The sialyltransferase ST3Gal-IV guides murine T-cell progenitors to the thymus.

T lymphocytes are important players in beneficial and detrimental immune responses. In contrast to other lymphocyte populations that develop in the bone marrow, T-cell precursors need to migrate to the thymus for further development. The interaction of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) is crucial for thymic entry of T-cell precursors during settings of T-cell lineage reconstitution. PSGL-1 has to be sialylated to function as a ligand for P-selectin, and the sialyltransferase ST3Gal-IV might play a critical role in this process. We therefore investigated the role of ST3Gal-IV for T-cell development using competitive mixed bone marrow chimeric mice. We found that ST3Gal-IV is dispensable for homing and engraftment of hematopoietic precursors in the bone marrow. However, ST3Gal-IV deficiency affects seeding of the thymus by early T-cell progenitors, leading to impaired restoration of the peripheral T-cell compartment. This defect could be restored by ectopic retroviral expression of ST3Gal-IV in hematopoietic stem cells derived from ST3Gal-IV-deficient donor mice. Our findings show that ST3Gal-IV plays a critical and nonredundant role for efficient T-cell lineage reconstitution after bone marrow transplantation.

Sialylation on O-glycans protects platelets from clearance by liver Kupffer cells.

Most platelet membrane proteins are modified by mucin-type core 1-derived glycans (O-glycans). However, the biological importance of O-glycans in platelet clearance is unclear. Here, we generated mice with a hematopoietic cell-specific loss of O-glycans (HC C1galt1-/- ). These mice lack O-glycans on platelets and exhibit reduced peripheral platelet numbers. Platelets from HC C1galt1-/- mice show reduced levels of α-2,3-linked sialic acids and increased accumulation in the liver relative to wild-type platelets. The preferential accumulation of HC C1galt1-/- platelets in the liver was reduced in mice lacking the hepatic asialoglycoprotein receptor [Ashwell-Morell receptor (AMR)]. However, we found that Kupffer cells are the primary cells phagocytosing HC C1galt1-/- platelets in the liver. Our results demonstrate that hepatic AMR promotes preferential adherence to and phagocytosis of desialylated and/or HC C1galt1-/- platelets by the Kupffer cell through its C-type lectin receptor CLEC4F. These findings provide insights into an essential role for core 1 O-glycosylation of platelets in their clearance in the liver.

Deficiency of the sialyltransferase St3Gal4 reduces Ccl5-mediated myeloid cell recruitment and arrest: short communication.

RATIONALE: Sialylation by α2,3-sialyltransferases has been shown to be a crucial glycosylation step in the generation of functional selectin ligands. Recent evidence suggests that sialylation also affects the binding of chemokines to their corresponding receptor. OBJECTIVE: Because the chemokine receptors for Ccl5 and Ccl2 are important in atherogenic recruitment of neutrophils and monocytes, we here investigated the role of α2,3-sialyltransferase IV (ST3Gal-IV) in Ccl5- and Ccl2-mediated myeloid cell arrest and further studied its relevance in a mouse model of atherosclerosis. METHODS AND RESULTS: St3Gal4-deficient myeloid cells showed a reduced binding of Ccl5 and an impaired Ccl5-triggered integrin activation. Correspondingly, Ccl5-induced arrest on tumor necrosis factor-α-stimulated endothelium was almost completely abrogated, as observed in flow chamber adhesion assays and during ex vivo perfusion or intravital microscopy of carotid arteries. Moreover, Ccl5-triggered neutrophil and monocyte extravasation into the peritoneal cavity was severely reduced in St3Gal4(-/-) mice. In contrast, St3Gal4 deficiency did not significantly affect Ccl2 binding and only marginally decreased Ccl2-induced flow arrest of myeloid cells. In agreement with the crucial role of leukocyte accumulation in atherogenesis, and the importance of Ccl5 chemokine receptors mediating myeloid cell recruitment to atherosclerotic vessels, St3Gal4 deficiency drastically reduced the size, stage, and inflammatory cell content of atherosclerotic lesions in Apoe(-/-) mice on high-fat diet. CONCLUSIONS: In summary, these findings identify ST3Gal-IV as a promising target to reduce inflammatory leukocyte recruitment and arrest.

Quantitative selection and parallel characterization of aptamers.

Aptamers are promising affinity reagents that are potentially well suited for high-throughput discovery, as they are chemically synthesized and discovered via completely in vitro selection processes. Recent advancements in selection, sequencing, and the use of modified bases have improved aptamer quality, but the overall process of aptamer generation remains laborious and low-throughput. This is because binding characterization remains a critical bottleneck, wherein the affinity and specificity of each candidate aptamer are measured individually in a serial manner. To accelerate aptamer discovery, we devised the Quantitative Parallel Aptamer Selection System (QPASS), which integrates microfluidic selection and next-generation sequencing with in situ-synthesized aptamer arrays, enabling simultaneous measurement of affinity and specificity for thousands of candidate aptamers in parallel. After using QPASS to select aptamers for the human cancer biomarker angiopoietin-2 (Ang2), we in situ synthesized arrays of the selected sequences and obtained equilibrium dissociation constants (Kd) for every aptamer in parallel. We thereby identified over a dozen high-affinity Ang2 aptamers, with Kd as low as 20.5 ± 7.3 nM. The same arrays enabled us to quantify binding specificity for these aptamers in parallel by comparing relative binding of differentially labeled target and nontarget proteins, and by measuring their binding affinity directly in complex samples such as undiluted serum. Finally, we show that QPASS offers a compelling avenue for exploring structure-function relationships for large numbers of aptamers in parallel by coupling array-based affinity measurements with next-generation sequencing data to identify nucleotides and motifs within the aptamer that critically affect Ang2 binding.

Core2 O-glycan structure is essential for the cell surface expression of sucrase isomaltase and dipeptidyl peptidase-IV during intestinal cell differentiation.

Alterations in glycosylation play an important role during intestinal cell differentiation. Here, we compared expression of mucin-type O-glycan synthases from proliferating and differentiated HT-29 and Caco-2 cells. Mucin-type O-glycan structures were analyzed at both stages by mass spectrometry. Core2 ß1,6-N-acetylglucosaminyltransferase-2 (C2GnT-2) was markedly increased in differentiated HT-29 and Caco-2 cells, but the core3 structure was hardly detectable. To determine whether such differential expression of mucin-type O-glycan structures has physiological significance in intestinal cell differentiation, expression of sucrase isomaltase (SI) and dipeptidyl-peptidase IV (DPP-IV), two well known intestinal differentiation markers, was examined. Interestingly, the fully glycosylated mature form of SI was decreased in C2GnT-2 knock-out mice but not in core2 N-acetylglucosaminyltransferase-3 (C2GnT-3) nulls. In addition, expression of SI and DPP-IV was dramatically reduced in C2GnT-1-3 triple knock-out mice. These patterns were confirmed by RNAi analysis; C2GnT-2 knockdown significantly reduced cell surface expression of SI and DPP-IV in Caco-2 cells. Similarly, overexpression of the core3 structure in HT-29 cells attenuated cell surface expression of both enzymes. These findings indicate that core3 O-glycan structure regulates cell surface expression of SI and DPP-IV and that core2 O-glycan is presumably an essential mucin-type O-glycan structure found in both molecules in vivo. Finally, goblet cells in the upper part of the crypt showed impaired maturation in the core2 O-glycan-deficient mice. These studies are the first to clearly identify functional mucin-type O-glycan structures modulating cell surface expression of SI and DPP-IV during the intestinal cell differentiation.

Alpha2,6-sialic acid on platelet endothelial cell adhesion molecule (PECAM) regulates its homophilic interactions and downstream antiapoptotic signaling.

Antiangiogenesis therapies are now part of the standard repertoire of cancer therapies, but the mechanisms for the proliferation and survival of endothelial cells are not fully understood. Although endothelial cells are covered with a glycocalyx, little is known about how endothelial glycosylation regulates endothelial functions. Here, we show that alpha2,6-sialic acid is necessary for the cell-surface residency of platelet endothelial cell adhesion molecule (PECAM), a member of the immunoglobulin superfamily that plays multiple roles in cell adhesion, mechanical stress sensing, antiapoptosis, and angiogenesis. As a possible underlying mechanism, we found that the homophilic interactions of PECAM in endothelial cells were dependent on alpha2,6-sialic acid. We also found that the absence of alpha2,6-sialic acid down-regulated the tyrosine phosphorylation of PECAM and recruitment of Src homology 2 domain-containing protein-tyrosine phosphatase 2 and rendered the cells more prone to mitochondrion-dependent apoptosis, as evaluated using PECAM- deficient endothelial cells. The present findings open up a new possibility that modulation of glycosylation could be one of the promising strategies for regulating angiogenesis.

Physiological and glycomic characterization of N-acetylglucosaminyltransferase-IVa and -IVb double deficient mice.

N-Acetylglucosaminyltransferase-IV (GnT-IV) has two isoenzymes, GnT-IVa and GnT-IVb, which initiate the GlcNAcbeta1-4 branch synthesis on the Manalpha1-3 arm of the N-glycan core thereby increasing N-glycan branch complexity and conferring endogenous lectin binding epitopes. To elucidate the physiological significance of GnT-IV, we engineered and characterized GnT-IVb-deficient mice and further generated GnT-IVa/-IVb double deficient mice. In wild-type mice, GnT-IVa expression is restricted to gastrointestinal tissues, whereas GnT-IVb is broadly expressed among organs. GnT-IVb deficiency induced aberrant GnT-IVa expression corresponding to the GnT-IVb distribution pattern that might be attributed to increased Ets-1, which conceivably activates the Mgat4a promoter, and thereafter preserved apparent GnT-IV activity. The compensative GnT-IVa expression might contribute to amelioration of the GnT-IVb-deficient phenotype. GnT-IVb deficiency showed mild phenotypic alterations in hematopoietic cell populations and hemostasis. GnT-IVa/-IVb double deficiency completely abolished GnT-IV activity that resulted in the disappearance of the GlcNAcbeta1-4 branch on the Manalpha1-3 arm that was confirmed by MALDI-TOF MS and GC-MS linkage analyses. Comprehensive glycomic analyses revealed that the abundance of terminal moieties was preserved in GnT-IVa/-IVb double deficiency that was due to the elevated expression of glycosyltransferases regarding synthesis of terminal moieties. Thereby, this may maintain the expression of glycan ligands for endogenous lectins and prevent cellular dysfunctions. The fact that the phenotype of GnT-IVa/-IVb double deficiency largely overlapped that of GnT-IVa single deficiency can be attributed to the induced glycomic compensation. This is the first report that mammalian organs have highly organized glycomic compensation systems to preserve N-glycan branch complexity.

Dual roles for hepatic lectin receptors in the clearance of chilled platelets.

Rapid chilling causes glycoprotein-Ib (GPIb) receptors to cluster on blood platelets. Hepatic macrophage beta(2) integrin binding to beta-N-acetylglucosamine (beta-GlcNAc) residues in the clusters leads to rapid clearance of acutely chilled platelets after transfusion. Although capping the beta-GlcNAc moieties by galactosylation prevents clearance of short-term-cooled platelets, this strategy is ineffective after prolonged refrigeration. We report here that prolonged refrigeration increased the density and concentration of exposed galactose residues on platelets such that hepatocytes, through Ashwell-Morell receptor binding, become increasingly involved in platelet removal. Macrophages rapidly removed a large fraction of transfused platelets independent of their storage conditions. With prolonged platelet chilling, hepatocyte-dependent clearance further diminishes platelet recovery and survival after transfusion. Inhibition of chilled platelet clearance by both beta(2) integrin and Ashwell-Morell receptors may afford a potentially simple method for storing platelets in the cold.

Core2 1-6-N-glucosaminyltransferase-I is crucial for the formation of atherosclerotic lesions in apolipoprotein E-deficient mice.

OBJECTIVE: Core2 1-6-N-glucosaminyltransferase-I (C2GlcNAcT-I) modification of adhesion molecules is required for optimal binding to target ligands. The objective of this study was to determine the role of C2GlcNAcT-I in the recruitment of Ly-6C(hi) monocytes to atherosclerotic lesions and in lesion formation in mice. METHODS AND RESULTS: In a whole-blood binding assay, Ly-6C(hi) monocytes and certain lymphocytes and natural killer cells from wild-type mice bound to P- and E-selectin. C2GlcNAcT-I deficiency abrogated leukocyte binding to P- and E-selectin in this assay as well as in an in vitro flow chamber assay. Moreover, C2GlcNAcT-I deficiency decreased Ly-6C(hi) monocyte interactions with atherosclerotic arteries under physiological flow conditions and also inhibited monocyte recruitment to the peritoneal cavity in mice challenged with thioglycollate. In apolipoprotein E-deficient (apoE(-/-)) mice, lack of C2GlcNAcT-I resulted in fewer and smaller atherosclerotic lesions in mouse aortas. Atherosclerosis was also suppressed in C2GlcNAcT-I(-/-)/apoE(-/-) chimeric mice transplanted with C2GlcNAcT-I(+/+) bone marrow cells. CONCLUSIONS: C2GlcNAcT-I in both leukocytes and blood vessel wall cells contributes to leukocyte recruitment to the arterial wall. C2GlcNAcT-I deficiency leads to the formation of small, macrophage-poor, and collagen-rich atherosclerotic lesions.

Initiation of protein O glycosylation by the polypeptide GalNAcT-1 in vascular biology and humoral immunity.

Core-type protein O glycosylation is initiated by polypeptide N-acetylgalactosamine (GalNAc) transferase (ppGalNAcT) activity and produces the covalent linkage of serine and threonine residues of proteins. More than a dozen ppGalNAcTs operate within multicellular organisms, and they differ with respect to expression patterns and substrate selectivity. These distinctive features imply that each ppGalNAcT may differentially modulate regulatory processes in animal development, physiology, and perhaps disease. We found that ppGalNAcT-1 plays key roles in cell and glycoprotein selective functions that modulate the hematopoietic system. Loss of ppGalNAcT-1 activity in the mouse results in a bleeding disorder which tracks with reduced plasma levels of blood coagulation factors V, VII, VIII, IX, X, and XII. ppGalNAcT-1 further supports leukocyte trafficking and residency in normal homeostatic physiology as well as during inflammatory responses, in part by providing a scaffold for the synthesis of selectin ligands expressed by neutrophils and endothelial cells of peripheral lymph nodes. Animals lacking ppGalNAcT-1 are also markedly impaired in immunoglobulin G production, coincident with increased germinal center B-cell apoptosis and reduced levels of plasma B cells. These findings reveal that the initiation of protein O glycosylation by ppGalNAcT-1 provides a distinctive repertoire of advantageous functions that support vascular responses and humoral immunity.

Structural and mechanistic features of protein O glycosylation linked to CD8+ T-cell apoptosis.

CD8+ T-cell apoptosis is essential for the contraction phase of the immune response, yet the initiating signals and precise pathways involved are unresolved. The ST3Gal-I sialyltransferase is a candidate mechanistic component and catalyzes sialic acid addition to core 1 O-glycans during protein O glycosylation. ST3Gal-I inactivation or enzymatic removal of its product renders CD8+ T cells, but not CD4+ T cells, susceptible to apoptosis by differential cross-linking of O-glycoproteins in the absence of interleukin-2 and T-cell receptor (TCR) signaling. This results in caspase activation, DNA fragmentation, and phosphatidylserine externalization prior to cell death. We further show that ST3Gal-I function is regulated by a posttranscriptional mechanism operating distal to Golgi core 2 O glycosylation and is invariably linked to CD8+ T-cell contraction following viral (lymphocytic choriomeningitis virus) infection and bacterial (staphylococcal enterotoxin B) antigen immunization. The mechanism does not involve the ST3Gal-I substrate CD43 or core 2 O-glycan induction and overcomes the ability of Bcl-2 to inhibit the contraction phase in vivo. Loss of ST3Gal-I function further reduces Bim-deficient CD8+ T-cell accumulation without diminishing apoptotic sensitivity. We propose that an endogenous lectin activates an apoptotic pathway constructed in CD8+ T cells following TCR stimulation and enables contraction upon attenuation of immune signaling.

Essential role of RGS-PX1/sorting nexin 13 in mouse development and regulation of endocytosis dynamics.

RGS-PX1 (also known as sorting nexin 13) is a member of both the regulator of G protein signaling (RGS) and sorting nexin (SNX) protein families. Biochemical and cell culture studies have shown that RGS-PX1/SNX13 attenuates Galphas-mediated signaling through its RGS domain and regulates endocytic trafficking and degradation of the epidermal growth factor receptor. To understand the functions of RGS-PX1/SNX13 in vivo, we generated mice carrying targeted mutations of Snx13 and found that systemic Snx13-null mice were embryonic lethal around midgestation. Snx13-null embryos had significant overall growth retardation and defects in neural tube closure, blood vessel formation, and the formation of the placental labyrinthine layer. Moreover, the Snx13-null visceral yolk sac endoderm cells showed dramatic changes in the organization of endocytic compartments, abundant autophagic vacuoles, and abnormal localization of several endocytic markers, including megalin, a receptor for nutrients and proteins; ARH, a coat protein that binds megalin; LAMP2; and LC3. These changes suggest that Snx13-null embryos are defective in nutrient uptake and transport, which may contribute to the other developmental abnormalities observed. Taken together, our findings demonstrate an essential role for RGS-PX1/SNX13 in mouse development and provide previously undescribed insights into its cellular function in the regulation of endocytosis dynamics.

ST6Gal-I restrains CD22-dependent antigen receptor endocytosis and Shp-1 recruitment in normal and pathogenic immune signaling.

The ST6Gal-I sialyltransferase produces Siglec ligands for the B-cell-specific CD22 lectin and sustains humoral immune responses. Using multiple experimental approaches to elucidate the mechanisms involved, we report that ST6Gal-I deficiency induces immunoglobulin M (IgM) antigen receptor endocytosis in the absence of immune stimulation. This coincides with increased antigen receptor colocalization with CD22 in both clathrin-deficient and clathrin-enriched membrane microdomains concurrent with diminished tyrosine phosphorylation of Igalpha/beta, Syk, and phospholipase C-gamma2 upon immune activation. Codeficiency with CD22 restores IgM antigen receptor half-life at the cell surface in addition to reversing alterations in membrane trafficking and immune signaling. Diminished immune responses due to ST6Gal-I deficiency further correlate with constitutive recruitment of Shp-1 to CD22 in unstimulated B cells independent of Lyn tyrosine kinase activity and prevent autoimmune disease pathogenesis in the Lyn-deficient model of systemic lupus erythematosus, resulting in a significant extension of life span. Protein glycosylation by ST6Gal-I restricts access of antigen receptors and Shp-1 to CD22 and operates by a CD22-dependent mechanism that decreases the basal rate of IgM antigen receptor endocytosis in altering the threshold of B-cell immune activation.

Neuronal LRP1 functionally associates with postsynaptic proteins and is required for normal motor function in mice.

The LDL receptor-related protein 1 (LRP1) is a multifunctional cell surface receptor that is highly expressed on neurons. Neuronal LRP1 in vitro can mediate ligand endocytosis, as well as modulate signal transduction processes. However, little is known about its role in the intact nervous system. Here, we report that mice that lack LRP1 selectively in differentiated neurons develop severe behavioral and motor abnormalities, including hyperactivity, tremor, and dystonia. Since their central nervous systems appear histoanatomically normal, we suggest that this phenotype is likely attributable to abnormal neurotransmission. This conclusion is supported by studies of primary cultured neurons that show that LRP1 is present in close proximity to the N-methyl-D-aspartate (NMDA) receptor in dendritic synapses and can be coprecipitated with NMDA receptor subunits and the postsynaptic density protein PSD-95 from neuronal cell lysates. Moreover, treatment with NMDA, but not dopamine, reduces the interaction of LRP1 with PSD-95, indicating that LRP1 participates in transmitter-dependent postsynaptic responses. Together, these findings suggest that LRP1, like other ApoE receptors, can modulate synaptic transmission in the brain.

Sialyltransferase ST8Sia-II assembles a subset of polysialic acid that directs hippocampal axonal targeting and promotes fear behavior.

Polysialic acid (PSA) is a post-translational protein modification that is widely expressed among neural cell types during development. Found predominantly on the neural cell adhesion molecule (NCAM), PSA becomes restricted to regions of neurogenesis and neuroplasticity in the adult. In the mammalian genome, two polysialyltransferases termed ST8Sia-II and ST8Sia-IV have been hypothesized to be responsible for the production of PSA in vivo. Approaches to discover PSA function have involved the application of endoneuraminidase-N to remove PSA and genetic manipulations in the mouse to deplete either NCAM or ST8Sia-IV. Here we report the production and characterization of mice deficient in the ST8Sia-II polysialyltransferase. We observed alterations in brain PSA expression unlike those observed in mice lacking ST8Sia-IV. This included a PSA deficit in regions of neurogenesis but without changes in the frequency of mitotic neural progenitor cells. In further contrast with ST8Sia-IV deficiency, loss of ST8Sia-II did not impair hippocampal synaptic plasticity but instead resulted in the misguidance of infrapyramidal mossy fibers and the formation of ectopic synapses in the hippocampus. Consistent with studies of animal models bearing these morphological changes, ST8Sia-II-deficient mice exhibited higher exploratory drive and reduced behavioral responses to Pavlovian fear conditioning. PSA produced by the ST8Sia-II polysialyltransferase modifies memory and behavior processes that are distinct from the neural roles reported for ST8Sia-IV. This genetic partitioning of PSA formation engenders discrete neurological processes and reveals that this post-translational modification forms the predominant basis for the multiple functions attributed to the NCAM glycoprotein.

Cloning and characterization of a new human UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase, designated pp-GalNAc-T13, that is specifically expressed in neurons and synthesizes GalNAc alpha-serine/threonine antigen.

To date, 10 members of the UDP-N-acetyl-alpha-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (pp-GalNAc-T) family have been cloned and analyzed in human. In this study, we cloned and analyzed a novel human pp-GalNAc-T from an NT2 cell cDNA library, and we named it pp-GalNAc-T13. In amino acid sequences, pp-GalNAc-T13 was highly homologous, showing 84.3% identity, to pp-GalNAc-T1. Real time PCR analysis revealed pp-GalNAc-T13 to be highly and restrictively expressed in the brain and present at very low or undetectable levels in other tissues, in contrast to the ubiquitous expression of pp-GalNAc-T1. pp-GalNAc-T13 was abundantly expressed in all neuroblastoma cells examined and primary cultured neurons but not in glioblastoma cells and primary cultured astrocytes. pp-GalNAc-T13 exhibited much stronger activity to transfer GalNAc to mucin peptides, such as Muc5Ac and MUC7, than did pp-GalNAc-T1. In addition, pp-GalNAc-T13 differed in substrate specificity to pp-GalNAc-T1. pp-GalNAc-T13 was able to form a triplet Tn epitope, three consecutive GalNAc-Ser/Thr structures, on peptides encoded in syndecan-3, a proteoglycan expressed in neurons. pp-GalNAc-T13-deficient mice have been established in a previous work. Immunohistochemical study showed a remarkable decrease in Tn antigen expression in the cerebellum of the pp-GalNAc-T13 knockout mouse. pp-GalNAc-T13 would be a major enzyme responsible for the synthesis of O-glycan and specifically the Tn antigen epitope in neurons.

Sialyltransferase specificity in selectin ligand formation.

Selectin ligands are glycan structures that participate in leukocyte trafficking and inflammation. At least 6 ST3Gal sialyltransferases (I-VI) have been identified that may contribute to selectin ligand formation. However, it is not known which of these sialyltransferases are involved in vivo and whether they may differentially regulate selectin function. We have produced and characterized mice genetically deficient in ST3Gal-I, ST3Gal-II, ST3Gal-III, and ST3Gal-IV. Unlike mice bearing severe defects in selectin ligand formation, there was no finding of leukocytosis with these single ST3Gal deficiencies. Among neutrophils, only ST3Gal-IV was found to play a role in the synthesis of selectin ligands. In vitro rolling of marrow-derived neutrophils on E- or P-selectins presented by Chinese hamster ovary cells was reduced in the absence of ST3Gal-IV. However, in a tumor necrosis factor alpha (TNF-alpha)-induced inflammation model in vivo, no defect among P-selectin ligands was observed. Nevertheless, the number of leukocytes rolling on postcapillary venules in an E-selectin-dependent manner was decreased while E-selectin-dependent rolling velocity was increased. We propose that multiple ST3Gal sialyltransferases contribute to selectin ligand formation, as none of these ST3Gal deficiencies recapitulated the degree of E- and P-selectin ligand deficit observed on neuraminidase treatment of intact neutrophils. Our findings indicate a high degree of functional specificity among sialyltransferases and a substantial role for ST3Gal-IV in selectin ligand formation.

Generation of mice deficient for macrophage galactose- and N-acetylgalactosamine-specific lectin: limited role in lymphoid and erythroid homeostasis and evidence for multiple lectins.

Macrophage receptors function in pattern recognition for the induction of innate immunity, in cellular communication to mediate the regulation of adaptive immune responses, and in the clearance of some glycosylated cells or glycoproteins from the circulation. They also function in homeostasis by initiating the engulfment of apoptotic cells. Evidence has suggested that macrophage receptors function to recognize cells that are destined for programmed cell death but not yet overtly apoptotic. We have examined the function of a macrophage receptor specific for unsialylated glycoproteins, known as the mouse macrophage galactose- and N-acetylgalactosamine-specific lectin (mMGL) (Ii et al., J. Biol. Chem. 265:11295-11298, 1990; Sato et al., J. Biochem. [Tokyo] 111:331-336, 1992; Yamamoto et al., Biochemistry 33:8159-8166, 1994). With targeted disruption, we tested whether mMGL is necessary for macrophage function, controlled thymic development, the loss of activated CD8 T cells, and the turnover of red blood cells. Evidence indicates that mMGL may play a nonessential role in several of these macrophage functions. Experiments are presented that indicate the existence of another galactose- and N-acetylgalactosamine-recognizing lectin distinct from mMGL. This may explain the absence of a strong phenotype in mMGL-deficient mice.