is removed last, as was revealed by differential sensitivity to mannosidase inhibitors 36 . The additive effect of combined EDEM1 and ERManI in vitro (Fig.…”

Synthetic trisaccharides reveal discrimination ofendo-glycosidic linkages byexo-acting α-1,2-mannosidases in the endoplasmic reticulum

“…As a tissue for the extraction of the ER fraction, we selected the senescence-accelerated mouse prone 6 (SAMP6) 33 liver, which we have previously used for the analysis of the activity of ER α-1,2-mannosidases using an M9-type synthetic glycan substrate. 18,34 Extraction of the ER fraction from the SAMP6 liver was performed by centrifugal fractionation. 18,34 Because α-mannosidase is also present in the Golgi apparatus, 35 the ER/Golgi content of each centrifugal fraction was verified by western blotting using both ER and Golgi marker proteins as indicators.…”

“…18,34 Extraction of the ER fraction from the SAMP6 liver was performed by centrifugal fractionation. 18,34 Because α-mannosidase is also present in the Golgi apparatus, 35 the ER/Golgi content of each centrifugal fraction was verified by western blotting using both ER and Golgi marker proteins as indicators. This proved that the extracted ER fraction had no Golgi contamination (Fig.…”

“…12,13 The completely folded M9-glycoprotein and misfolded M9glycoprotein have their outermost mannose trimmed by ER α-1,2-mannosidases [ER mannosidase I (ERManI), 14 ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1), 15 EDEM2, 16 and EDEM3 17 ] to produce glycoproteins with the Man 8A GlcNAc 2 (M8A), Man 8B GlcNAc 2 (M8B) or Man 8C GlcNAc 2 (M8C) glycan. 18,19 The M8B-glycoprotein is recognized by the cargo-receptor ER-Golgi intermediate compartment 53 kDa protein (ERGIC53), 20 which transports completely folded glycoproteins from the ER to the Golgi apparatus. Thus, the M8B glycan functions as a secretion signal of properly folded glycoproteins.…”

Metabolic syndrome perturbs deglucosylation and reglucosylation in the glycoprotein folding cycle

“…Nascent glycoproteins are synthesized and folded into their correct conformations in the endoplasmic reticulum (ER). During this process, high‐mannose‐type glycan moieties on the protein scaffolds function as signals for retention, acceleration of folding, folding quality control, and the secretion and degradation of glycoproteins [1–4]. Particularly important steps of the quality control system are deglucosylation and reglucosylation during the processing of N‐glycans to form high‐mannose‐type glycans (Fig.…”