Pregnancy enables antibody protection against intracellular infection.

Adaptive immune components are thought to exert non-overlapping roles in antimicrobial host defence, with antibodies targeting pathogens in the extracellular environment and T cells eliminating infection inside cells1,2. Reliance on antibodies for vertically transferred immunity from mothers to babies may explain neonatal susceptibility to intracellular infections3,4. Here we show that pregnancy-induced post-translational antibody modification enables protection against the prototypical intracellular pathogen Listeria monocytogenes. Infection susceptibility was reversed in neonatal mice born to preconceptually primed mothers possessing L. monocytogenes-specific IgG or after passive transfer of antibodies from primed pregnant, but not virgin, mice. Although maternal B cells were essential for producing IgGs that mediate vertically transferred protection, they were dispensable for antibody acquisition of protective function, which instead required sialic acid acetyl esterase5 to deacetylate terminal sialic acid residues on IgG variable-region N-linked glycans. Deacetylated L. monocytogenes-specific IgG protected neonates through the sialic acid receptor CD226,7, which suppressed IL-10 production by B cells leading to antibody-mediated protection. Consideration of the maternal-fetal dyad as a joined immunological unit reveals protective roles for antibodies against intracellular infection and fine-tuned adaptations to enhance host defence during pregnancy and early life.

Sialic acid in the regulation of blood cell production, differentiation and turnover.

Sialic acid is a unique sugar moiety that resides in the distal and most accessible position of the glycans on mammalian cell surface and extracellular glycoproteins and glycolipids. The potential for sialic acid to obscure underlying structures has long been postulated, but the means by which such structural changes directly affect biological processes continues to be elucidated. Here, we appraise the growing body of literature detailing the importance of sialic acid for the generation, differentiation, function and death of haematopoietic cells. We conclude that sialylation is a critical post-translational modification utilized in haematopoiesis to meet the dynamic needs of the organism by enforcing rapid changes in availability of lineage-specific cell types. Though long thought to be generated only cell-autonomously within the intracellular ER-Golgi secretory apparatus, emerging data also demonstrate previously unexpected diversity in the mechanisms of sialylation. Emphasis is afforded to the mechanism of extrinsic sialylation, whereby extracellular enzymes remodel cell surface and extracellular glycans, supported by charged sugar donor molecules from activated platelets.

Computational studies on glycosaminoglycan recognition of sialyl transferases.

Despite decades of research, glycosaminoglycans (GAGs) have not been known to interact with sialyl transferases (STs). Using our in-house combinatorial virtual library screening (CVLS) technology, we studied seven human isoforms, including ST6GAL1, ST6GAL2, ST3GAL1, ST3GAL3, ST3GAL4, ST3GAL5, and ST3GAL6, and predicted that GAGs, especially heparan sulfate (HS), are likely to differentially bind to STs. Exhaustive CVLS and molecular dynamics studies suggested that the common hexasaccharide sequence of HS preferentially recognized ST6GAL1 in a site overlapping the binding site of the donor substrate CMP-Sia. Interestingly, CVLS did not ascribe any special role for the rare 3-O-sulfate modification of HS in ST6GAL1 recognition. The computational predictions were tested using spectrofluorimetric studies, which confirmed preferential recognition of HS over other GAGs. A classic chain length-dependent binding of GAGs to ST6GAL1 was observed with polymeric HS displaying a tight affinity of ~65 nM. Biophysical studies also confirmed a direct competition between CMP-Sia and an HS oligosaccharide and CS polysaccharide for binding to ST6GAL1. Overall, our novel observation that GAGs bind to ST6GAL1 with high affinity and compete with the donor substrate is likely to be important because modulation of sialylation of glycan substrates on cells has considerable physiological/pathological consequences. Our work also brings forth the possibility of developing GAG-based chemical probes of ST6GAL1.

Bacterial colonization and TH17 immunity are shaped by intestinal sialylation in neonatal mice.

Interactions between the neonate host and its gut microbiome are central to the development of a healthy immune system. However, the mechanisms by which animals alter early colonization of microbiota for their benefit remain unclear. Here, we investigated the role of early-life expression of the α2,6-sialyltransferase ST6GAL1 in microbiome phylogeny and mucosal immunity. Fecal, upper respiratory, and oral microbiomes of pups expressing or lacking St6gal1 were analyzed by 16S rRNA sequencing. At weaning, the fecal microbiome of St6gal1-KO mice had reduced Clostridiodes, Coprobacillus, and Adlercreutzia, but increased Helicobacter and Bilophila. Pooled fecal microbiomes from syngeneic donors were transferred to antibiotic-treated wild-type mice, before analysis of recipient mucosal immune responses by flow cytometry, RT-qPCR, microscopy, and ELISA. Transfer of St6gal1-KO microbiome induced a mucosal Th17 response, with expression of T-bet and IL-17, and IL-22-dependent gut lengthening. Early life intestinal sialylation was characterized by RT-qPCR, immunoblot, microscopy, and sialyltransferase enzyme assays in genetic mouse models at rest or with glucocorticoid receptor modulators. St6gal1 expression was greatest in the duodenum, where it was mediated by the P1 promoter and efficiently inhibited by dexamethasone. Our data show that the inability to produce α2,6-sialyl ligands contributes to microbiome-dependent Th17 inflammation, highlighting a pathway by which the intestinal glycosylation regulates mucosal immunity.

Extracellular ST6GAL1 regulates monocyte-macrophage development and survival.

Interaction of immune cells with the systemic environment is necessary for the coordinated development and execution of immune responses. Monocyte-macrophage lineage cells reside at the junction of innate and adaptive immunity. Previously we reported that the sialyltransferase ST6GAL1 in the extracellular milieu modulates B cell development and IgG production, granulocyte production, and attenuates acute airway inflammation to bacterial challenge in mouse models. Here, we report that extracellular ST6GAL1 also elicits profound responses in monocyte-macrophage lineage cells. We show that recombinant ST6GAL1 adheres to subsets of thioglycolate-elicited inflammatory cells in the mouse peritoneum and to cultured human monocyte THP-1 cells. Exposure of the inflammatory cells to recombinant ST6GAL1 elicited wholesale changes in the gene expression profile of primary mouse myeloid cells; most notable was the striking up-regulation of monocyte-macrophage and monocyte-derived dendritic cell development pathway signature genes and transcription factors PU.1, NFκB and their target genes, driving increased monocyte-macrophage population and survival ex vivo. In the cultured human monocyte cells, the essential cell surface receptor of the monocyte-macrophage lineage, the M-CSF receptor (M-CSF-R, Csfr1) was a target of extracellular ST6GAL1 catalytic activity. Extracellular ST6GAL1 activated the M-CSF-R and initiated intracellular signaling events, namely, the nuclear translocation of NFκB subunit p65, and phosphorylation of ERK 1/2 and AKT. The findings implicate extracellular ST6GAL1 in monocyte development by a mechanism initiated at the cell surface and support an emerging paradigm of an extracellular glycan-modifying enzyme as a central regulator coordinating immune hematopoietic cell development and function.

The sialyltransferase ST6GAL1 protects against radiation-induced gastrointestinal damage.

High-dose irradiation poses extreme risk of mortality from acute damage to the hematopoietic compartment and gastrointestinal tract. While bone marrow transplantation can reestablish the hematopoietic compartment, a more imminent risk of death is posed by gastrointestinal acute radiation syndrome (GI-ARS), for which there are no FDA-approved medical countermeasures. Although the mechanisms dictating the severity of GI-ARS remain incompletely understood, sialylation by ST6GAL1 has been shown to protect against radiation-induced apoptosis in vitro. Here, we used a C57BL/6 St6gal1-KO mouse model to investigate the contribution of ST6GAL1 to susceptibility to total body irradiation in vivo. Twelve gray total body ionizing γ-irradiation (TBI) followed by bone marrow transplant is not lethal to wild-type mice, but St6gal1-KO counterparts succumbed within 7 d. Both St6gal1-KO and wild-type animals exhibited damage to the GI epithelium, diarrhea and weight loss, but these symptoms became progressively more severe in the St6gal1-KO animals while wild-type counterparts showed signs of recovery by 120 h after TBI. Increased apoptosis in the GI tracts of St6gal1-KO mice and the absence of regenerative crypts were also observed. Together, these observations highlight an important role for ST6GAL1 in protection and recovery from GI-ARS in vivo.

Extrinsic sialylation is dynamically regulated by systemic triggers in vivo.

Recent reports have documented that extracellular sialyltransferases can remodel both cell-surface and secreted glycans by a process other than the canonical cell-autonomous glycosylation that occurs within the intracellular secretory apparatus. Despite association of the abundance of these extracellular sialyltransferases, particularly ST6Gal-1, with disease states such as cancer and a variety of inflammatory conditions, the prevalence of this extrinsic glycosylation pathway in vivo remains unknown. Here we observed no significant extrinsic sialylation in resting mice, suggesting that extrinsic sialylation is not a constitutive process. However, extrinsic sialylation in the periphery could be triggered by inflammatory challenges, such as exposure to ionizing radiation or to bacterial lipopolysaccharides. Sialic acids from circulating platelets were used in vivo to remodel target cell surfaces. Platelet activation was minimally sufficient to elicit extrinsic sialylation, as demonstrated with the FeCl3 model of mesenteric artery thrombosis. Although extracellular ST6Gal-1 supports extrinsic sialylation, other sialyltransferases are present in systemic circulation. We also observed in vivo extrinsic sialylation in animals deficient in ST6Gal-1, demonstrating that extrinsic sialylation is not mediated exclusively by ST6Gal-1. Together, these observations form an emerging picture of glycans biosynthesized by the canonical cell-autonomous glycosylation pathway, but subjected to remodeling by extracellular glycan-modifying enzymes.

ST3Gal-4 is the primary sialyltransferase regulating the synthesis of E-, P-, and L-selectin ligands on human myeloid leukocytes.

The precise glycosyltransferase enzymes that mediate selectin-ligand biosynthesis in human leukocytes are unknown. This knowledge is important because selectin-mediated cell tethering and rolling is a critical component of both normal immune response and various vascular disorders. We evaluated the role of 3 α(2,3)sialyltransferases, ST3Gal-3, -4, and -6, which act on the type II N-Acetyllactosamine structure (Galß1,4GlcNAc) to create sialyl Lewis-X (sLe(X)) and related sialofucosylated glycans on human leukocytes of myeloid lineage. These genes were either silenced using lentiviral short hairpin RNA (shRNA) or functionally ablated using the clustered regularly interspaced short palindromic repeat/Cas9 technology. The results show that ST3Gal-4, but not ST3Gal-3 or -6, is the major sialyltransferase regulating the biosynthesis of E-, P-, and L-selectin ligands in humans. Reduction in ST3Gal-4 activity lowered cell-surface HECA-452 epitope expression by 75% to 95%. Glycomics profiling of knockouts demonstrate an almost complete loss of the sLe(X) epitope on both leukocyte N- and O-glycans. In cell-adhesion studies, ST3Gal-4 knockdown/knockout cells displayed 90% to 100% reduction in tethering and rolling density on all selectins. ST3Gal-4 silencing in neutrophils derived from human CD34(+) hematopoietic stem cells also resulted in 80% to 90% reduction in cell adhesion to all selectins. Overall, a single sialyltransferase regulates selectin-ligand biosynthesis in human leukocytes, unlike mice where multiple enzymes contribute to this function.

Remodeling of marrow hematopoietic stem and progenitor cells by non-self ST6Gal-1 sialyltransferase.

Glycans occupy the critical cell surface interface between hematopoietic cells and their marrow niches. Typically, glycosyltransferases reside within the intracellular secretory apparatus, and each cell autonomously generates its own cell surface glycans. In this study, we report an alternate pathway to generate cell surface glycans where remotely produced glycosyltransferases remodel surfaces of target cells and for which endogenous expression of the cognate enzymes is not required. Our data show that extracellular ST6Gal-1 sialyltransferase, originating mostly from the liver and released into circulation, targets marrow hematopoietic stem and progenitor cells (HSPCs) and mediates the formation of cell surface α2,6-linked sialic acids on HSPCs as assessed by binding to the specific lectins Sambucus nigra agglutinin and Polysporus squamosus lectin and confirmed by mass spectrometry. Marrow HSPCs, operationally defined as the Lin-c-Kit+ and Lin-Sca-1+c-Kit+ populations, express negligible endogenous ST6Gal-1. Animals with reduced circulatory ST6Gal-1 have marrow Lin-Sca-1+c-Kit+ cells with reduced S. nigra agglutinin reactivity. Bone marrow chimeras demonstrated that α2,6-sialylation of HSPCs is profoundly dependent on circulatory ST6Gal-1 status of the recipients and independent of the ability of HSPCs to express endogenous ST6Gal-1. Biologically, HSPC abundance in the marrow is inversely related to circulatory ST6Gal-1 status, and this relationship is recapitulated in the bone marrow chimeras. We propose that remotely produced, rather than the endogenously expressed, ST6Gal-1 is the principal modifier of HSPC glycans for α2,6-sialic acids. In so doing, liver-produced ST6Gal-1 may be a potent systemic regulator of hematopoiesis.

Platelets support extracellular sialylation by supplying the sugar donor substrate.

Sizable pools of freely circulating glycosyltransferases are in blood, but understanding their physiologic contributions has been hampered because functional sources of sugar donor substrates needed to drive extracellular glycosylation have not been identified. The blood-borne ST6Gal-1 produced and secreted by the liver is the most noted among the circulatory glycosyltransferases, and decorates marrow hematopoietic progenitor cells with α2,6-linked sialic acids and restricts blood cell production. Platelets, upon activation, secrete a plethora of bioactive molecules including pro- and anti-inflammatory mediators. Cargos of sugar donor substrates for glycosyltransferase activity have also been reported in platelets. Here, we implemented a cell-based system to interrogate platelets for their ability to deliver effectively the sugar donor substrate for extracellular ST6Gal-1 to function. We report that thrombin-activated platelets, at physiologic concentration and pH, can efficiently and effectively substitute for CMP-sialic acid in extracellular ST6Gal-1-mediated sialylation of target cell surfaces. Activated platelets can also supply the sialic acid donor to sialylate the synthetic acceptor, Gal(ß1,4)GlcNAcα-o-benzyl, with the product Sia(α2,6)Gal(ß1,4)GlcNAcα-o-benzyl structurally confirmed by LC/MS. Platelet-secreted donor substrate was recovered in the 100,000 × g sediment, strongly suggesting the association of this otherwise soluble substrate, putatively CMP-sialic acid, within platelet microparticles. Sequestration within microparticles may facilitate delivery of glycosylation substrate at effective dosages to sites of extracellular glycosylation while minimizing excessive dilution.

Silencing α1,3-fucosyltransferases in human leukocytes reveals a role for FUT9 enzyme during E-selectin-mediated cell adhesion.

Leukocyte adhesion during inflammation is initiated by the binding of sialofucosylated carbohydrates expressed on leukocytes to endothelial E/P-selectin. Although the glycosyltransferases (glycoTs) constructing selectin-ligands have largely been identified using knock-out mice, important differences may exist between humans and mice. To address this, we developed a systematic lentivirus-based shRNA delivery workflow to create human leukocytic HL-60 cell lines that lack up to three glycoTs. Using this, the contributions of all three myeloid α1,3-fucosyltransferases (FUT4, FUT7, and FUT9) to selectin-ligand biosynthesis were evaluated. The cell adhesion properties of these modified cells to L-, E-, and P-selectin under hydrodynamic shear were compared with bone marrow-derived neutrophils from Fut4(-/-)Fut7(-/-) dual knock-out mice. Results demonstrate that predominantly FUT7, and to a lesser extent FUT4, forms the selectin-ligand at the N terminus of leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) in humans and mice. Here, 85% reduction in leukocyte interaction was observed in human FUT4(-)7(-) dual knockdowns on P/L-selectin substrates. Unlike Fut4(-/-)Fut7(-/-) mouse neutrophils, however, human knockdowns lacking FUT4 and FUT7 only exhibited partial reduction in rolling interaction on E-selectin. In this case, the third α1,3-fucosyltransferase FUT9 played an important role because leukocyte adhesion was reduced by 50-60% in FUT9-HL-60, 70-80% in dual knockdown FUT7(-)9(-) cells, and ∼85% in FUT4(-)7(-)9(-) triple knockdowns. Gene silencing results are in agreement with gain-of-function experiments where all three fucosyltransferases conferred E-selectin-mediated rolling in HEK293T cells. This study advances new tools to study human glycoT function. It suggests a species-specific role for FUT9 during the biosynthesis of human E-selectin ligands.

VEGF-FGF Signaling Activates Quiescent CD63+ Liver Stem Cells to Proliferate and Differentiate.

Understanding the liver stem cells (LSCs) holds great promise for new insights into liver diseases and liver regeneration. However, the heterogenicity and plasticity of liver cells have made it controversial. Here, by employing single-cell RNA-sequencing technology, transcriptome features of Krt19+ bile duct lineage cells isolated from Krt19CreERT; Rosa26R-GFP reporter mouse livers are examined. Distinct biliary epithelial cells which include adult LSCs, as well as their downstream hepatocytes and cholangiocytes are identified. Importantly, a novel cell surface LSCs marker, CD63, as well as CD56, which distinguished active and quiescent LSCs are discovered. Cell expansion and bi-potential differentiation in culture demonstrate the stemness ability of CD63+ cells in vitro. Transplantation and lineage tracing of CD63+ cells confirm their contribution to liver cell mass in vivo upon injury. Moreover, CD63+CD56+ cells are proved to be activated LSCs with vigorous proliferation ability. Further studies confirm that CD63+CD56- quiescent LSCs express VEGFR2 and FGFR1, and they can be activated to proliferation and differentiation through combination of growth factors: VEGF-A and bFGF. These findings define an authentic adult liver stem cells compartment, make a further understanding of fate regulation on LSCs, and highlight its contribution to liver during pathophysiologic processes.

Anti-inflammatory IgG production requires functional P1 promoter in ß-galactoside α2,6-sialyltransferase 1 (ST6Gal-1) gene.

The anti-inflammatory properties associated with intravenous immunoglobulin therapy require the sialic acid modification of the N-glycan of the Fc domain of IgG. Sialylation of the Fc fragment is mediated by ß-galactoside α2,6-sialyltransferase 1 (ST6Gal-1), acting on the Gal(ß4)GlcNAc terminal structure of the biantennary N-glycans on the Fc domain. However, little is known regarding the in vivo regulation of Fc sialylation and its role in the progression of inflammatory processes. Here, we report that decreased Fc sialylation of circulatory IgG accompanies the acute phase response elicited by turpentine exposure or upon acute exposure to either nontypeable Haemophilus influenzae or ovalbumin. However, Fc sialylation was increased 3-fold from the base line upon transition to chronic inflammation by repeated exposure to challenge. The P1 promoter of the ST6Gal-1 gene is critical for Fc sialylation, but P1 does not drive ST6Gal-1 expression in B cells. The Siat1ΔP1 mouse, with a dysfunctional P1 promoter, was unable to produce sialylated Fc in the systemic circulation, despite the presence of Gal(ß4)GlcNAc termini on the Fc glycans. The major contribution of P1 action is to synthesize ST6Gal-1 enzymes that are deposited into the systemic circulation. The data strongly indicate that this pool of extracellular ST6Gal-1 in the blood impacts the sialylation of IgG Fc and that defective Fc sialylation is likely a major contributing mechanism for the proinflammatory tendencies previously noted in Siat1ΔP1 animals.

Fluorinated per-acetylated GalNAc metabolically alters glycan structures on leukocyte PSGL-1 and reduces cell binding to selectins.

Novel strategies to control the binding of adhesion molecules belonging to the selectin family are required for the treatment of inflammatory diseases. We tested the possibility that synthetic monosaccharide analogs can compete with naturally occurring sugars to alter the O-glycan content on human leukocyte cell surface selectin-ligand, P-selectin glycoprotein ligand-1 (PSGL-1). Resulting reduction in the sialyl Lewis-X-bearing epitopes on this ligand may reduce cell adhesion. Consistent with this hypothesis, 50muM per-acetylated 4F-GalNAc added to the growth media of promyelocytic HL-60 cells reduced the expression of the cutaneous lymphocyte associated-antigen (HECA-452 epitope) by 82% within 2 cell doubling cycles. Cell binding to all 3 selectins (L-, E-, and P-selectin) was reduced in vitro. 4F-GalNAc was metabolically incorporated into PSGL-1, and this was accompanied by an approximately 20% reduction in PSGL-1 glycan content. A 70% to 85% reduction in HECA-452 binding epitope and N-acetyl lactosamine content in PSGL-1 was also noted on 4F-GalNAc addition. Intravenous 4F-GalNAc infusion reduced leukocyte migration to the peritoneum in a murine model of thioglycolate-induced peritonitis. Thus, the compound has pharmacologic activity. Overall, the data suggest that 4F-GalNAc may be applied as a metabolic inhibitor to reduce O-linked glycosylation, sialyl Lewis-X formation, and leukocyte adhesion via the selectins.

Deficiency in ST6GAL1, one of the two α2,6-sialyltransferases, has only a minor effect on the pathogenesis of prion disease.

Prion diseases are a group of fatal neurodegenerative diseases caused by misfolding of the normal cellular form of the prion protein or PrPC, into a disease-associated self-replicating state or PrPSc. PrPC and PrPSc are posttranslationally modified with N-linked glycans, in which the terminal positions occupied by sialic acids residues are attached to galactose predominantly via α2-6 linkages. The sialylation status of PrPSc is an important determinant of prion disease pathogenesis, as it dictates the rate of prion replication and controls the fate of prions in an organism. The current study tests whether a knockout of ST6Gal1, one of the two mammalian sialyltransferases that catalyze the sialylation of glycans via α2-6 linkages, reduces the sialylation status of PrPSc and alters prion disease pathogenesis. We found that a global knockout of ST6Gal1 in mice significantly reduces the α2-6 sialylation of the brain parenchyma, as determined by staining with Sambucus Nigra agglutinin. However, the sialylation of PrPSc remained stable and the incubation time to disease increased only modestly in ST6Gal1 knockout mice (ST6Gal1-KO). A lack of significant changes in the PrPSc sialylation status and prion pathogenesis is attributed to the redundancy in sialylation and, in particular, the plausible involvement of a second member of the sialyltransferase family that sialylate via α2-6 linkages, ST6Gal2.

Extracellular sialyltransferase st6gal1 in breast tumor cell growth and invasiveness.

The sialyltransferase ST6GAL1 that adds α2-6 linked sialic acids to N-glycans of cell surface and secreted glycoproteins is prominently associated with many human cancers. Tumor-native ST6GAL1 promotes tumor cell behaviors such as invasion and resistance to cell stress and chemo- and radio-treatments. Canonically, ST6GAL1 resides in the intracellular secretory apparatus and glycosylates nascent glycoproteins in biosynthetic transit. However, ST6GAL1 is also released into the extracellular milieu and extracellularly remodels cell surface and secreted glycans. The impact of this non-canonical extrinsic mechanism of ST6GAL1 on tumor cell pathobiology is not known. We hypothesize that ST6GAL1 action is the combined effect of natively expressed sialyltransferase acting cell-autonomously within the ER-Golgi complex and sialyltransferase from extracellular origins acting extrinsically to remodel cell-surface glycans. We found that shRNA knockdown of intrinsic ST6GAL1 expression resulted in decreased ST6GAL1 cargo in the exosome-like vesicles as well as decreased breast tumor cell growth and invasive behavior in 3D in vitro cultures. Extracellular ST6GAL1, present in cancer exosomes or the freely soluble recombinant sialyltransferase, compensates for insufficient intrinsic ST6GAL1 by boosting cancer cell proliferation and increasing invasiveness. Moreover, we present evidence supporting the existence novel but yet uncharacterized cofactors in the exosome-like particles that potently amplify extrinsic ST6GAL1 action, highlighting a previously unknown mechanism linking this enzyme and cancer pathobiology. Our data indicate that extracellular ST6GAL1 from remote sources can compensate for cellular ST6GAL1-mediated aggressive tumor cell proliferation and invasive behavior and has great clinical potential for extracellular ST6GAL1 as these molecules are in the extracellular space should be easily accessible targets.

Role for hepatic and circulatory ST6Gal-1 sialyltransferase in regulating myelopoiesis.

Recent findings have established a role for the ST6Gal-1 sialyltransferase in modulating inflammatory cell production during Th1 and Th2 responses. ST6Gal-1 synthesizes the Sia(alpha2,6) to Gal(beta1,4)GlcNAc linkage on glycoproteins on cell surfaces and in systemic circulation. Engagement of P1, one of six promoter/regulatory regions driving murine ST6Gal-1 gene expression, generates the ST6Gal-1 for myelopoietic regulation. P1 utilization, however, is restricted to the liver and silent in hematopoietic cells. We considered the possibility that myelopoiesis is responsive to the sialylation of liver-derived circulatory glycoproteins, such that reduced alpha2,6-sialylation results in elevated myelopoiesis. However, 2-dimensional differential in gel electrophoresis (2D-DIGE) analysis disclosed only minimal alterations in the sialylation of sera glycoproteins of ST6Gal-1-deficient mice when compared with wild-type controls, either at baseline or during an acute phase response when the demand for sialylation is greatest. Furthermore, sera from ST6Gal-1-deficient animals did not enhance myelopoietic activity in ex vivo colony formation assays. Whereas there was only minimal consequence to the alpha2,6-sialylation of circulatory glycoproteins, ablation of the P1 promoter did result in strikingly depressed levels of ST6Gal-1 released into systemic circulation. Therefore, we considered the alternative possibility that myelopoiesis may be regulated not by the hepatic sialyl glycoproteins, but by the ST6Gal-1 that was released directly into circulation. Supporting this, ex vivo colony formation was notably attenuated upon introduction of physiologic levels of ST6Gal-1 into the culture medium. Our data support the idea that circulatory ST6Gal-1, mostly of hepatic origin, limits myelopoiesis by a mechanism independent of hepatic sialylation of serum glycoproteins.

Blood-Borne ST6GAL1 Regulates Immunoglobulin Production in B Cells.

Humoral immunity is an effective but metabolically expensive defense mechanism. It is unclear whether systemic cues exist to communicate the dynamic need for antigen presentation and immunoglobulin production. Here, we report a novel role for the liver-produced, acute phase reactant ST6GAL1 in IgG production. B cell expression of ST6GAL1, a sialyltransferase mediating the attachment of α2,6-linked sialic acids on N-glycans, is classically implicated in the dysregulated B cell development and immunoglobulin levels of St6gal1-deficient mice. However, the blood-borne pool of ST6GAL1, upregulated during systemic inflammation, can also extrinsically modify leukocyte cell surfaces. We show that B cell independent, extracellular ST6GAL1 enhances B cell IgG production and increases blood IgG titers. B cells of mice lacking the hepatocyte specific St6gal1 promoter have reduced sialylation of cell surface CD22 and CD45 and produce less IgG upon stimulation. Sialylation of B cells by extracellular ST6GAL1 boosts expression of IgM, IgD, and CD86, proliferation, and IgG production in vitro. In vivo, elevation of blood ST6GAL1 enhances B cell development and systemic IgG in a CD22-dependent manner. Our data point to a function of an extracellular glycosyltransferase in promoting humoral immunity. Manipulation of systemic ST6GAL1 may represent an effective therapeutic approach for humoral insufficiency.

Macrophages Expressing GALC Improve Peripheral Krabbe Disease by a Mechanism Independent of Cross-Correction.

Many therapies for lysosomal storage disorders rely on cross-correction of lysosomal enzymes. In globoid cell leukodystrophy (GLD), mutations in GALC cause psychosine accumulation, inducing demyelination, a neuroinflammatory "globoid" reaction and neurodegeneration. The efficiency of GALC cross-correction in vivo, the role of the GALC substrate galactosylceramide, and the origin of psychosine are poorly understood. Using a novel GLD model, we show that cross-correction does not occur efficiently in vivo and that Galc-deficient Schwann cells autonomously produce psychosine. Furthermore, macrophages require GALC to degrade myelin, as Galc-deficient macrophages are transformed into globoid cells by exposure to galactosylceramide and produce a more severe GLD phenotype. Finally, hematopoietic stem cell transplantation in patients reduces globoid cells in nerves, suggesting that the phagocytic response of healthy macrophages, rather than cross-correction, contributes to the therapeutic effect. Thus, GLD may be caused by at least two mechanisms: psychosine-induced demyelination and secondary neuroinflammation from galactosylceramide storage in macrophages.

Effect of sialic acid loss on dendritic cell maturation.

Sialic acids are key structural determinants and contribute to the functionality of a number of immune cell receptors. Previously, we demonstrated that differentiation of human dendritic cells (DCs) is accompanied by an increased expression of sialylated cell surface structures, putatively through the activity of the ST3Gal.I and ST6Gal.I sialyltransferases. Furthermore, DC endocytosis was reduced upon removal of the cell surface sialic acid residues by neuraminidase. In the present work, we evaluate the contribution of the sialic acid modifications in DC maturation. We demonstrate that neuraminidase-treated human DCs have increased expression of major histocompatibility complex (MHC) and costimulatory molecules, increased gene expression of specific cytokines and induce a higher proliferative response of T lymphocytes. Together, the data suggest that clearance of cell surface sialic acids contributes to the development of a T helper type 1 proinflammatory response. This postulate is supported by mouse models, where elevated MHC class II and increased maturation of specific DC subsets were observed in DCs harvested from ST3Gal.I(-/-) and ST6Gal.I(-/-) mice. Moreover, important qualitative differences, particularly in the extent of reduced endocytosis and in the peripheral distribution of DC subsets, existed between the ST3Gal.I(-/-) and ST6Gal.I(-/-) strains. Together, the data strongly suggest not only a role of cell surface sialic acid modifications in maturation and functionality of DCs, but also that the sialic acid linkages created by different sialyltransferases are functionally distinct. Consequently, with particular relevance to DC-based therapies, cell surface sialylation, mediated by individual sialyltransferases, can influence the immunogenicity of DCs upon antigen loading.