Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the ...

National Research Council Canada

N- Glycosylation of proteins is recognized as one of the most common post-translational modifications. Until recently it was believed that N-glycosylation occurred exclusively in eukaryotes until the discovery of the general protein glycosylation pathway (Pgl) in Campylobacter jejuni. We have developed a new glycomics strategy based on lectin-affinity capture of lipid-linked oligosaccharides (LLOs) coupled to capillary electrophoresis mass spectrometry. The LLO intermediates of the C. jejuni Pgl pathway were used to validate the methodology and to better characterize the bacterial model system for protein N-glycosylation. This method provides a rapid, non-radioactive approach for the characterization of intermediates in polysaccharide biosynthesis and is a useful tool for glycoengineering efforts in bacteria.

Characterization of lipid-linked oligosaccharides by mass spectrometry.

The development of glycomics increasingly requires the detection and quantification of large numbers of glycans, which is only partially achieved by current glycomics approaches. Taking advantage of selected reaction monitoring to enhance both sensitivity and selectivity, we report here a strategy termed targeted glycomics that enables highly sensitive and consistent identification and quantification of diverse glycans across multiple samples at the same time. In this proof-of-principle study, we validated the method by analyzing global N-glycans expressed in different systems: single proteins, cancer cells, and serum samples. A dynamic range of three orders of magnitude was obtained for the detection of all five glycans released from ribonuclease B. The limit of detection of 80 attomole for Man(9)GlcNAc(2) demonstrated the excellent sensitivity of the method. The capability of the strategy to identify diverse glycans was demonstrated by identification and detection of 162 different glycans and isomers from pancreatic cancer cells. The sensitivity of the method was illustrated further by the ability to detect eight glycans from 250 cancer cells and five glycans released from 100 cancer cells. In serum obtained from rabbits fed control diet or diet enriched with 2% cholesterol, differences to 42 glycans were accurately measured and this indicates that this strategy might find use in studies of biomarker discovery and validation.

Targeted glycomics by selected reaction monitoring for highly sensitive glycan compositional analysis.

N-acylethanolamines (NAEs) are endogenous lipid-based signaling molecules best known for their role in the endocannabinoid system in mammals, but they are also known to play roles in signaling pathways in plants. The regulation of NAEs in vivo is partly accomplished by the enzyme fatty acid amide hydrolase (FAAH), which hydrolyses NAEs to ethanolamine and their corresponding fatty acid. Inhibition of FAAH has been shown to increase the levels of NAEs in vivo and to produce desirable phenotypes. This has led to the development of pharmaceutical-based therapies for a variety of conditions targeting FAAH. Recently, our group identified a functional FAAH homolog in Dictyostelium discoideum, leading to our hypothesis that D. discoideum also possesses NAEs. In this study, we provide a further characterization of FAAH and identify NAEs in D. discoideum for the first time. We also demonstrate the ability to modulate their levels in vivo through the use of a semispecific FAAH inhibitor and confirm that these NAEs are FAAH substrates through in vitro studies. We believe the demonstration of the in vivo modulation of NAE levels suggests that D. discoideum could be a good simple model organism in which to study NAE-mediated signaling.

Identification of N-acylethanolamines in Dictyostelium discoideum and confirmation of their hydrolysis by fatty acid amide hydrolase.

The emergence of ceftriaxone-resistant strains of Neisseria gonorrhoeae may herald an era of untreatable gonorrhea. Vaccines against this infection are urgently needed. The 2C7 epitope is a conserved oligosaccharide (OS) structure, a part of lipooligosaccharide (LOS) on N gonorrhoeae. The epitope is expressed by 94% of gonococci that reside in the human genital tract (in vivo) and by 95% of first passaged isolates. Absence of the 2C7 epitope shortens the time of gonococcal carriage in a mouse model of genital infection. To circumvent the limitations of saccharide immunogens in producing long lived immune responses, previously we developed a peptide mimic (called PEP1) as an immunologic surrogate of the 2C7-OS epitope and reconfigured it into a multi-antigenic peptide, (MAP1). To test vaccine efficacy of MAP1, female BALB/c mice were passively immunized with a complement-dependent bactericidal monoclonal antibody specific for the 2C7 epitope or were actively immunized with MAP1. Mice immunized with MAP1 developed a TH1-biased anti-LOS IgG antibody response that was also bactericidal. Length of carriage was shortened in immune mice; clearance occurred in 4 days in mice passively administered 2C7 antibody vs. 6 days in mice administered control IgG3λ mAb in one experiment (p = 0.03) and 6 vs. 9 days in a replicate experiment (p = 0.008). Mice vaccinated with MAP1 cleared infection in 5 days vs. 9 days in mice immunized with control peptide (p = 0.0001 and p = 0.0002, respectively in two replicate experiments). Bacterial burden was lower over the course of infection in passively immunized vs. control mice in both experiments (p = 0.008 and p = 0.0005); burdens were also lower in MAP1 immunized mice vs. controls (p<0.0001) and were inversely related to vaccine antibodies induced in the vagina (p = 0.043). The OS epitope defined by mAb 2C7 may represent an effective vaccine target against gonorrhea, which is rapidly becoming incurable with currently available antibiotics. 

Immunization against a saccharide epitope accelerates clearance of experimental gonococcal infection.

Alzheimer's disease (AD) is characterized by extracellular deposits of amyloid-β (Aβ) in the brain. ABCA7 is highly expressed in the brain and a susceptibility gene for late-onset AD (LOAD). The minor alleles at two ABCA7 single-nucleotide polymorphisms (SNPs), rs3764650 (T>G; intron13) and rs3752246 at a predicted myristoylation site (C>G; exon33; p.Gly1527Ala), are significantly associated with LOAD risk; however, the mechanism of this association is unknown. Functional consequences of both SNPs were examined in HEK293 and CHO cells stably expressing AβPPSwe. Luciferase reporter assays in HEK293 cells suggested that intron13 carrying rs3764650 major T-allele (int13-T) possessed promoter-enhancing capabilities. Co-transfection experiments with hABCA7 and int13-T resulted in significantly increased ABCA7 protein level relative to that with int13-G. Expression of hABCA7 carrying rs3752246 risk allele led to increases in secreted Aβ40 and Aβ42 and β-secretase activity in CHO- and HEK-AβPPSwe cells. Hydroxymyristic acid treatment of cells expressing hABCA7 carrying the rs3752246 major G allele resulted in increased β-secretase activity and levels of Aβ, suggesting that lack of myristoylation contributes to the observed cell-phenotypes. Molecular weight determination, by gel-electrophoresis and mass spectrometry, of hABCA7 peptides spanning position 1527 showed loss of post-translational modification in the risk-allele peptide. These results suggest that decreased expression, or impaired function, of ABCA7 may contribute to AD pathology.

Genetic Variations in ABCA7 Can Increase Secreted Levels of Amyloid-β40 and Amyloid-β42 Peptides and ABCA7 Transcription in Cell Culture Models.

Mass spectrometry (MS) has played a vital role across a broad range of fields and applications in proteomics. The development of high-resolution MS has significantly advanced biology in areas such as protein structure, function, post-translational modification and global protein dynamics. The two most widely used MS ionization techniques in proteomics are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). ESI typically yields multiple charge values for each molecular mass and an isotopic cluster for each nominal mass-to-charge (m/z) value. Although MALDI mass spectra typically contain only singly charged ions, overlapping isotope patterns can be problematic for accurate mass measurement. To overcome these challenges of overlapping isotope patterns associated with complex samples in MS-based proteomics research, deconvolution strategies are being used. This manuscript describes a wide variety of deconvolution strategies, including de-isotoping and de-charging processes, deconvolution of co-eluting isomers or peptides with different sequences in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes, and data analysis in intact protein mass determination, ion mobility MS, native MS, and hydrogen/deuterium exchange MS. It concludes with a discussion of future prospects in the development of bioinformatics and potential new applications in proteomics.

Deconvolution in mass spectrometry based proteomics.

Neoantigen-based therapeutic vaccines have a high potential impact on tumor eradication and patient survival. Mass spectrometry (MS)-based immunopeptidomics has the capacity to identify tumor-associated epitopes and pinpoint mutation-bearing major histocompatibility complex (MHC)-binding peptides. This approach presents several challenges, including the identification of low-abundance peptides. In addition, MHC peptides have much lower MS/MS identification rates than tryptic peptides due to their shorter sequence and lack of basic amino acid at C-termini. In this study, we report the development and application of a novel chemical derivatization strategy that combines the analysis of native, dimethylated, and alkylamidated peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to expand the coverage of the MHC peptidome. The results revealed that dimethylation increases hydrophobicity and ionization efficiency of MHC class I peptides, while alkylamidation significantly improves the fragmentation by producing more y-ions during MS/MS fragmentation. Thus, the combination of dimethylation and alkylamidation enabled the identification of peptides that could not be identified from the analysis of their native form. Using this strategy, we identified 3148 unique MHC I peptides from HCT 116 cell lines, compared to only 1388 peptides identified in their native form. Among these, 10 mutation-bearing peptides were identified with high confidence, indicating that this chemical derivatization strategy is a promising approach for neoantigen discovery in clinical applications.

Chemical Derivatization Strategy for Extending the Identification of MHC Class I Immunopeptides.

 and  are the most common causes of bacterial gastroenteritis in the world. Ganglioside mimicry by  lipooligosaccharide (LOS) is the triggering factor of Guillain-Barré syndrome (GBS), an acute polyneuropathy. Sialyltransferases from glycosyltransferase family 42 (GT-42) are essential for the expression of ganglioside mimics in  Recently, two novel GT-42 genes,  and , have been identified in  Despite being present in ∼11% of currently available  genomes, the biological role of  and  is unknown. In the present investigation, mutation studies with two strains expressing either  or  were performed and mass spectrometry was used to investigate differences in the chemical composition of LOS. Attempts were made to identify donor and acceptor molecules using  activity tests with recombinant GT-42 enzymes. Here we show that CstIV and CstV are involved in  LOS biosynthesis. In particular,  is associated with LOS sialylation, while  is linked to the addition of a diacetylated nonulosonic acid residue. Despite the fact that  a major foodborne pathogen, its glycobiology has been largely neglected. The genetic makeup of the  lipooligosaccharide biosynthesis locus was largely unknown until recently.  harbors a large set of genes associated with lipooligosaccharide biosynthesis, including genes for several putative glycosyltransferases involved in the synthesis of sialylated lipooligosaccharide in  In the present study,  was found to express lipooligosaccharide structures containing sialic acid and other nonulosonate acids. These findings have a strong impact on our understanding of  ecology, host-pathogen interaction, and pathogenesis.

Genetics behind the Biosynthesis of Nonulosonic Acid-Containing Lipooligosaccharides in Campylobacter coli.

Porous graphic carbon chromatography (PGC) has a different mechanism in the retention of tryptic peptides compared with reversed-phase chromatography and in this study we show that coupling PGC with tandem mass spectrometry offer advantages for the quantitation of phosphorylation stoichiometry and characterization of site-specific glycosylation.

Online porous graphic carbon chromatography coupled with tandem mass spectrometry for post-translational modification analysis.

Lentiviral vectors (LVs) are a powerful tool for gene and cell therapy and human embryonic kidney cells (HEK293) have been extensively used as a platform for production of these vectors. Like most cells and cellular tissues, HEK293 cells release extracellular vesicles (EVs). EVs released by cells share similar size, biophysical characteristics and even a biogenesis pathway with cell-produced enveloped viruses, making it a challenge to efficiently separate EVs from LVs. Thus, EVs co-purified with LVs during downstream processing, are considered "impurities" in the context of gene and cell therapy. A greater understanding of EVs co-purifying with LVs is needed to enable improved downstream processing. To that end, EVs from an inducible lentivirus producing cell line were studied under two conditions: non-induced and induced. EVs were identified in both conditions, with their presence confirmed by transmission electron microscopy and Western blot. EV cargos from each condition were then further characterized by a multi-omic approach. Nineteen proteins were identified by mass spectrometry as potential EV markers to differentiate EVs in LV preparations. Lipid composition of EV preparations before and after LV induction showed similar enrichment in phosphatidylserine. RNA cargos in EVs showed enrichment in transcripts involved in viral processes and binding functions. These findings provide insights on the product profile of lentiviral preparations and could support the development of improved separation strategies aimed at removing co-produced EVs.

Jacek Stupak

Human Health Therapeutics Research Center

National Research Council Canada

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Jacek Stupak

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Human Health Therapeutics Research Center

National Research Council Canada

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Human Health Therapeutics Research Center