What comes next in glycobiology.

Glycans, with their variable compositions and highly dynamic conformations, vastly expand the heterogeneity of whatever factor or cell they are attached to. These properties make them crucial contributors to biological function and organismal health and also very difficult to study. That may be changing as we look to the future of glycobiology.

Analysis of the Healthy Platelet Proteome Identifies a New Form of Domain-Specific O-Fucosylation.

Platelet activation induces the secretion of proteins that promote platelet aggregation and inflammation. However, detailed analysis of the released platelet proteome is hampered by platelets' tendency to preactivate during their isolation and a lack of sensitive protocols for low abundance releasate analysis. Here, we detail the most sensitive analysis to date of the platelet releasate proteome with the detection of >1300 proteins. Unbiased scanning for posttranslational modifications within releasate proteins highlighted O-glycosylation as being a major component. For the first time, we detected O-fucosylation on previously uncharacterized sites including multimerin-1 (MMRN1), a major alpha granule protein that supports platelet adhesion to collagen and is a carrier for platelet factor V. The N-terminal elastin microfibril interface (EMI) domain of MMRN1, a key site for protein-protein interaction, was O-fucosylated at a conserved threonine within a new domain context. Our data suggest that either protein O-fucosyltransferase 1, or a novel protein O-fucosyltransferase, may be responsible for this modification. Mutating this O-fucose site on the EMI domain led to a >50% reduction of MMRN1 secretion, supporting a key role of EMI O-fucosylation in MMRN1 secretion. By comparing releasates from resting and thrombin-treated platelets, 202 proteins were found to be significantly released after high-dose thrombin stimulation. Complementary quantification of the platelet lysates identified >3800 proteins, which confirmed the platelet origin of releasate proteins by anticorrelation analysis. Low-dose thrombin treatment yielded a smaller subset of significantly regulated proteins with fewer secretory pathway enzymes. The extensive platelet proteome resource provided here (larancelab.com/platelet-proteome) allows identification of novel regulatory mechanisms for drug targeting to address platelet dysfunction and thrombosis.

Glycoproteome remodeling and organelle-specific N-glycosylation accompany neutrophil granulopoiesis.

Neutrophils store microbicidal glycoproteins in cytosolic granules to fight intruding pathogens, but their granule distribution and formation mechanism(s) during granulopoiesis remain unmapped. Herein, we comprehensively profile the neutrophil N-glycoproteome with spatiotemporal resolution by analyzing four key types of intracellular organelles isolated from blood-derived neutrophils and during their maturation from bone marrow-derived progenitors using a glycomics-guided glycoproteomics approach. Interestingly, the organelles of resting neutrophils exhibited distinctive glycophenotypes including, most strikingly, highly truncated N-glycans low in α2,6-sialylation and Lewis fucosylation decorating a diverse set of microbicidal proteins (e.g., myeloperoxidase, azurocidin, neutrophil elastase) in the azurophilic granules. Excitingly, proteomics and transcriptomics data from discrete myeloid progenitor stages revealed that profound glycoproteome remodeling underpins the promyelocytic-to-metamyelocyte transition and that the glycophenotypic differences are driven primarily by dynamic changes in protein expression and less by changes within the glycosylation machinery. Notable exceptions were the oligosaccharyltransferase subunits responsible for initiation of N-glycoprotein biosynthesis that were strongly expressed in early myeloid progenitors correlating with relatively high levels of glycosylation of the microbicidal proteins in the azurophilic granules. Our study provides spatiotemporal insights into the complex neutrophil N-glycoproteome featuring intriguing organelle-specific N-glycosylation patterns formed by dynamic glycoproteome remodeling during the early maturation stages of the myeloid progenitors.

Position-specific N- and O-glycosylation of the reactive center loop impacts neutrophil elastase-mediated proteolysis of corticosteroid-binding globulin.

Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues through proteolysis of an exposed reactive center loop (RCL) by neutrophil elastase (NE). We previously demonstrated that RCL-localized Asn347-linked N-glycans impact NE proteolysis, but a comprehensive structure-function characterization of the RCL glycosylation is still required to better understand CBG glycobiology. Herein, we first performed RCL-centric glycoprofiling of serum-derived CBG to elucidate the Asn347-glycans and then used molecular dynamics simulations to study their impact on NE proteolysis. Importantly, we also identified O-glycosylation (di/sialyl T) across four RCL sites (Thr338/Thr342/Thr345/Ser350) of serum CBG close to the NE-targeted Val344-Thr345 cleavage site. A restricted N- and O-glycan co-occurrence pattern on the RCL involving exclusively Asn347 and Thr338 glycosylation was experimentally observed and supported in silico by modeling of a CBG-GalNAc-transferase (GalNAc-T) complex with various RCL glycans. GalNAc-T2 and GalNAc-T3 abundantly expressed by liver and gall bladder, respectively, showed in vitro a capacity to transfer GalNAc (Tn) to multiple RCL sites suggesting their involvement in RCL O-glycosylation. Recombinant CBG was then used to determine roles of RCL O-glycosylation through longitudinal NE-centric proteolysis experiments, which demonstrated that both sialoglycans (disialyl T) and asialoglycans (T) decorating Thr345 inhibit NE proteolysis. Synthetic RCL O-glycopeptides expanded on these findings by showing that Thr345-Tn and Thr342-Tn confer strong and moderate protection against NE cleavage, respectively. Molecular dynamics substantiated that short Thr345-linked O-glycans abrogate NE interactions. In conclusion, we report on biologically relevant CBG RCL glycosylation events, which improve our understanding of mechanisms governing cortisol delivery to inflamed tissues.

Comprehensive Glycoprofiling of Oral Tumors Associates N-Glycosylation With Lymph Node Metastasis and Patient Survival.

While altered protein glycosylation is regarded a trait of oral squamous cell carcinoma (OSCC), the heterogeneous and dynamic glycoproteome of tumor tissues from OSCC patients remain unmapped. To this end, we here employ an integrated multi-omics approach comprising unbiased and quantitative glycomics and glycoproteomics applied to a cohort of resected primary tumor tissues from OSCC patients with (n = 19) and without (n = 12) lymph node metastasis. While all tumor tissues displayed relatively uniform N-glycome profiles suggesting overall stable global N-glycosylation during disease progression, altered expression of six sialylated N-glycans was found to correlate with lymph node metastasis. Notably, glycoproteomics and advanced statistical analyses uncovered altered site-specific N-glycosylation revealing previously unknown associations with several clinicopathological features. Importantly, the glycomics and glycoproteomics data unveiled that comparatively high abundance of two core-fucosylated and sialylated N-glycans (Glycan 40a and Glycan 46a) and one N-glycopeptide from fibronectin were associated with low patient survival, while a relatively low abundance of N-glycopeptides from both afamin and CD59 were also associated with poor survival. This study provides insight into the complex OSCC tissue N-glycoproteome, thereby forming an important resource to further explore the underpinning disease mechanisms and uncover new prognostic glycomarkers for OSCC.

The Complexity and Dynamics of the Tissue Glycoproteome Associated With Prostate Cancer Progression.

The complexity and dynamics of the immensely heterogeneous glycoproteome of the prostate cancer (PCa) tumor microenvironment remain incompletely mapped, a knowledge gap that impedes our molecular-level understanding of the disease. To this end, we have used sensitive glycomics and glycoproteomics to map the protein-, cell-, and tumor grade-specific N- and O-glycosylation in surgically removed PCa tissues spanning five histological grades (n = 10/grade) and tissues from patients with benign prostatic hyperplasia (n = 5). Quantitative glycomics revealed PCa grade-specific alterations of the oligomannosidic-, paucimannosidic-, and branched sialylated complex-type N-glycans, and dynamic remodeling of the sialylated core 1- and core 2-type O-glycome. Deep quantitative glycoproteomics identified ∼7400 unique N-glycopeptides from 500 N-glycoproteins and ∼500 unique O-glycopeptides from nearly 200 O-glycoproteins. With reference to a recent Tissue and Blood Atlas, our data indicate that paucimannosidic glycans of the PCa tissues arise mainly from immune cell-derived glycoproteins. Furthermore, the grade-specific PCa glycosylation arises primarily from dynamics in the cellular makeup of the PCa tumor microenvironment across grades involving increased oligomannosylation of prostate-derived glycoproteins and decreased bisecting GlcNAcylation of N-glycans carried by the extracellular matrix proteins. Furthermore, elevated expression of several oligosaccharyltransferase subunits and enhanced N-glycoprotein site occupancy were observed associated with PCa progression. Finally, correlations between the protein-specific glycosylation and PCa progression were observed including increased site-specific core 2-type O-glycosylation of collagen VI. In conclusion, integrated glycomics and glycoproteomics have enabled new insight into the complexity and dynamics of the tissue glycoproteome associated with PCa progression generating an important resource to explore the underpinning disease mechanisms.

Integrated Glycoproteomics Identifies a Role of N-Glycosylation and Galectin-1 on Myogenesis and Muscle Development.

Many cell surface and secreted proteins are modified by the covalent addition of glycans that play an important role in the development of multicellular organisms. These glycan modifications enable communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signaling. Aberrant protein glycosylation has been associated with the development of several muscular diseases, suggesting essential glycan- and lectin-mediated functions in myogenesis and muscle development, but our molecular understanding of the precise glycans, catalytic enzymes, and lectins involved remains only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during a time-course of myogenesis in cell culture. We observed wide-spread changes in the abundance of several important lectins and enzymes facilitating glycan biosynthesis. Glycomics-based quantification of released N-linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown digalactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labeling and analysis of enriched glycopeptides with multiple fragmentation approaches identified glycoproteins modified by these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins, most notably the upregulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation, suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in N-glycosylation and the upregulation of galectin-1 during postnatal skeletal muscle development were observed in mice. Treatment of new-born mice with recombinant adeno-associated viruses to overexpress galectin-1 in the musculature resulted in enhanced muscle mass. Our data form a valuable resource to further understand the glycobiology of myogenesis and will aid the development of intervention strategies to promote healthy muscle development or regeneration.

Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase.

Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remains elusive. To this end, we have characterised the structure-biosynthesis-activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturing, mature, granule-separated and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, molecular modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.

N-acetyl-ß-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative noncanonical truncation pathway in human neutrophils.

We recently discovered that human neutrophils express immunomodulatory glycoproteins carrying unusual and highly truncated paucimannosidic N-glycans (Man1-3GlcNAc2Fuc0-1), but their biosynthesis remains elusive. Guided by the well-characterized truncation pathway in invertebrates and plants in which the N-acetyl-ß-D-hexosaminidase (Hex) isoenzymes catalyze paucimannosidic protein (PMP) formation, we here set out to test if the homologous human Hex α and ß subunits encoded by HEXA and HEXB drive a similar truncation pathway in human neutrophils. To this end, we performed quantitative glycomics and glycoproteomics of several CRISPR-Cas9-edited Hex-disrupted neutrophil-like HL-60 mutants (HEXA-KO and HEXB-KO) and matching unedited cell lines. Hex disruption was validated using next-generation sequencing, enzyme-linked immunosorbent assay (ELISA), quantitative proteomics and Hex activity assays. Excitingly, all Hex-disrupted mutants displayed significantly reduced levels of paucimannosylation, particularly Man2-3GlcNAc2Fuc1, relative to unedited HL-60 suggesting that both HEXA and HEXB contribute to PMP formation via a hitherto unexplored truncation pathway in neutrophils. Quantitative N-glycomics indeed demonstrated reduced utilization of a putative noncanonical truncation pathway in favor of the canonical elongation pathway in all Hex-disrupted mutants relative to unedited controls. Quantitative glycoproteomics recapitulated the truncation-to-elongation switch in all Hex-disrupted mutants and showed a greater switch for N-glycoproteins cotrafficking with Hex to the azurophilic granules of neutrophils such as myeloperoxidase. Finally, we supported the Hex-PMP relationship by documenting that primary neutrophils isolated from an early-onset Sandhoff disease patient (HEXB-/-) displayed dramatically reduced paucimannosylation relative to neutrophils from an age-matched unaffected donor. We conclude that both human Hex α and ß mediate PMP formation via a putative noncanonical truncation pathway in neutrophils.

Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery.

Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoiesis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called "targeting by timing." Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand the glycosylation process during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation, and N- and O-glycans present in each compartment were analyzed by LC-MS. We found abundant paucimannosidic N-glycans and lack of O-glycans in the early-formed azurophil granules, whereas the later-formed specific and gelatinase granules and secretory vesicles contained complex N- and O-glycans with remarkably elongated N-acetyllactosamine repeats with Lewis epitopes. Immunoblotting and histochemical analysis confirmed the expression of Lewis X and sialyl-Lewis X in the intracellular granules and on the cell surface, respectively. Many glycans identified are unique to neutrophils, and their complexity increased progressively from azurophil granules to specific granules and then to gelatinase granules, suggesting temporal changes in the glycosylation machinery indicative of "glycosylation by timing" during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step toward a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function.

Towards structure-focused glycoproteomics.

Facilitated by advances in the separation sciences, mass spectrometry and informatics, glycoproteomics, the analysis of intact glycopeptides at scale, has recently matured enabling new insights into the complex glycoproteome. While diverse quantitative glycoproteomics strategies capable of mapping monosaccharide compositions of N- and O-linked glycans to discrete sites of proteins within complex biological mixtures with considerable sensitivity, quantitative accuracy and coverage have become available, developments supporting the advancement of structure-focused glycoproteomics, a recognised frontier in the field, have emerged. Technologies capable of providing site-specific information of the glycan fine structures in a glycoproteome-wide context are indeed necessary to address many pending questions in glycobiology. In this review, we firstly survey the latest glycoproteomics studies published in 2018-2020, their approaches and their findings, and then summarise important technological innovations in structure-focused glycoproteomics. Our review illustrates that while the O-glycoproteome remains comparably under-explored despite the emergence of new O-glycan-selective mucinases and other innovative tools aiding O-glycoproteome profiling, quantitative glycoproteomics is increasingly used to profile the N-glycoproteome to tackle diverse biological questions. Excitingly, new strategies compatible with structure-focused glycoproteomics including novel chemoenzymatic labelling, enrichment, separation, and mass spectrometry-based detection methods are rapidly emerging revealing glycan fine structural details including bisecting GlcNAcylation, core and antenna fucosylation, and sialyl-linkage information with protein site resolution. Glycoproteomics has clearly become a mainstay within the glycosciences that continues to reach a broader community. It transpires that structure-focused glycoproteomics holds a considerable potential to aid our understanding of systems glycobiology and unlock secrets of the glycoproteome in the immediate future.

The Hitchhiker's guide to glycoproteomics.

Protein glycosylation is one of the most common post-translational modifications that are essential for cell function across all domains of life. Changes in glycosylation are considered a hallmark of many diseases, thus making glycoproteins important diagnostic and prognostic biomarker candidates and therapeutic targets. Glycoproteomics, the study of glycans and their carrier proteins in a system-wide context, is becoming a powerful tool in glycobiology that enables the functional analysis of protein glycosylation. This 'Hitchhiker's guide to glycoproteomics' is intended as a starting point for anyone who wants to explore the emerging world of glycoproteomics. The review moves from the techniques that have been developed for the characterisation of single glycoproteins to technologies that may be used for a successful complex glycoproteome characterisation. Examples of the variety of approaches, methodologies, and technologies currently used in the field are given. This review introduces the common strategies to capture glycoprotein-specific and system-wide glycoproteome data from tissues, body fluids, or cells, and a perspective on how integration into a multi-omics workflow enables a deep identification and characterisation of glycoproteins - a class of biomolecules essential in regulating cell function.

Structural basis for the recognition of nectin-like protein-5 by the human-activating immune receptor, DNAM-1.

Nectin and nectin-like (Necl) adhesion molecules are broadly overexpressed in a wide range of cancers. By binding to these adhesion molecules, the immunoreceptors DNAX accessory molecule-1 (DNAM-1), CD96 molecule (CD96), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT) play a crucial role in regulating the anticancer activities of immune effector cells. However, within this axis, it remains unclear how DNAM-1 recognizes its cognate ligands. Here, we determined the structure of human DNAM-1 in complex with nectin-like protein-5 (Necl-5) at 2.8 Å resolution. Unexpectedly, we found that the two extracellular domains (D1-D2) of DNAM-1 adopt an unconventional "collapsed" arrangement that is markedly distinct from those in other immunoglobulin-based immunoreceptors. The DNAM-1/Necl-5 interaction was underpinned by conserved lock-and-key motifs located within their respective D1 domains, but also included a distinct interface derived from DNAM-1 D2. Mutation of the signature DNAM-1 "key" motif within the D1 domain attenuated Necl-5 binding and natural killer cell-mediated cytotoxicity. Altogether, our results have implications for understanding the binding mode of an immune receptor family that is emerging as a viable candidate for cancer immunotherapy.

High-resolution longitudinal N- and O-glycoprofiling of human monocyte-to-macrophage transition.

Protein glycosylation impacts the development and function of innate immune cells. The glycophenotypes and the glycan remodelling associated with the maturation of macrophages from monocytic precursor populations remain incompletely described. Herein, label-free porous graphitised carbon-liquid chromatography-tandem mass spectrometry (PGC-LC-MS/MS) was employed to profile with high resolution the N- and O-glycome associated with human monocyte-to-macrophage transition. Primary blood-derived CD14+ monocytes were differentiated ex vivo in the absence of strong anti- and proinflammatory stimuli using a conventional 7-day granulocyte-macrophage colony-stimulating factor differentiation protocol with longitudinal sampling. Morphology and protein expression monitored by light microscopy and proteomics validated the maturation process. Glycomics demonstrated that monocytes and macrophages display similar N-glycome profiles, comprising predominantly paucimannosidic (Man1-3GlcNAc2Fuc0-1, 22.1-30.8%), oligomannosidic (Man5-9GlcNAc2, 29.8-35.7%) and α2,3/6-sialylated complex-type N-glycans with variable core fucosylation (27.6-39.1%). Glycopeptide analysis validated conjugation of these glycans to human proteins, while quantitative proteomics monitored the glycoenzyme expression levels during macrophage differentiation. Significant interperson glycome variations were observed suggesting a considerable physiology-dependent or heritable heterogeneity of CD14+ monocytes. Only few N-glycome changes correlated with the monocyte-to-macrophage transition across donors including decreased core fucosylation and reduced expression of mannose-terminating (paucimannosidic-/oligomannosidic-type) N-glycans in macrophages, while lectin flow cytometry indicated that more dramatic cell surface glycan remodelling occurs during maturation. The less heterogeneous core 1-rich O-glycome showed a minor decrease in core 2-type O-glycosylation but otherwise remained unchanged with macrophage maturation. This high-resolution glycome map underpinning normal monocyte-to-macrophage transition, the most detailed to date, aids our understanding of the molecular makeup pertaining to two vital innate immune cell types and forms an important reference for future glycoimmunological studies.

Tissue Proteome Signatures Associated with Five Grades of Prostate Cancer and Benign Prostatic Hyperplasia.

The histology-based Gleason score (GS) of prostate cancer (PCa) tissue biopsy is the most accurate predictor of disease aggressiveness and an important measure to guide treatment strategies and patient management. The variability associated with PCa tumor sampling and the subjective determination of the GS are challenges that limit accurate diagnostication and prognostication. Thus, novel molecular signatures are needed to distinguish between indolent and aggressive forms of PCa for better patient management and outcomes. Herein, label-free LC-MS/MS proteomics is used to profile the proteome of 50 PCa tissues spanning five grade groups (n = 10 per group) relative to tissues from individuals with benign prostatic hyperplasia (BPH). Over 2000 proteins are identified albeit at different levels between and within the patient groups, revealing biological processes associated with specific grades. A panel of 11 prostate-derived proteins including IGKV3D-20, RNASET2, TACC2, ANXA7, LMOD1, PRCP, GYG1, NDUFV1, H1FX, APOBEC3C, and CTSZ display the potential to stratify patients from low and high PCa grade groups. Parallel reaction monitoring of the same sample cohort validate the differential expression of LMOD1, GYG1, IGKV3D-20, and RNASET2. The four proteins associated with low and high PCa grades reported here warrant further exploration as candidate biomarkers for PCa aggressiveness.

Protein Paucimannosylation Is an Enriched N-Glycosylation Signature of Human Cancers.

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics-centric study investigates a possible link between protein paucimannosylation, an under-studied class of human N-glycosylation [Man1-3 GlcNAc2 Fuc0-1 ], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non-cancerous specimens are profiled from 467 published and unpublished PGC-LC-MS/MS N-glycome datasets collected over a decade. PMGs, particularly Man2-3 GlcNAc2 Fuc1 , are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0-50.2%). Analyses of paired (tumor/non-tumor) and stage-stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N-acetyl-ß-hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.

Post-Column Make-Up Flow (PCMF) Enhances the Performance of Capillary-Flow PGC-LC-MS/MS-Based Glycomics.

Deep characterization of biologically relevant glycans remains challenging. Porous graphitized carbon-liquid chromatography tandem mass spectrometry (PGC-LC-MS/MS) enables the quantitative elucidation of glycan fine structures. However, the early PGC-LC elution of smaller glycans (tri-, tetra-, and pentasaccharides) at low organic solvent content hampers their detection. In efforts to improve the glycan profiling sensitivity and accuracy, we present a new capillary-flow PGC-LC-MS/MS-based configuration comprising a post-column make-up flow (PCMF) that supplies an ion-promoting organic solvent to separated glycans prior to their detection by MS. The analytical performance of this setup was systematically evaluated against our existing capillary-flow PGC-LC-MS/MS platform (Jensen et al., Nat. Protoc. 2012, 7, 1299). Specifically, the ion intensities and signal-to-noise ratios of various classes of nonderivatized glycans from N- and O-glycoproteins and fructooligosaccharide mixtures were compared using methanol (MeOH)-, isopropanol (IPA)-, and acetonitrile (ACN)-based PCMF at various concentrations. In particular, ACN- and IPA-based PCMF dramatically increased the signal response across all glycan types (30- to 100-fold), improved the MS/MS spectral quality, and reduced the quantitative glycoprofile variation between replicates. In particular, the detection of the early eluting glycans benefitted from the PCMF. The highest sensitivity gains were achieved with the supplements of 100% ACN and IPA (equating to 57% (v/v) net concentration at the ion source) while neither compromising the favorable PGC-LC properties including the high peak capacity and glycan isomer separation nor changing the MS detection behavior. In conclusion, PCMF-based PGC-LC-MS/MS dramatically improves the glycomics sensitivity, coverage, and quantitative accuracy not least for the difficult-to-detect early eluting and low-abundance glycans detached from N- and O-glycoproteins.

Paucimannose-Rich N-glycosylation of Spatiotemporally Regulated Human Neutrophil Elastase Modulates Its Immune Functions.

Human neutrophil elastase (HNE) is an important N-glycosylated serine protease in the innate immune system, but the structure and immune-modulating functions of HNE N-glycosylation remain undescribed. Herein, LC-MS/MS-based glycan, glycopeptide and glycoprotein profiling were utilized to first determine the heterogeneous N-glycosylation of HNE purified from neutrophil lysates and then from isolated neutrophil granules of healthy individuals. The spatiotemporal expression of HNE during neutrophil activation and the biological importance of its N-glycosylation were also investigated using immunoblotting, cell surface capture, native MS, receptor interaction, protease inhibition, and bacteria growth assays. Site-specific HNE glycoprofiling demonstrated that unusual paucimannosidic N-glycans, particularly Manα1,6Manß1,4GlcNAcß1,4(Fucα1,6)GlcNAcß, predominantly occupied Asn124 and Asn173. The equally unusual core fucosylated monoantenna complex-type N-sialoglycans also decorated these two fully occupied sites. In contrast, the mostly unoccupied Asn88 carried nonfucosylated paucimannosidic N-glycans probably resulting from low glycosylation site solvent accessibility. Asn185 was not glycosylated. Subcellular- and site-specific glycoprofiling showed highly uniform N-glycosylation of HNE residing in distinct neutrophil compartments. Stimulation-induced cell surface mobilization demonstrated a spatiotemporal regulation, but not cell surface-specific glycosylation signatures, of HNE in activated human neutrophils. The three glycosylation sites of HNE were located distal to the active site indicating glycan functions other than interference with HNE enzyme activity. Functionally, the paucimannosidic HNE glycoforms displayed preferential binding to human mannose binding lectin compared with the HNE sialoglycoforms, suggesting a glycoform-dependent involvement of HNE in complement activation. The heavily N-glycosylated HNE protease inhibitor, α1-antitrypsin, displayed concentration-dependent complex formation and preferred glycoform-glycoform interactions with HNE. Finally, both enzymatically active HNE and isolated HNE N-glycans demonstrated low micromolar concentration-dependent growth inhibition of clinically-relevant Pseudomonas aeruginosa, suggesting some bacteriostatic activity is conferred by the HNE N-glycans. Taken together, these observations support that the unusual HNE N-glycosylation, here reported for the first time, is involved in modulating multiple immune functions central to inflammation and infection.

N-glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research.

Studies of protein N-glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one of the 12 enzymes required for normal N-glycan maturation in the glycosylation machinery. The inactivation of the individual genes resulted in altered N-glycan patterns as documented using mass spectrometry and glycan-recognising antibodies, indicating successful identification of null mutations in the target glyco-genes. For example, both mass spectrometry and immunoblotting experiments suggest that proteins derived from the α1,3-fucosyltransferase (Lj3fuct) mutant completely lacked α1,3-core fucosylation. Mass spectrometry also suggested that the Lotus japonicus convicilin 2 was one of the main glycoproteins undergoing differential expression/N-glycosylation in the mutants. Demonstrating the functional importance of glycosylation, reduced growth and seed production phenotypes were observed for the mutant plants lacking functional mannosidase I, N-acetylglucosaminyltransferase I, and α1,3-fucosyltransferase, even though the relative protein composition and abundance appeared unaffected. The strength of our N-glycosylation mutant platform is the broad spectrum of resulting glycoprotein profiles and altered physiological phenotypes that can be produced from single, double, triple and quadruple mutants. This platform will serve as a valuable tool for elucidating the functional role of protein N-glycosylation in plants. Furthermore, this technology can be used to generate stable plant mutant lines for biopharmaceutical production of glycoproteins displaying relative homogeneous and mammalian-like N-glycosylation features.

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease.

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.