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The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein syntaxin-5 (Stx5) is essential for Golgi transport. In humans, the STX5 mRNA encodes two protein isoforms, Stx5 Long (Stx5L) from the first starting methionine and Stx5 Short (Stx5S) from an alternative starting methionine at position 55. In this study, we identify a human disorder caused by a single missense substitution in the second starting methionine (p.M55V), resulting in complete loss of the short isoform. Patients suffer from an early fatal multisystem disease, including severe liver disease, skeletal abnormalities and abnormal glycosylation. Primary human dermal fibroblasts isolated from these patients show defective glycosylation, altered Golgi morphology as measured by electron microscopy, mislocalization of glycosyltransferases, and compromised ER-Golgi trafficking. Measurements of cognate binding SNAREs, based on biotin-synchronizable forms of Stx5 (the RUSH system) and Förster resonance energy transfer (FRET), revealed that the short isoform of Stx5 is essential for intra-Golgi transport. Alternative starting codons of Stx5 are thus linked to human disease, demonstrating that the site of translation initiation is an important new layer of regulating protein trafficking. Mutations in genes critical for proper intra-Golgi transport can cause human syndromes due to defects in glycosylation of proteins. Here, the authors identify a human variant of Syntaxin-5 that causes fatal multisystem disease and mislocalization of glycosyltransferases due to altered Golgi transport.

Variants in SLC35C1 underlie leucocyte adhesion deficiency (LADII) or congenital disorder of glycosylation type 2c (CDGIIc), an autosomal recessive disorder of fucosylation. This immunodeficiency syndrome is generally characterized by severe recurrent infections, Bombay blood group, reduced growth and intellectual disability (ID). Features are all caused by an inability to generate key fucosylated molecules due to a defective transport of GDP-fucose into the Golgi. Here we report the use of exome sequencing to identify biallelic variants in SLC35C1 (c.501_503delCTT, p.(Phe168del) and c.891T > G, p.(Asn297Lys)) in an individual with short stature and ID. Retrospective clinical examination based on the genetic findings revealed increased otitis media as the only immunological feature present in this child. Biochemical analysis of patient serum identified a clear but mild decrease in protein fucosylation. Modelling all described missense mutations on a SLC35C1 protein model showed pathogenic substitutions localise to close to the dimer interface, providing insight into the possible pathophysiology of non-synonymous causative variants identified in patients. Our evidence confirms this is the second family presenting with only a subset of features and broadens the clinical presentation of this syndrome. Of note, both families segregated a common allele (p.Phe168del), suggesting there could be an associated genotype-phenotype relationship for specific variants. Based on two out of 14 reported families not presenting with the characteristic features of SLC35C1-CDG, we suggest there is clinical utility in considering this gene in patients with short stature and ID.

Hemolytic uremic syndrome caused by an invasive Streptococcus pneumoniae infection (SP-HUS) is a rare and severe disease that primarily affects children under two years of age. The pathophysiology of SP-HUS remains poorly understood, and treatment is largely supportive. Complement factor H (FH) is a key regulator of the alternative pathway of the complement system. It has been hypothesized that loss of sialic acids from FH’s N-glycans may impair its regulatory functions, thereby potentially leading to complement-mediated endothelial cell damage in SP-HUS. In this study, we investigated the N-glycosylation patterns of FH across three N-glycosylation sites for four SP-HUS patients and compared it to healthy controls using LC-MS/MS-based glycopeptide profiling. We identified significant changes in FH glycosylation during the acute phase of SP-HUS, including an increased presence of N-glycans lacking sialic acids, galactose and N-acetylglucosamine (GlcNAc) relative to the controls. This abnormal glycosylation was most prominent during the acute phase in all patients and showed partial or complete normalization during remission. Interestingly, despite these major glycosylation changes, functional assays revealed no significant impairment in the complement regulatory activity of FH, as measured by its ability to facilitate C3b degradation and to prevent complement-mediated hemolysis of sheep erythrocytes. In conclusion, our findings show that FH’s N-glycosylation is severely altered in the acute phase in SP-HUS patients, comprising more than just the loss of sialic acids. However, these changes do not directly affect FH’s complement regulatory function. These results highlight the complex yet poorly understood role of N-glycosylation during infection, and the contribution of FH’s N-glycans to complement (dys)regulation and disease pathogenesis.

Andrea Superti-Furga, Ron Wevers, Clara van Karnebeek, Luisa Bonafé and colleagues identify mutations in NANS , which encodes the sialic acid synthase, in nine individuals with severe infantile-onset developmental delay and skeletal dysplasia. They describe abnormal metabolites accumulating because of deficient NANS enzyme activity and show that impaired sialic acid synthesis in zebrafish perturbs skeletal development, which can partially be rescued by supplementation with exogenous sialic acid. We identified biallelic mutations in NANS , the gene encoding the synthase for N -acetylneuraminic acid (NeuNAc; sialic acid), in nine individuals with infantile-onset severe developmental delay and skeletal dysplasia. Patient body fluids showed an elevation in N -acetyl- D -mannosamine levels, and patient-derived fibroblasts had reduced NANS activity and were unable to incorporate sialic acid precursors into sialylated glycoproteins. Knockdown of nansa in zebrafish embryos resulted in abnormal skeletal development, and exogenously added sialic acid partially rescued the skeletal phenotype. Thus, NANS-mediated synthesis of sialic acid is required for early brain development and skeletal growth. Normal sialylation of plasma proteins was observed in spite of NANS deficiency. Exploration of endogenous synthesis, nutritional absorption, and rescue pathways for sialic acid in different tissues and developmental phases is warranted to design therapeutic strategies to counteract NANS deficiency and to shed light on sialic acid metabolism and its implications for human nutrition.

The glycosylation of proteins plays an important role in neurological development and disease. Glycoproteomic studies on cerebrospinal fluid (CSF) are a valuable tool to gain insight into brain glycosylation and its changes in disease. However, it is important to consider that most proteins in CSFs originate from the blood and enter the CSF across the blood–CSF barrier, thus not reflecting the glycosylation status of the brain. Here, we apply a glycoproteomics method to human CSF, focusing on differences between brain- and blood-derived proteins. To facilitate the analysis of the glycan site occupancy, we refrain from glycopeptide enrichment. In healthy individuals, we describe the presence of heterogeneous brain-type N-glycans on prostaglandin H2-D isomerase alongside the dominant plasma-type N-glycans for proteins such as transferrin or haptoglobin, showing the tissue specificity of protein glycosylation. We apply our methodology to patients diagnosed with various genetic glycosylation disorders who have neurological impairments. In patients with severe glycosylation alterations, we observe that heavily truncated glycans and a complete loss of glycans are more pronounced in brain-derived proteins. We speculate that a similar effect can be observed in other neurological diseases where a focus on brain-derived proteins in the CSF could be similarly beneficial to gain insight into disease-related changes.

Real-time database searching allows for simpler and automated proteomics workflows as it eliminates technical bottlenecks in high-throughput experiments. Most importantly, it enables results-dependent acquisition (RDA), where search results can be used to guide data acquisition during acquisition. This is especially beneficial for glycoproteomics since the wide range of physicochemical properties of glycopeptides lead to a wide range of optimal acquisition parameters. We established here the GlycoPaSER prototype by extending the Parallel Search Engine in Real-time (PaSER) functionality for real-time glycopeptide identification from fragmentation spectra. Glycopeptide fragmentation spectra were decomposed into peptide and glycan moiety spectra using common N-glycan fragments. Each moiety was subsequently identified by a specialized algorithm running in real-time. GlycoPaSER can keep up with the rate of data acquisition for real-time analysis with similar performance to other glycoproteomics software and produces results that are in line with the literature reference data. The GlycoPaSER prototype presented here provides the first proof-of-concept for real-time glycopeptide identification that unlocks the future development of RDA technology to transcend data acquisition.

Glycoproteins play important roles in numerous physiological processes and are often implicated in disease. Analysis of site-specific protein glycobiology through glycoproteomics is evolving rapidly in recent years thanks to hardware and software innovations. Particularly, the introduction of Parallel Accumulation Serial Fragmentation (PASEF) on hybrid trapped ion mobility time-of-flight mass spectrometry instruments combined deep proteome sequencing with separation of (near-)isobaric precursor ions or converging isotope envelopes through ion mobility separation. However, reported use of PASEF in integrated glycoproteomics workflows to comprehensively capture the glycoproteome is still limited. To this end, we developed an integrated methodology using the timsTOF Pro 2 to enhance N-glycopeptide identifications in complex mixtures. We systematically optimized the ion optics tuning, collision energies, mobility isolation width and the use of dopant-enriched nitrogen gas (DEN). Thus, we obtained a marked increase in unique glycopeptide identification rates compared to standard proteomics settings showcasing our results on a large set of glycopeptides. With short liquid chromatography gradients of 30 minutes, we increased the number of unique N-glycopeptide identifications in human plasma samples from around 100 identifications under standard proteomics condition to up to 1500 with our optimized glycoproteomics approach, highlighting the need for tailored optimizations to obtain comprehensive data.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Glycoproteins play important roles in numerous physiological processes and are often implicated in disease. Analysis of site-specific protein glycobiology through glycoproteomics is evolving rapidly in recent years thanks to hardware and software innovations. Particularly, the introduction of Parallel Accumulation Serial Fragmentation (PASEF) on hybrid trapped ion mobility time-of-flight mass spectrometry instruments combined deep proteome sequencing with separation of (near-)isobaric precursor ions or converging isotope envelopes through ion mobility separation. However, reported use of PASEF in integrated glycoproteomics workflows to comprehensively capture the glycoproteome is still limited. To this end, we devel-oped an integrated methodology using the timsTOF Pro 2 to enhance N-glycopeptide identifications in complex mixtures. We systematically optimized the ion optics tuning, collision energies, mobility isolation width and the use of dopant-enriched nitrogen gas (DEN). Thus, we obtained a marked increase in unique glycopeptide identification rates compared to standard proteomics settings showcasing our results on a large set of glycopeptides. With short liquid chromatography gradients of 30 minutes, we increased the number of unique N-glycopeptide identifications in human plasma samples from around 100 identifications under standard proteomics condition to up to 1500 with our optimized glycoproteomics approach, highlight-ing the need for tailored optimizations to obtain comprehensive data.

Nat. Genet. 48, 777–784 (2016); published online 23 May 2016; corrected after print 6 March 2017 In the version of this article initially published, the name of author Torben Heise was given incorrectly as Thorben Heisse, and the name of author Valérie Cormier-Daire was given incorrectly as Valerie Cormier.