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.

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.

Serum N-Glycomics Stratifies Bacteremic Patients Infected with Different Pathogens.

Bacteremia-i.e., the presence of pathogens in the blood stream-is associated with long-term morbidity and is a potential precursor condition to life-threatening sepsis. Timely detection of bacteremia is therefore critical to reduce patient mortality, but existing methods lack precision, speed, and sensitivity to effectively stratify bacteremic patients. Herein, we tested the potential of quantitative serum N-glycomics performed using porous graphitized carbon liquid chromatography tandem mass spectrometry to stratify bacteremic patients infected with Escherichia coli (n = 11), Staphylococcus aureus (n = 11), Pseudomonas aeruginosa (n = 5), and Streptococcus viridans (n = 5) from healthy donors (n = 39). In total, 62 N-glycan isomers spanning 41 glycan compositions primarily comprising complex-type core fucosylated, bisecting N-acetylglucosamine (GlcNAc), and α2,3-/α2,6-sialylated structures were profiled across all samples using label-free quantitation. Excitingly, unsupervised hierarchical clustering and principal component analysis of the serum N-glycome data accurately separated the patient groups. P. aeruginosa-infected patients displayed prominent N-glycome aberrations involving elevated levels of fucosylation and bisecting GlcNAcylation and reduced sialylation relative to other bacteremic patients. Notably, receiver operating characteristic analyses demonstrated that a single N-glycan isomer could effectively stratify each of the four bacteremic patient groups from the healthy donors (area under the curve 0.93-1.00). Thus, the serum N-glycome represents a new hitherto unexplored class of potential diagnostic markers for bloodstream infections.

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.

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.

Human protein paucimannosylation: cues from the eukaryotic kingdoms.

Paucimannosidic proteins (PMPs) are bioactive glycoproteins carrying truncated α- or ß-mannosyl-terminating asparagine (N)-linked glycans widely reported across the eukaryotic domain. Our understanding of human PMPs remains limited, despite findings documenting their existence and association with human disease glycobiology. This review comprehensively surveys the structures, biosynthetic routes and functions of PMPs across the eukaryotic kingdoms with the aim of synthesising an improved understanding on the role of protein paucimannosylation in human health and diseases. Convincing biochemical, glycoanalytical and biological data detail a vast structural heterogeneity and fascinating tissue- and subcellular-specific expression of PMPs within invertebrates and plants, often comprising multi-α1,3/6-fucosylation and ß1,2-xylosylation amongst other glycan modifications and non-glycan substitutions e.g. O-methylation. Vertebrates and protists express less-heterogeneous PMPs typically only comprising variable core fucosylation of bi- and trimannosylchitobiose core glycans. In particular, the Manα1,6Manß1,4GlcNAc(α1,6Fuc)ß1,4GlcNAcßAsn glycan (M2F) decorates various human neutrophil proteins reportedly displaying bioactivity and structural integrity demonstrating that they are not degradation products. Less-truncated paucimannosidic glycans (e.g. M3F) are characteristic glycosylation features of proteins expressed by human cancer and stem cells. Concertedly, these observations suggest the involvement of human PMPs in processes related to innate immunity, tumorigenesis and cellular differentiation. The absence of human PMPs in diverse bodily fluids studied under many (patho)physiological conditions suggests extravascular residence and points to localised functions of PMPs in peripheral tissues. Absence of PMPs in Fungi indicates that paucimannosylation is common, but not universally conserved, in eukaryotes. Relative to human PMPs, the expression of PMPs in plants, invertebrates and protists is more tissue-wide and constitutive yet, similar to their human counterparts, PMP expression remains regulated by the physiology of the producing organism and PMPs evidently serve essential functions in development, cell-cell communication and host-pathogen/symbiont interactions. In most PMP-producing organisms, including humans, the N-acetyl-ß-hexosaminidase isoenzymes and linkage-specific α-mannosidases are glycoside hydrolases critical for generating PMPs via N-acetylglucosaminyltransferase I (GnT-I)-dependent and GnT-I-independent truncation pathways. However, the identity and structure of many species-specific PMPs in eukaryotes, their biosynthetic routes, strong tissue- and development-specific expression, and diverse functions are still elusive. Deep exploration of these PMP features involving, for example, the characterisation of endogenous PMP-recognising lectins across a variety of healthy and N-acetyl-ß-hexosaminidase-deficient human tissue types and identification of microbial adhesins reactive to human PMPs, are amongst the many tasks required for enhanced insight into the glycobiology of human PMPs. In conclusion, the literature supports the notion that PMPs are significant, yet still heavily under-studied biomolecules in human glycobiology that serve essential functions and create structural heterogeneity not dissimilar to other human N-glycoprotein types. Human PMPs should therefore be recognised as bioactive glycoproteins that are distinctly different from the canonical N-glycoprotein classes and which warrant a more dedicated focus in glycobiological research.