The glycoproteome remains severely understudied because of significant analytical challenges associated with glycoproteomics, the system-wide analysis of intact glycopeptides. This review introduces ...important structural aspects of protein N-glycosylation and summarizes the latest technological developments and applications in LC-MS/MS-based qualitative and quantitative N-glycoproteomics. These maturing technologies provide unique structural insights into the N-glycoproteome and its synthesis and regulation by complementing existing methods in glycoscience. Modern glycoproteomics is now sufficiently mature to initiate efforts to capture the molecular complexity displayed by the N-glycoproteome, opening exciting opportunities to increase our understanding of the functional roles of protein N-glycosylation in human health and disease.
Just as Koch's postulates formed the foundation of early infectious disease study, Stanley Falkow's molecular Koch's postulates define best practice in determining whether a specific gene contributes ...to virulence of a pathogen. Fundamentally, these molecular postulates state that if a gene is involved in virulence, its removal will compromise virulence. Likewise, its reintroduction should restore virulence to the mutant. These approaches are widely employed in Cryptococcus neoformans, where gene deletion via biolistic transformation is a well-established technique. However, the complementation of these mutants is less straightforward. Currently, one of three approaches will be taken: the gene is reintroduced at the original locus, the gene is reintroduced into a random site in the genome, or the mutant is not complemented at all. Depending on which approach is utilized, the mutant may be complemented but other genes are potentially disrupted in the process. To counter the drawbacks of the current approaches to complementation we have created a new tool to assist in this key step in the study of a gene's role in virulence. We have identified and characterized a small gene-free region in the C. neoformans genome dubbed the "safe haven", and constructed a plasmid vector that targets DNA constructs to this preselected site. The plasmid vector integrates with high frequency, effectively complementing a mutant strain without disrupting adjacent genes. qRT-PCR of the flanking genes on either side of the safe haven site following integration of the targeting vector revealed no changes in their expression, and no secondary phenotypes were observed in a range of phenotypic assays including an intranasal murine infection model. Combined, these data confirm that we have successfully created a much-needed molecular resource for the Cryptococcus community, enabling the reliable fulfillment of the molecular Koch's postulates.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In August 2022, a novel henipavirus (HNV) named Langya virus (LayV) was isolated from patients with severe pneumonic disease in China. This virus is closely related to Mòjiāng virus (MojV), and both ...are divergent from the bat-borne HNV members, Nipah (NiV) and Hendra (HeV) viruses. The spillover of LayV is the first instance of a HNV zoonosis to humans outside of NiV and HeV, highlighting the continuing threat this genus poses to human health. In this work, we determine the prefusion structures of MojV and LayV F proteins via cryogenic electron microscopy to 2.66 and 3.37 Å, respectively. We show that despite sequence divergence from NiV, the F proteins adopt an overall similar structure but are antigenically distinct as they do not react to known antibodies or sera. Glycoproteomic analysis revealed that while LayV F is less glycosylated than NiV F, it contains a glycan that shields a site of vulnerability previously identified for NiV. These findings explain the distinct antigenic profile of LayV and MojV F, despite the extent to which they are otherwise structurally similar to NiV. Our results carry implications for broad-spectrum HNV vaccines and therapeutics, and indicate an antigenic, yet not structural, divergence from prototypical HNVs.
N-glycosylation plays an essential role in regulating protein folding and function in eukaryotic cells. Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH) ...has proven useful as a data independent acquisition (DIA) MS method for analysis of glycoproteins and their glycan modifications. By separating the entire m/z range into consecutive isolation windows, DIA-MS allows comprehensive MS data acquisition and high-sensitivity detection of molecules of interest. Variable width DIA windows allow optimal analyte measurement, as peptide ions are not evenly distributed across the full m/z range. However, the m/z distribution of glycopeptides is different to that of unmodified peptides because of their large glycan structures. Here, we improved the performance of DIA glycoproteomics by using variable width windows optimized for glycopeptides. This method allocates narrow windows at m/z ranges rich in glycopeptides, improving analytical specificity and performance. We show that related glycoforms must fall in separate windows to allow accurate glycopeptide measurement. We demonstrate the utility of the method by comparing the cell wall glycoproteomes of wild-type and N-glycan biosynthesis deficient yeast and showing improved measurement of glycopeptides with different glycan structures. Our results highlight the importance of appropriately optimized DIA methods for measurement of post-translationally modified peptides.
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•Glycopeptides have a distinct m/z distribution to unmodified peptides.•Variable window SWATH optimized for glycopeptides improves performance.•Data Independent Acquisition methods should be optimized for modified peptides.
Carbohydrate analyses are often challenging due to the structural complexity of these molecules, as well as the lack of suitable analytical tools for distinguishing the vast number of possible ...isomers. The coupled technique, ion mobility-mass spectrometry (IM-MS), has been in use for two decades for the analysis of complex biomolecules, and in recent years it has emerged as a powerful technique for the analysis of carbohydrates. For carbohydrates, most studies have focused on the separation and characterization of isomers in biological samples. IM-MS is capable of separating isomeric ions by drift time, and further characterizing them by mass analysis. Applications of IM-MS in carbohydrate analysis are extremely useful and important for understanding many biological mechanisms and for the determination of disease states, although efforts are still needed for higher sensitivity and resolution.
Glycan macro- and microheterogeneity have profound impacts on protein folding and function. This heterogeneity can be regulated by physiological or environmental factors. However, unregulated ...heterogeneity can lead to disease, and mutations in the glycosylation process cause a growing number of Congenital Disorders of Glycosylation. We systematically studied how mutations in the N-glycosylation pathway lead to defects in mature proteins using all viable Saccharomyces cerevisiae strains with deletions in genes encoding Endoplasmic Reticulum lumenal mannosyltransferases (Alg3, Alg9, and Alg12), glucosyltransferases (Alg6, Alg8, and Die2/Alg10), or oligosaccharyltransferase subunits (Ost3, Ost5, and Ost6). To measure the changes in glycan macro- and microheterogeneity in mature proteins caused by these mutations we developed a SWATH-mass spectrometry glycoproteomics workflow. We measured glycan structures and occupancy on mature cell wall glycoproteins, and relative protein abundance, in the different mutants. All mutants showed decreased glycan occupancy and altered cell wall proteomes compared with wild-type cells. Mutations in earlier mannosyltransferase or glucosyltransferase steps of glycan biosynthesis had stronger hypoglycosylation phenotypes, but glucosyltransferase defects were more severe. ER mannosyltransferase mutants displayed substantial global changes in glycan microheterogeneity consistent with truncations in the glycan transferred to protein in these strains. Although ER glucosyltransferase and oligosaccharyltransferase subunit mutants broadly showed no change in glycan structures, ost3Δ cells had shorter glycan structures at some sites, consistent with increased protein quality control mannosidase processing in this severely hypoglycosylating mutant. This method allows facile relative quantitative glycoproteomics, and our results provide insights into global regulation of site-specific glycosylation.
Nontypeable Haemophilus influenzae (NTHi) is a leading cause of respiratory tract infections worldwide and continues to be a global health burden. Adhesion and colonization of host cells are crucial ...steps in bacterial pathogenesis, and in many strains of NTHi, the interaction with the host is mediated by the high molecular weight adhesins HMW1A and HMW2A. These adhesins are N-glycoproteins that are modified by cytoplasmic glycosyltransferases HMW1C and HMW2C. Phase variation in the number of short sequence repeats in the promoters of hmw1A and hmw2A directly affects their expression. Here, we report the presence of similar variable repeat elements in the promoters of hmw1C and hmw2C in diverse NTHi isolates. In an ex vivo assay, we systematically altered the substrate and glycosyltransferase expression and showed that both of these factors quantitatively affected the site-specific efficiency of glycosylation on HMW-A. This represents a novel mechanism through which phase variation can generate diversity in the quantitative extent of site-specific post-translational modifications on antigenic surface proteins. Glycosylation occupancy was incomplete at many sites, variable between sites, and generally lower close to the C-terminus of HMW-A. We investigated the causes of this variability. As HMW-C glycosylates HMW-A in the cytoplasm, we tested how secretion affected glycosylation on HMW-A and showed that retaining HMW-A in the cytoplasm indeed increased glycosylation occupancy across the full length of the protein. Site-directed mutagenesis showed that HMW-C had no inherent preference for glycosylating asparagines in NxS or NxT sequons. This work provides key insights into factors contributing to the heterogenous modifications of NTHi HMW-A adhesins, expands knowledge of NTHi population diversity and pathogenic capability, and is relevant to vaccine design for NTHi and related pathogens.
Autophagy is reported to be an important innate immune defense against the intracellular bacterial pathogen Group A Streptococcus (GAS). However, the GAS strains examined to date belong to serotypes ...infrequently associated with human disease. We find that the globally disseminated serotype M1T1 clone of GAS can evade autophagy and replicate efficiently in the cytosol of infected cells. Cytosolic M1T1 GAS (strain 5448), but not M6 GAS (strain JRS4), avoids ubiquitylation and recognition by the host autophagy marker LC3 and ubiquitin-LC3 adaptor proteins NDP52, p62, and NBR1. Expression of SpeB, a streptococcal cysteine protease, is critical for this process, as an isogenic M1T1 ΔspeB mutant is targeted to autophagy and attenuated for intracellular replication. SpeB degrades p62, NDP52, and NBR1 in vitro and within the host cell cytosol. These results uncover a proteolytic mechanism utilized by GAS to escape the host autophagy pathway that may underpin the success of the M1T1 clone.
•M1T1 group A Streptococcus (GAS) evade autophagy and replicate intracellularly•M1T1 GAS evade ubiquitylation and host ubiquitin-LC3 adaptors NBR1, p62, and NDP52•M1T1 ΔspeB is targeted to autophagy and has reduced intracellular replication•GAS cysteine protease SpeB degrades ubiquitin-LC3 adaptors in vitro and in vivo
The dense glycan shield is an essential feature of the SARS-CoV-2 spike (S) architecture, key to immune evasion and to the activation of the prefusion conformation. Recent studies indicate that the ...occupancy and structures of the SARS-CoV-2 S glycans depend not only on the nature of the host cell, but also on the structural stability of the trimer; a point that raises important questions about the relative competence of different glycoforms. Moreover, the functional role of the glycan shield in the SARS-CoV-2 pathogenesis suggests that the evolution of the sites of glycosylation is potentially intertwined with the evolution of the protein sequence to affect optimal activity. Our results from multi-microsecond molecular dynamics simulations indicate that the type of glycosylation at N234, N165 and N343 greatly affects the stability of the receptor binding domain (RBD) open conformation, and thus its exposure and accessibility. Furthermore, our results suggest that the loss of glycosylation at N370, a newly acquired modification in the SARS-CoV-2 S glycan shield's topology, may have contributed to increase the SARS-CoV-2 infectivity as we find that
N
-glycosylation at N370 stabilizes the closed RBD conformation by binding a specific cleft on the RBD surface. We discuss how the absence of the N370 glycan in the SARS-CoV-2 S frees the RBD glycan binding cleft, which becomes available to bind cell-surface glycans, and potentially increases host cell surface localization.
The
N
-glycans structures affect the mechanistic properties of the SARS-CoV-2 S, fine-tuning the glycoprotein. The evolution of the glycan shield led to the loss of N370 glycosylation in SARS-CoV-2 S, where the RBD cleft can bind host-cell glycans.
Beer brewing is an ancient process that is now an established industrial-scale bioprocess. However, critical aspects of the protein biochemistry underlying brewing are both poorly understood and ...surprisingly complex. We used SWATH-MS proteomics to study the mashing stage of beer brewing, identified an extreme diversity of proteoforms caused by abundant physiological proteolysis, and used methods based on proteome thermal profiling to show that these posttranslational modifications control the dynamic beer brewing proteome.
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Highlights
•SWATH-MS profiles protein abundance and modification during mashing in beer brewing.•Proteolysis due to barley proteases leads to extreme proteoform diversity.•Sequence-specific proteolytic clipping of proteins controls their stability.
Mashing is a key step in beer brewing in which starch and proteins are solubilized from malted barley in a hot water extraction and digested to oligomaltose and free amino nitrogen. We used SWATH-MS to measure the abundance and site-specific modifications of proteins throughout a small-scale pale ale mash. Proteins extracted from the malt at low temperatures early in the mash decreased precipitously in abundance at higher temperatures late in the mash due to temperature/time-induced unfolding and aggregation. We validated these observations using experimental manipulation of time and temperature parameters in a microscale pale ale mash. Correlation analysis of temperature/time-dependent abundance showed that sequence and structure were the main features that controlled protein abundance profiles. Partial proteolysis by barley proteases was common early in the mash. The resulting proteolytically clipped proteins were particularly sensitive and were preferentially lost at high temperatures late in the mash, while intact proteins remained soluble. The beer brewing proteome is therefore driven by the interplay between protein solubilization and proteolysis, which are in turn determined by barley variety, growth conditions, and brewing process parameters.
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