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.

Antieosinophil Antibodies Alone or in Combination with Antineutrophil Cytoplasmic Antibodies (ANCA) Detected in Different Autoimmune Conditions.

Circulating antieosinophil antibodies (AEOSA) have been associated with various autoimmune conditions affecting the liver, kidneys, lungs, and joints but are not part of routine clinical diagnostics. While analyzing human sera for antineutrophil cytoplasmic antibodies (ANCA) by indirect immunofluorescence (IIF) on granulocytes, 0.8% of analyzed samples were found to be reactive with eosinophils. Our aim was to determine the diagnostic relevance and antigenic specificity of AEOSA. AEOSA were seen either in combination with an myeloperoxidase (MPO)-positive p-ANCA (44%; AEOSA+/ANCA+) or on their own (56%; AEOSA+/ANCA-). AEOSA/ANCA positivity was seen in patients with thyroid disease (44%) or vasculitis (31%), while AEOSA+/ANCA- pattern was more common in patients with autoimmune disorders of the gastrointestinal tract and/or liver. Eosinophil peroxidase (EPX) was the main target recognized in 66% of the AEOSA+ sera by enzyme-linked immunosorbent assay (ELISA). Eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) were also identified as target antigens but less frequently and only in combination with EPX. In conclusion, we confirmed that EPX is a major target of AEOSA, illustrating the high antigenic potential of EPX. Our results also demonstrate the presence of concomitant AEOSA/ANCA positivity in a defined patient group. Further research should aim to elucidate the association of AEOSA with autoimmunity.

A rare CTSC mutation in Papillon-Lefèvre Syndrome results in abolished serine protease activity and reduced NET formation but otherwise normal neutrophil function.

Papillon-Lefèvre Syndrome (PLS) is an autosomal recessive monogenic disease caused by loss-of-function mutations in the CTSC gene, thus preventing the synthesis of the protease Cathepsin C (CTSC) in a proteolytically active form. CTSC is responsible for the activation of the pro-forms of the neutrophil serine proteases (NSPs; Elastase, Proteinase 3 and Cathepsin G), suggesting its involvement in a variety of neutrophil functions. In PLS neutrophils, the lack of CTSC protease activity leads to inactivity of the NSPs. Clinically, PLS is characterized by an early, typically pre-pubertal, onset of severe periodontal pathology and palmoplantar hyperkeratosis. However, PLS is not considered an immune deficiency as patients do not typically suffer from recurrent and severe (bacterial and fungal) infections. In this study we investigated an unusual CTSC mutation in two siblings with PLS, a 503A>G substitution in exon 4 of the CTSC gene, expected to result in an amino acid replacement from tyrosine to cysteine at position 168 of the CTSC protein. Both patients bearing this mutation presented with pronounced periodontal pathology. The characteristics and functions of neutrophils from patients homozygous for the 503A>G CTSC mutation were compared to another previously described PLS mutation (755A>T), and a small cohort of healthy volunteers. Neutrophil lysates from patients with the 503A>G substitution lacked CTSC protein and did not display any CTSC or NSP activity, yet neutrophil counts, morphology, priming, chemotaxis, radical production, and regulation of apoptosis were without any overt signs of alteration. However, NET formation upon PMA-stimulation was found to be severely depressed, but not abolished, in PLS neutrophils.

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.

CEBPE-Mutant Specific Granule Deficiency Correlates With Aberrant Granule Organization and Substantial Proteome Alterations in Neutrophils.

Specific granule deficiency (SGD) is a rare disorder characterized by abnormal neutrophils evidenced by reduced granules, absence of granule proteins, and atypical bilobed nuclei. Mutations in CCAAT/enhancer-binding protein-ε (CEBPE) are one molecular etiology of the disease. Although C/EBPε has been studied extensively, the impact of CEBPE mutations on neutrophil biology remains elusive. Here, we identified two SGD patients bearing a previously described heterozygous mutation (p.Val218Ala) in CEBPE. We took this rare opportunity to characterize SGD neutrophils in terms of granule distribution and protein content. Granules of patient neutrophils were clustered and polarized, suggesting that not only absence of specific granules but also defects affecting other granules contribute to the phenotype. Our analysis showed that remaining granules displayed mixed protein content and lacked several glycoepitopes. To further elucidate the impact of mutant CEBPE, we performed detailed proteomic analysis of SGD neutrophils. Beside an absence of several granule proteins in patient cells, we observed increased expression of members of the linker of nucleoskeleton and cytoskeleton complex (nesprin-2, vimentin, and lamin-B2), which control nuclear shape. This suggests that absence of these proteins in healthy individuals might be responsible for segmented shapes of neutrophilic nuclei. We further show that the heterozygous mutation p.Val218Ala in CEBPE causes SGD through prevention of nuclear localization of the protein product. In conclusion, we uncover that absence of nuclear C/EBPε impacts on spatiotemporal expression and subsequent distribution of several granule proteins and further on expression of proteins controlling nuclear shape.

Similarities and differences between the responses induced in human phagocytes through activation of the medium chain fatty acid receptor GPR84 and the short chain fatty acid receptor FFA2R.

GPR84 is a recently de-orphanized member of the G-protein coupled receptor (GPCR) family recognizing medium chain fatty acids, and has been suggested to play important roles in inflammation. Due to the lack of potent and selective GPR84 ligands, the basic knowledge related to GPR84 functions is very limited. In this study, we have characterized the GPR84 activation profile and regulation mechanism in human phagocytes, using two recently developed small molecules that specifically target GPR84 agonistically (ZQ16) and antagonistically (GLPG1205), respectively. Compared to our earlier characterization of the short chain fatty acid receptor FFA2R which is functionally expressed in neutrophils but not in monocytes, GPR84 is expressed in both cell types and in monocyte-derived macrophages. In neutrophils, the GPR84 agonist had an activation profile very similar to that of FFA2R. The GPR84-mediated superoxide release was low in naïve cells, but the response could be significantly primed by TNFα and by the actin cytoskeleton disrupting agent Latrunculin A. Similar to that of FFA2R, a desensitization mechanism bypassing the actin cytoskeleton was utilized by GPR84. All ZQ16-mediated cellular responses were sensitive to GLPG1205, confirming the GPR84-dependency. Finally, our data of in vivo transmigrated tissue neutrophils indicate that both GPR84 and FFA2R are involved in neutrophil recruitment processes in vivo. In summary, we show functional similarities but also some important differences between GPR84 and FFA2R in human phagocytes, thus providing some mechanistic insights into GPR84 regulation in blood neutrophils and cells recruited to an aseptic inflammatory site in vivo.

The balance in the delivery of ER components and the vacuolar proton pump to the phagosome depends on myosin IK in Dictyostelium.

In Dictyostelium, the cytoskeletal proteins Actin binding protein 1 (Abp1) and the class I myosin MyoK directly interact and couple actin dynamics to membrane deformation during phagocytosis. Together with the kinase PakB, they build a regulatory switch that controls the efficiency of uptake of large particles. As a basis for further functional dissection, exhaustive phagosome proteomics was performed and established that about 1300 proteins participate in phagosome biogenesis. Then, quantitative and comparative proteomic analysis of phagosome maturation was performed to investigate the impact of the absence of MyoK or Abp1. Immunoblots and two-dimensional differential gel electrophoresis of phagosomes isolated from myoK-null and abp1-null cells were used to determine the relative abundance of proteins during the course of maturation. Immunoblot profiling showed that absence of Abp1 alters the maturation profile of its direct binding partners such as actin and the Arp2/3 complex, suggesting that Abp1 directly regulates actin dynamics at the phagosome. Comparative two-dimensional differential gel electrophoresis analysis resulted in the quantification of mutant-to-wild type abundance ratios at all stages of maturation for over one hundred identified proteins. Coordinated temporal changes in these ratio profiles determined the classification of identified proteins into functional groups. Ratio profiling revealed that the early delivery of ER proteins to the phagosome was affected by the absence of MyoK and was coupled to a reciprocal imbalance in the delivery of the vacuolar proton pump and Rab11 GTPases. As direct functional consequences, a delayed acidification and a reduced intraphagosomal proteolysis were demonstrated in vivo in myoK-null cells. In conclusion, the absence of MyoK alters the balance of the contributions of the ER and an endo-lysosomal compartment, and slows down phagosome acidification as well as the speed and efficiency of particle degradation inside the phagosome.

Dynamin A, Myosin IB and Abp1 couple phagosome maturation to F-actin binding.

The role of actin, class I myosins and dynamin in endocytic uptake processes is well characterized, but their role during endo-phagosomal membrane trafficking and maturation is less clear. In Dictyostelium, knockout of myosin IB (myoB) leads to a defect in membrane protein recycling from endosomes back to the plasma membrane. Here, we show that actin plays a central role in the morphology and function of the endocytic pathway. Indeed, latrunculin B (LatB) induces endosome tubulation, a phenotype also observed in dynamin A (dymA)-null cells. Knockout of dymA impairs phagosome acidification, whereas knockout of myoB delays reneutralization, a phenotype mimicked by a low dose of LatB. As a read out for actin-dependent processes during maturation, we monitored the capacity of purified phagosomes to bind F-actin in vitro, and correlated this with the presence of actin-binding and membrane-trafficking proteins. Phagosomes isolated from myoB-null cells showed an increased binding to F-actin, especially late phagosomes. In contrast, early phagosomes from dymA-null cells showed reduced binding to F-actin while late phagosomes were unaffected. We provide evidence that Abp1 is the main F-actin-binding protein in this assay and is central for the interplay between DymA and MyoB during phagosome maturation.

Molecular characterization of the evolution of phagosomes.

Amoeba use phagocytosis to internalize bacteria as a source of nutrients, whereas multicellular organisms utilize this process as a defense mechanism to kill microbes and, in vertebrates, initiate a sustained immune response. By using a large-scale approach to identify and compare the proteome and phosphoproteome of phagosomes isolated from distant organisms, and by comparative analysis over 39 taxa, we identified an 'ancient' core of phagosomal proteins around which the immune functions of this organelle have likely organized. Our data indicate that a larger proportion of the phagosome proteome, compared with the whole cell proteome, has been acquired through gene duplication at a period coinciding with the emergence of innate and adaptive immunity. Our study also characterizes in detail the acquisition of novel proteins and the significant remodeling of the phagosome phosphoproteome that contributed to modify the core constituents of this organelle in evolution. Our work thus provides the first thorough analysis of the changes that enabled the transformation of the phagosome from a phagotrophic compartment into an organelle fully competent for antigen presentation.

A myosin IK-Abp1-PakB circuit acts as a switch to regulate phagocytosis efficiency.

Actin dynamics and myosin (Myo) contractile forces are necessary for formation and closure of the phagocytic cup. In Dictyostelium, the actin-binding protein Abp1 and myosin IK are enriched in the closing cup and especially at an actin-dense constriction furrow formed around the neck of engulfed budded yeasts. This phagocytic furrow consists of concentric overlapping rings of MyoK, Abp1, Arp3, coronin, and myosin II, following an order strikingly reminiscent of the overall organization of the lamellipodium of migrating cells. Mutation analyses of MyoK revealed that both a C-terminal farnesylation membrane anchor and a Gly-Pro-Arg domain that interacts with profilin and Abp1 were necessary for proper localization in the furrow and efficient phagocytosis. Consequently, we measured the binding affinities of these interactions and unraveled further interactions with profilins, dynamin A, and PakB. Due to the redundancy of the interaction network, we hypothesize that MyoK and Abp1 are restricted to regulatory roles and might affect the dynamic of cup progression. Indeed, phagocytic uptake was regulated antagonistically by MyoK and Abp1. MyoK is phosphorylated by PakB and positively regulates phagocytosis, whereas binding of Abp1 negatively regulates PakB and MyoK. We conclude that a MyoK-Abp1-PakB circuit acts as a switch regulating phagocytosis efficiency of large particles.

Monitoring time-dependent maturation changes in purified phagosomes from Dictyostelium discoideum.

The amoeba Dictyostelium discoideum is an established model to study phagocytosis. The sequence of events leading to the internalization and degradation of a particle is conserved in D. discoideum compared to metazoan cells. As its small haploid genome has been sequenced, it is now amenable to genome-wide analysis including organelle proteomics. Therefore, we adapted to Dictyostelium the classical protocol to purify phagosomes formed by ingestion of latex beads particles. The pulse-chase protocol detailed here gives easy access to pure, intact, and synchronized phagosomes from representative stages of the entire process of phagosome maturation. Recently, this protocol was used to generate individual temporal profiles of proteins and lipids during phagosome maturation generating a proteomic fingerprint of six maturation stages (1). In addition, immunolabeling of phagosomes on a coverslip was developed to visualize and quantitate antigen distribution at the level of individual phagosomes.

ISG20L2, a novel vertebrate nucleolar exoribonuclease involved in ribosome biogenesis.

Proteomics analyses of human nucleoli provided molecular bases for an understanding of the multiple functions fulfilled by these nuclear domains. However, the biological roles of about 100 of the identified proteins are unpredictable. The present study describes the functional characterization of one of these proteins, ISG20L2. We demonstrate that ISG20L2 is a 3' to 5' exoribonuclease involved in ribosome biogenesis at the level of 5.8 S rRNA maturation, more specifically in the processing of the 12 S precursor rRNA. The use of truncated forms of ISG20L2 demonstrated that its N-terminal half promotes the nucleolar localization and suggested that its C-terminal half bears the exoribonuclease activity. Identification of the binding partners of ISG20L2 confirmed its involvement in the biogenesis of the large ribosomal subunit. These results strongly support the notion that, in human, as it was demonstrated in yeast, 5.8 S rRNA maturation requires several proteins in addition to the exosome complex. Furthermore this observation greatly sustains the idea that the extremely conserved need for correctly processed rRNAs in vertebrates and yeast is achieved by close but different mechanisms.

Preparation of intact, highly purified phagosomes from Dictyostelium.

Phagocytosis plays a fundamental role in the immune system for the defense against invading microorganisms and the clearing of apoptotic and cancerous cells. The common amoeba Dictyostelium discoideum is a recognized model for professional immune phagocytes and is now commonly used to study host-pathogen interactions. Dictyostelium is genetically and biochemically tractable and is a most versatile experimental system. The classical protocol for purifying phagosomes formed by ingestion of latex beads particles has been adapted to Dictyostelium. It was improved in yield, purity, and synchronicity, allowing isolation of milligram amounts of phagosomal proteins and lipids. This method has been used successfully to highlight membrane trafficking and phagosome maturation. Here, we present a step-by-step protocol including detailed notes necessary for ensuring access to a large number of highly synchronized phagosomes of high purity and integrity.