Immunopeptidomic Analysis Reveals That Deamidated HLA-bound Peptides Arise Predominantly from Deglycosylated Precursors.

The presentation of post-translationally modified (PTM) peptides by cell surface HLA molecules has the potential to increase the diversity of targets for surveilling T cells. Although immunopeptidomics studies routinely identify thousands of HLA-bound peptides from cell lines and tissue samples, in-depth analyses of the proportion and nature of peptides bearing one or more PTMs remains challenging. Here we have analyzed HLA-bound peptides from a variety of allotypes and assessed the distribution of mass spectrometry-detected PTMs, finding deamidation of asparagine or glutamine to be highly prevalent. Given that asparagine deamidation may arise either spontaneously or through enzymatic reaction, we assessed allele-specific and global motifs flanking the modified residues. Notably, we found that the N-linked glycosylation motif NX(S/T) was highly abundant across asparagine-deamidated HLA-bound peptides. This finding, demonstrated previously for a handful of deamidated T cell epitopes, implicates a more global role for the retrograde transport of nascently N-glycosylated polypeptides from the ER and their subsequent degradation within the cytosol to form HLA-ligand precursors. Chemical inhibition of Peptide:N-Glycanase (PNGase), the endoglycosidase responsible for the removal of glycans from misfolded and retrotranslocated glycoproteins, greatly reduced presentation of this subset of deamidated HLA-bound peptides. Importantly, there was no impact of PNGase inhibition on peptides not containing a consensus NX(S/T) motif. This indicates that a large proportion of HLA-I bound asparagine deamidated peptides are generated from formerly glycosylated proteins that have undergone deglycosylation via the ER-associated protein degradation (ERAD) pathway. The information herein will help train deamidation prediction models for HLA-peptide repertoires and aid in the design of novel T cell therapeutic targets derived from glycoprotein antigens.

N-Glycanase 1 Transcriptionally Regulates Aquaporins Independent of Its Enzymatic Activity.

Patients with pathogenic mutations in NGLY1 cannot make tears and have global developmental delay and liver dysfunction. Traditionally, NGLY1 cleaves intact N-glycans from misfolded, retrotranslocated glycoproteins before proteasomal degradation. We demonstrate that Ngly1-null mouse embryonic fibroblasts, NGLY1 knockout human cells, and patient fibroblasts are resistant to hypotonic lysis. Ngly1-deficient mouse embryonic fibroblasts swell slower and have reduced aquaporin1 mRNA and protein expression. Ngly1 knockdown and overexpression confirms that Ngly1 regulates aquaporin1 and hypotonic cell lysis. Patient fibroblasts and NGLY1 knockout cells show reduced aquaporin11 mRNA, supporting NGLY1 as regulating expression of multiple aquaporins across species. Complementing Ngly1-deficient cells with catalytically inactive NGLY1 (p.Cys309Ala) restores normal hypotonic lysis and aquaporin1 protein. We show that transcription factors Atf1/Creb1 regulate aquaporin1 and that the Atf1/Creb1 signaling pathway is disrupted in Ngly1-deficient mouse embryonic fibroblasts. These results identify a non-enzymatic, regulatory function of NGLY1 in aquaporin transcription, possibly related to alacrima and neurological symptoms.

Stress and interferon signalling-mediated apoptosis contributes to pleiotropic anticancer responses induced by targeting NGLY1.

BACKGROUND: Although NGLY1 is known as a pivotal enzyme that catalyses the deglycosylation of denatured glycoproteins, information regarding the responses of human cancer and normal cells to NGLY1 suppression is limited. METHODS: We examined how NGLY1 expression affects viability, tumour growth, and responses to therapeutic agents in melanoma cells and an animal model. Molecular mechanisms contributing to NGLY1 suppression-induced anticancer responses were revealed by systems biology and chemical biology studies. Using computational and medicinal chemistry-assisted approaches, we established novel NGLY1-inhibitory small molecules. RESULTS: Compared with normal cells, NGLY1 was upregulated in melanoma cell lines and patient tumours. NGLY1 knockdown caused melanoma cell death and tumour growth retardation. Targeting NGLY1 induced pleiotropic responses, predominantly stress signalling-associated apoptosis and cytokine surges, which synergise with the anti-melanoma activity of chemotherapy and targeted therapy agents. Pharmacological and molecular biology tools that inactivate NGLY1 elicited highly similar responses in melanoma cells. Unlike normal cells, melanoma cells presented distinct responses and high vulnerability to NGLY1 suppression. CONCLUSION: Our work demonstrated the significance of NGLY1 in melanoma cells, provided mechanistic insights into how NGLY1 inactivation leads to eradication of melanoma with limited impact on normal cells, and suggested that targeting NGLY1 represents a novel anti-melanoma strategy.

ERADication of EDEM1 occurs by selective autophagy and requires deglycosylation by cytoplasmic peptide N-glycanase.

ER degradation-enhancing α-mannosidase-like 1 protein (EDEM1) is involved in the routing of misfolded glycoproteins for degradation in the cytoplasm. Previously, we reported that EDEM1 leaves the endoplasmic reticulum via non-COPII vesicles (Zuber et al. in Proc Natl Acad Sci USA 104:4407-4412, 2007) and becomes degraded by basal autophagy (Le Fourn et al. in Cell Mol Life Sci 66:1434-1445, 2009). However, it is unknown which type of autophagy is involved. Likewise, how EDEM1 is targeted to autophagosomes remains elusive. We now show that EDEM1 is degraded by selective autophagy. It colocalizes with the selective autophagy cargo receptors p62/SQSTM1, neighbor of BRCA1 gene 1 (NBR1) and autophagy-linked FYVE (Alfy) protein, and becomes engulfed by autophagic isolation membranes. The interaction with p62/SQSTM1 and NBR1 is required for routing of EDEM1 to autophagosomes since it can be blocked by short inhibitory RNA knockdown of the cargo receptors. Furthermore, p62/SQSTM1 interacts only with deglycosylated EDEM1 that is also ubiquitinated. The deglycosylation of EDEM1 occurs by the cytosolic peptide N-glycanase and is a prerequisite for interaction and aggregate formation with p62/SQSTM1 as demonstrated by the effect of peptide N-glycanase inhibitors on the formation of protein aggregates. Conversely, aggregation of p62/SQSTM1 and EDEM1 occurs independent of cytoplasmic histone deacetylase. These data provide novel insight into the mechanism of autophagic degradation of the ER-associated protein degradation (ERAD) component EDEM1 and disclose hitherto unknown parallels with the clearance of cytoplasmic aggregates of misfolded proteins by selective autophagy.

Irreversible inhibitors and activity-based probes as research tools in chemical glycobiology.

In this review, we will discuss the enzymes that are involved in the synthesis and degradation of glycoconjugates and we will give an overview of the inhibitors and activity-based probes (ABPs) that have been used to study these. Following discussion of some general aspects of the biosynthesis and degradation of N-linked glycoproteins, attention is focused on the enzymes that hydrolyze the protein-carbohydrate linkage, peptide N-glycanase and glycosylasparaginase and their mechanism. We then focus on the biosynthesis of O-linked glycoproteins and glycolipids and in particular on the enzymes that hydrolyze the interglycosidic linkages in these, the glycosidases. Some important mechanism-based glycosidase inhibitors that form a covalent bond with the targeted enzyme(s), their corresponding ABPs and their application to study this class of enzymes are highlighted. Finally, alternative pathways for degradation of glycoconjugates and an ABP-based strategy to study these will be discussed.

Systematic synthesis and inhibitory activity of haloacetamidyl oligosaccharide derivatives toward cytoplasmic peptide:N-glycanase.

A series of glycosyl haloacetamides were synthesized as potential inhibitors of cytoplasmic peptide:N-glycanase (PNGase), an enzyme that removes N-glycans from misfolded glycoproteins. Chloro-, bromo-, and iodoacetamidyl chitobiose and chitotetraose derivatives exhibited a significant inhibitory activity. No inhibitory activity was observed with of fluoroacetamididyl derivatives. Moreover, N-acetylglucosamine derivatives, beta-chloropropionamidyl chitobiose, and chloroacetamidyl cellooligosaccharide derivatives did not show any activity. These results underscore the importance of the N-acetyl groups of chitobiose for PNGase recognition. In addition, reactivity and position of the leaving group at the reducing end are also important factors.

Synthesis and biological evaluation of a chitobiose-based peptide N-glycanase inhibitor library.

Peptide N-glycanase (PNGase), the enzyme responsible for the deglycosylation of N-linked glycoproteins, has an active site related to that of cysteine proteases. Chitiobiose was equipped with electrophilic traps often used in cysteine protease inhibitors, and the resulting compounds were evaluated as PNGase inhibitors. We found that the electrophilic trap of the inhibitor has a great influence on the potency of the compounds with the chloromethyl ketone inhibitor being the first potent C-glycoside-based PNGase inhibitor.

Fluorescently labeled inhibitor for profiling cytoplasmic peptide:N-glycanase.

Cytoplasmic peptide:N-glycanase (PNGase) is an enzyme that removes N-glycans from misfolded glycoproteins. The function of cytoplasmic PNGase plays a significant role in the degradation of misfolded glycoproteins, which is critical for cell viability. Recently, we reported that haloacetoamidyl derivatives of high-mannose-type oligosaccharides selectively modify the catalytic cysteine of cytoplasmic PNGase and serve as its specific inhibitor. Interestingly, a drastically simplified chloroacetamidyl chitobiose derivative [(GlcNAc)(2)-ClAc] was also reactive to PNGase. In our work, it was conjugated to a hydrophobic fluorophore in order to render (GlcNAc)(2)-ClAc cells permeable. We demonstrated that this compound [BODIPY-(GlcNAc)(2)-ClAc] specifically binds to cytoplasmic PNGase from budding yeast (Png1). To date, only Z-VAD-fmk is known as an inhibitor of PNGase. BODIPY-(GlcNAc)(2)-ClAc and Z-VAD-fmk share the same binding site on Png1, while BODIPY-(GlcNAc)(2)-ClAc has markedly stronger inhibitory activity. The functional analysis of PNGase using Z-VAD-fmk should be carefully interpreted because of its intrinsic property as a caspase inhibitor. In sharp contrast, chloroacetamidyl chitobiose was not reactive to caspase. In addition, BODIPY-(GlcNAc)(2)-ClAc did not bind either chitobiose-binding lectins or PNGase from other sources. Moreover, fluorescent microscopy clearly showed that BODIPY-(GlcNAc)(2)-ClAc was efficiently introduced into cells. These results suggest that this compound could be an in vivo inhibitor of cytoplasmic PNGase.

Site-specific labeling of cytoplasmic peptide:N-glycanase by N,N'-diacetylchitobiose-related compounds.

Peptide:N-glycanase (PNGase) is the deglycosylating enzyme, which releases N-linked glycan chains from N-linked glycopeptides and glycoproteins. Recent studies have revealed that the cytoplasmic PNGase is involved in the degradation of misfolded/unassembled glycoproteins. This enzyme has a Cys, His, and Asp catalytic triad, which is required for its enzymatic activity and can be inhibited by "free" N-linked glycans. These observations prompted us to investigate the possible use of haloacetamidyl derivatives of N-glycans as potent inhibitors and labeling reagents of this enzyme. Using a cytoplasmic PNGase from budding yeast (Png1), Man9GlcNAc2-iodoacetoamide was shown to be a strong inhibitor of this enzyme. The inhibition was found to be through covalent binding of the carbohydrate to a single Cys residue on Png1, and the binding was highly selective. The mutant enzyme in which Cys191 of the catalytic triad was changed to Ala did not bind to the carbohydrate probe, suggesting that the catalytic Cys is the binding site for this compound. Precise determination of the carbohydrate attachment site by mass spectrometry clearly identified Cys191 as the site of covalent attachment. Molecular modeling of N,N'-diacetylchitobiose (chitobiose) binding to the protein suggests that the carbohydrate binding site is distinct from but adjacent to that of Z-VAD-fmk, a peptide-based inhibitor of this enzyme. These results suggest that cytoplasmic PNGase has a separate binding site for chitobiose and other carbohydrates, and haloacetamide derivatives can irreversibly inhibit that catalytic Cys in a highly specific manner.

Using a small molecule inhibitor of peptide: N-glycanase to probe its role in glycoprotein turnover.

Peptide:N-glycanase (PNGase) is ostensibly the sole enzyme responsible for deglycosylation of unfolded N-linked glycoproteins dislocated from the ER to the cytosol. Here we show the pan-caspase inhibitor, Z-VAD-fmk, to be an active site-directed irreversible inhibitor of yeast and mammalian PNGase at concentrations below those used to inhibit caspases in vivo. Through chemical synthesis we determined that the P1 residue, electrophile position, and leaving group are important structural parameters for PNGase inhibition. We show that Z-VAD-fmk inhibits PNGase in living cells and that degradation of class I MHC heavy chains and TCRalpha, in an identical cellular setting, is markedly different. Remarkably, proteasome-mediated turnover of class I MHC heavy chains proceeds even when PNGase is completely inhibited, suggesting that the function of PNGase may be to facilitate more efficient proteasomal proteolysis of N-linked glycoproteins through glycan removal.