Bacteroidales (syn. Bacteroidetes) are prominent members of the human gastrointestinal ecosystem mainly due to their efficient glycan-degrading machinery, organized into gene clusters known as polysaccharide utilization loci (PULs). A single PUL was reported for catabolism of high-mannose (HM) N-glycan glyco-polypeptides in the gut symbiont Bacteroides thetaiotaomicron, encoding a surface endo-β-N-acetylglucosaminidase (ENGase), BT3987. Here, we discover an ENGase from the GH18 family in B. thetaiotaomicron, BT1285, encoded in a distinct PUL with its own repertoire of proteins for catabolism of the same HM N-glycan substrate as that of BT3987. We employ X-ray crystallography, electron microscopy, mass spectrometry-based activity measurements, alanine scanning mutagenesis and a broad range of biophysical methods to comprehensively define the molecular mechanism by which BT1285 recognizes and hydrolyzes HM N-glycans, revealing that the stabilities and activities of BT1285 and BT3987 were optimal in markedly different conditions. BT1285 exhibits significantly higher affinity and faster hydrolysis of poorly accessible HM N-glycans than does BT3987. We also find that two HM-processing endoglycosidases from the human gut-resident Alistipes finegoldii display condition-specific functional properties. Altogether, our data suggest that human gut microbes employ evolutionary strategies to express distinct ENGases in order to optimally metabolize the same N-glycan substrate in the gastroinstestinal tract.

N-glycosylation is one of the most abundant posttranslational modifications of proteins, essential for many physiological processes, including protein folding, protein stability, oligomerization and aggregation, and molecular recognition events. Defects in the N-glycosylation pathway cause diseases that are classified as congenital disorders of glycosylation. The ability to manipulate protein N-glycosylation is critical not only to our fundamental understanding of biology but also for the development of new drugs for a wide range of human diseases. Chemoenzymatic synthesis using engineered endo-β-N-acetylglucosaminidases (ENGases) has been used extensively to modulate the chemistry of N-glycosylated proteins. However, defining the molecular mechanisms by which ENGases specifically recognize and process N-glycans remains a major challenge. Here we present the X-ray crystal structure of the ENGase EndoBT-3987 from Bacteroides thetaiotaomicron in complex with a hybrid-type glycan product. In combination with alanine scanning mutagenesis, molecular docking calculations and enzymatic activity measurements conducted on a chemically engineered monoclonal antibody substrate unveil two mechanisms for hybrid-type recognition and processing by paradigmatic ENGases. Altogether, the experimental data provide pivotal insight into the molecular mechanism of substrate recognition and specificity for GH18 ENGases and further advance our understanding of chemoenzymatic synthesis and remodeling of homogeneous N-glycan glycoproteins.

The analysis of N-glycan distributions in formalin-fixed, paraffin-embedded (FFPE) tissues by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is an effective approach for characterization of many disease states. As the workflow has matured and new technology emerged, approaches are needed to more efficiently characterize the isomeric structures of these N-glycans to expand on the specificity of their localization within tissue. Sialic acid chemical derivatization can be used to determine the isomeric linkage (α2,3 or α2,6) of sialic acids attached to N-glycans, while endoglycosidase F3 (Endo F3) can be enzymatically applied to preferentially release α1,6-linked core fucosylated glycans, further describing the linkage of fucose on N-glycans. Here we describe workflows where N-glycans are chemically derivatized to reveal sialic acid isomeric linkages, combined with a dual-enzymatic approach of endoglycosidase F3 and PNGase F to further elucidate fucosylation isomers on the same tissue section.

Mismatch negativity (MMN) is a differential electrophysiological response measuring cortical adaptability to unpredictable stimuli. MMN is consistently attenuated in patients with psychosis. However, the genetics of MMN are uncharted, limiting the validation of MMN as a psychosis endophenotype. Here, we perform a transcriptome-wide association study of 728 individuals, which reveals 2 genes (FAM89A and ENGASE) whose expression in cortical tissues is associated with MMN. Enrichment analyses of neurodevelopmental expression signatures show that genes associated with MMN tend to be overexpressed in the frontal cortex during prenatal development but are significantly downregulated in adulthood. Endophenotype ranking value calculations comparing MMN and three other candidate psychosis endophenotypes (lateral ventricular volume and two auditory-verbal learning measures) find MMN to be considerably superior. These results yield promising insights into sensory processing in the cortex and endorse the notion of MMN as a psychosis endophenotype.

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.

Specific alterations in N-linked glycans, such as core fucosylation, are associated with many cancers and other disease states. Because of the many possible anomeric linkages associated with fucosylated N-glycans, determination of specific anomeric linkages and the site of fucosylation (i.e., core vs outer arm) can be difficult to elucidate. A new MALDI mass spectrometry imaging workflow in formalin-fixed clinical tissues is described using recombinant endoglycosidase F3 (Endo F3), an enzyme with a specific preference for cleaving core-fucosylated N-glycans attached to glycoproteins. In contrast to the broader substrate enzyme peptide-N-glycosidase F (PNGaseF), Endo F3 cleaves between the two core N-acetylglucosamine residues at the protein attachment site. On tissues, this results in a mass shift of 349.137 a.m.u. for core-fucosylated N-glycans when compared to N-glycans released with standard PNGaseF. Endo F3 can be used singly and in combination with PNGaseF digestion of the same tissue sections. Initial results in liver and prostate tissues indicate core-fucosylated glycans associated to specific tissue regions while still demonstrating a diverse mix of core- and outer arm-fucosylated glycans throughout all regions of tissue. By determining these specific linkages while preserving localization, more targeted diagnostic biomarkers for disease states are possible without the need for microdissection or solubilization of the tissue.

Viral glycoproteins are expressed by many viruses, and during infection they usually play very important roles, such as receptor attachment or membrane fusion. The mature virion of the white spot syndrome virus (WSSV) is unusual in that it contains no glycosylated proteins, and there are currently no reports of any glycosylation mechanisms in the pathogenesis of this virus. In this study, we cloned a glycosylase, mannosyl-glycoprotein endo-β-N-acetylglucosaminidase (ENGase, EC 3.2.1.96), from Penaeus monodon and found that it was significantly up-regulated in WSSV-infected shrimp. A yeast two-hybrid assay showed that PmENGase interacted with both structural and non-structural proteins, and GST-pull down and co-immunoprecipitation (Co-IP) assays confirmed its interaction with the envelope protein VP41B. In the WSSV challenge tests, the cumulative mortality and viral copy number were significantly decreased in the PmEngase-silenced shrimp, from which we conclude that shrimp glycosylase interacts with WSSV in a way that benefits the virus. Lastly, we speculate that the deglycosylation activity of PmENGase might account for the absence of glycosylated proteins in the WSSV virion.

Peptide-N-glycosidase F (PNGase F) is the most frequently used enzyme to release N-glycan from glycoproteins in glycomics; however, the releasing process using PNGase F is tedious and can range in duration from hours to overnight. Recently, efforts have been made to accelerate this enzymatic reaction, and they include the use of microwave irradiation, ultrahigh pressure, enzyme immobilization, and other techniques. Here, we developed a novel method combining the oriented immobilization of PNGase F on magnetic particles and microwave-assisted enzymatic digestion techniques to achieve highly efficient release of N-glycans. The oriented immobilization of PNGase F on magnetic particles utilizes the affinity of its co-expressed His-tag towards iminodiacetic acid-Nickel modified magnetic particles. Compared with non-oriented immobilization, the oriented immobilization of PNGase F exhibits several advantages including tolerance to high temperature (52 °C) and the ability to retain strong activity after more than five reuses. When used in combination with microwave irradiation, efficient N-glycan removal from ribonuclease B was achieved within 5 min. The proposed strategy was also used to release glycan from fetuin and human serum and has proven to provide a promising deglycosylation method for the characterization of protein glycosylation.

Endoplasmic reticulum (ER) stress occurs when the abundance of unfolded proteins in the ER exceeds the capacity of the folding machinery. Despite the expanding cadre of characterized cellular adaptations to ER stress, knowledge of the effects of ER stress on cellular physiology remains incomplete. We investigated the impact of ER stress on ER and inner nuclear membrane protein quality control mechanisms in Saccharomyces cerevisiae. We analyzed the turnover of substrates of four ubiquitin ligases (Doa10, Rkr1/Ltn1, Hrd1, and the Asi complex) and the metalloprotease Ste24 in induced models of ER stress. ER stress did not substantially impact Doa10 or Rkr1 substrates. However, Hrd1-mediated destruction of a protein that aberrantly engages the translocon (Deg1-Sec62) and substrates with luminal degradation signals was markedly impaired by ER stress; by contrast, Hrd1-dependent degradation of proteins with intramembrane degrons was largely unperturbed by ER stress. ER stress impaired the degradation of one of two Asi substrates analyzed and caused a translocon-clogging Ste24 substrate to accumulate in a form consistent with persistent translocon occupation. Degradation of Deg1-Sec62 in the absence of stress and stabilization during ER stress were independent of four ER stress-sensing pathways. Our results indicate ER stress differentially impacts degradation of protein quality control substrates, including those mediated by the same ubiquitin ligase. These observations suggest the existence of additional regulatory mechanisms dictating substrate selection during ER stress.

There are several methods, both chemical and enzymatic, to release N-linked glycans for structural characterization. One of the most common enzymatic release methods is the use of peptide:N-glycosidase F (PNGase F). A less expensive and quicker alternative has been reported for the release of N-linked glycans chemically using sodium hypochlorite (NaOCl), which hydrolyzes the peptide-glycan bond, yielding the intact glycan with a free reducing terminus. Here, we quantitatively analyzed the efficiency of the NaOCl release protocol compared with the PNGaseF release protocol for small-scale analysis (300 µg) using liquid chromatography-single reaction monitoring-mass spectrometry. We determined that the relative glycan composition of released N-linked glycans from the NaOCl protocol is similar to a typical PNGase F protocol, but the absolute recovery of N-linked glycans is significantly lower with the chemical procedure.