Cytosolic N-GlcNAc proteins are formed by the action of endo-β-N-acetylglucosaminidase

Abstract

NGLY1 is a widely conserved eukaryotic cytosolic deglycosylating enzyme involved in the endoplasmic reticulum-associated degradation (ERAD) process, which eliminates misfolded proteins through retrograde translocation and proteasomal degradation. A human genetic disorder called NGLY1-deficiency has been reported, indicating the functional importance of NGLY1 in humans. Evidence suggests that Ngly1-KO is embryonic lethal in mice, while additional deletion of the Engase gene, encoding another cytosolic deglycosylating enzyme (endo-β-N-acetylglucosaminidase; ENGase), partially rescued lethality. Upon compromised Ngly1 activity, ENGase-mediated deglycosylation of misfolded glycoproteins may cause excess formation of N-GlcNAc proteins in the cytosol, leading to detrimental effects in the mice. Whether endogenous N-GlcNAc proteins are really formed in Ngly1-KO cells/animals or not remains unclarified. Here, comprehensive identification of O- and N-GlcNAc proteins was carried out using purified cytosol from wild type, Ngly1-KO, Engase-KO, and Ngly1/Engase double KO mouse embryonic fibroblasts. It was revealed that while there is no dramatic change in the level of O-GlcNAc proteins among cells examined, there was a vast increase of N-GlcNAc proteins in Ngly1-KO cells upon proteasome inhibition. Importantly, few N-GlcNAc proteins were observed in Engase-KO or Ngly1/Engase double-KO cells, clearly indicating that the cytosolic ENGase is responsible for the formation of N-GlcNAc proteins. The excess formation of N-GlcNAc proteins may at least in part account for the pathogenesis of NGLY1-deficiency.

Keywords: ENGase; Glycoproteomics; N-GlcNAc; NGLY1; NGLY1-Deficiency; O-GlcNAc.

1.

A) Ngly1 and ENGase deglycosylating enzymes function in relation to endoplasmic reticulum associated degradation. Ngly1 removes N-linked glycans from retrotranslocated, misfolded glycoproteins before proteosomal degradation. ENGase cleaves between the two core GlcNAcs of previously released glycans in the cytosol. B) MEF (wild type, Ngly1-KO, Engase-KO, Ngly1/Engase double-KO) cells were permeabilized with 0.015% digitonin to selectively release cytosol. Tryptic glycopeptides were enriched using wheat germ agglutinin-based lectin weak affinity chromatography. Ubiquitinated peptides were enriched from LWAC flowthrough. Enriched peptides were analyzed by mass spectrometry.

2.

N-GlcNAc proteins are formed by the action of ENGase in the absence of Ngly1. A) Abundance of N-GlcNAc and extended N-glycopeptides in wild type, Ngly1-KO, Engase-KO, and Ngly1/Engase-KO treated with and without proteosomal inhibitor MG132. Peptide spectral matches were normalized to total spectra. Levels of extended N-linked glycopeptide-PSMs were relatively unchanged while the levels of N-GlcNAc modified PSMs were dramatically increased in Ngly1-KO cells upon proteosomal inhibition. B) Annotated ETD MS2 peaklist of N-GlcNAc modified peptide from Thrombospondin-1. C) Annotated HCD MS2 peaklist of K-GlyGly modified peptide from Thrombospondin-1.

3.

O-GlcNAcylation in MEF cells. A) O-GlcNAc-modified peptides represent nearly 90% of the glycopeptide PSMs across all backgrounds and treatments from glycopeptide-enriched cytosol. B) O-GlcNAc PSM abundance remained relatively equal across the different samples. C) Network map of O-GlcNAcylated proteins with the Uniprot keyword “Ubl Conjugation Pathway.” The outer ring color represents MG132 treatment while the inner circle color represents genetic background of the sample.