竹松 弘

CD22, one of the sialic acid-binding immunoglobulin-like lectins (Siglecs), regulates B lymphocyte signaling via its interaction with glycan ligands bearing the sequence Neu5Ac/Gcα(2→6)Gal. We have developed the synthetic sialoside GSC-718 as a ligand mimic for CD22 and identified it as a potent CD22 inhibitor. Although the synthesis of CD22-binding sialosides including GSC-718 has been reported by our group, the synthetic route was unfortunately not suitable for large-scale synthesis. In this study, we developed an improved scalable synthetic procedure for sialosides which utilized 1,5-lactam formation as a key step. The improved procedure yielded sialosides incorporating a series of aglycones at the C2 position. Several derivatives with substituted benzyl residues as aglycones were found to bind to mouse CD22 with affinity comparable to that of GSC-718. The new procedure developed in this study affords sialosides in sufficient quantities for cell-based assays, and will facilitate the search for promising CD22 inhibitors that have therapeutic potential.

Spermatid production is a complex regulatory process in which coordination between hormonal control and apoptosis plays a pivotal role in maintaining a balanced number of sperm cells. Apoptosis in spermatogenesis is controlled by pro-apoptotic and anti-apoptotic molecules. Hormones involved in the apoptotic process during spermatogenesis include gonadotrophins, sex hormones, and glucocorticoid (GC). GC acts broadly as an apoptosis inducer by binding to its receptor (glucocorticoid receptor: GR) during organ development processes, such as spermatogenesis. However, the downstream pathway induced in GC-GR signaling and the apoptotic process during spermatogenesis remains poorly understood. We reported previously that GC induces full-length glucocorticoid-induced transcript 1 (GLCCI1-long), which functions as an anti-apoptotic mediator in thymic T cell development. Here, we demonstrate that mature murine testis expresses a novel isoform of GLCCI1 protein (GLCCI1-short) in addition to GLCCI1-long. We demonstrate that GLCCI1-long is expressed in spermatocytes along with GR. In contrast, GLCCI1-short is primarily expressed in spermatids where GR is absent; instead, the estrogen receptor is expressed. GLCCI1-short also binds to LC8, which is a known mediator of the anti-apoptotic effect of GLCCI1-long. A luciferase reporter assay revealed that β-estradiol treatment synergistically increased Glcci1-short promotor-driven luciferase activity in Erα-overexpressing cells. Together with the evidence that the conversion of testosterone to estrogen is preceded by aromatase expression in spermatids, we hypothesize that estrogen induces GLCCI1-short, which, in turn, may function as a novel anti-apoptotic mediator in mature murine testis.

BACKGROUND: The cause of podocyte injury in idiopathic nephrotic syndrome (INS) remains unknown. Although recent evidence points to the role of B cells and autoimmunity, the lack of animal models mediated by autoimmunity limits further research. We aimed to establish a mouse model mimicking human INS by immunizing mice with Crb2, a transmembrane protein expressed at the podocyte foot process. METHODS: C3H/HeN mice were immunized with the recombinant extracellular domain of mouse Crb2. Serum anti-Crb2 antibody, urine protein-to-creatinine ratio, and kidney histology were studied. For signaling studies, a Crb2-expressing mouse podocyte line was incubated with anti-Crb2 antibody. RESULTS: Serum anti-Crb2 autoantibodies and significant proteinuria were detected 4 weeks after the first immunization. The proteinuria reached nephrotic range at 9-13 weeks and persisted up to 29 weeks. Initial kidney histology resembled minimal change disease in humans, and immunofluorescence staining showed delicate punctate IgG staining in the glomerulus, which colocalized with Crb2 at the podocyte foot process. A subset of mice developed features resembling FSGS after 18 weeks. In glomeruli of immunized mice and in Crb2-expressing podocytes incubated with anti-Crb2 antibody, phosphorylation of ezrin, which connects Crb2 to the cytoskeleton, increased, accompanied by altered Crb2 localization and actin distribution. CONCLUSION: The results highlight the causative role of anti-Crb2 autoantibody in podocyte injury in mice. Crb2 immunization could be a useful model to study the immunologic pathogenesis of human INS, and may support the role of autoimmunity against podocyte proteins in INS.

Podocyte damage is a major pathological lesion leading to focal segmental glomerulosclerosis (FSGS). Podocytes damaged by cellular stress undergo hypertrophy to compensate for podocytopenia. It is known that cyclin-dependent kinase inhibitors induced by p53 ensure podocytes hypertrophy; however, its precise mechanism remains to be further investigated. In this study, we found that ubiquitin specific protease 40 (USP40) is a novel regulator of p53. Although USP40 knockout mice established in the present study revealed no abnormal kidney phenotype, intermediate filament Nestin was upregulated in the glomeruli, and was bound to and colocalized with USP40. We also found that USP40 deubiquitinated histidine triad nucleotide-binding protein 1 (HINT1), an inducer of p53. Gene knockdown experiments of USP40 in cultured podocytes revealed the reduction of HINT1 and p53 protein expression. Finally, in glomerular podocytes of mouse FSGS, upregulation of HINT1 occurred in advance of the proteinuria, which was followed by upregulation of USP40, p53 and Nestin. In conclusion, USP40 bound to Nestin deubiquitinates HINT1, and in consequence upregulates p53. These results provide additional insight into the pathological mechanism of podocyte hypertrophy in FSGS.

Germinal centers (GCs) are essential for antibody affinity maturation. GC B cells have a unique repertoire of cell surface glycans compared with naive B cells, yet functional roles for changes in glycosylation in the GC have yet to be ascribed. Detection of GCs by the antibody GL7 reflects a downregulation in ligands for CD22, an inhibitory co-receptor of the B cell receptor. To test a functional role for downregulation of CD22 ligands in the GC, we generate a mouse model that maintains CD22 ligands on GC B cells. With this model, we demonstrate that glycan remodeling plays a critical role in the maintenance of B cells in the GC. Sustained expression of CD22 ligands induces higher levels of apoptosis in GC B cells, reduces memory B cell and plasma cell output, and delays affinity maturation of antibodies. These defects are CD22 dependent, demonstrating that downregulation of CD22 ligands on B cells plays a critical function in the GC.

The protein tyrosine phosphatase CD45 plays a crucial role in B cell antigen receptor (BCR) signaling by activating Src family kinases. Cd45-/- mice show altered B cell development and a phenotype likely due to reduced steady-state signaling; however, Cd45-/- B cells show relatively normal BCR ligation-induced signaling. In our investigation of how BCR signaling was restored in Cd45-/- cells, we found that the coreceptor CD22 switched from an inhibitory to a stimulatory function in these cells. We disrupted the ability of CD22 to interact with its ligands in Cd45-/- B cells by generating Cd45-/-St6galI-/- mice, which cannot synthesize the glycan ligand of CD22, or by treating Cd45-/- B cells in vitro with the sialoside GSC718, which inhibits ligand binding to CD22. BCR ligation-induced signaling was reduced by ST6GalI deficiency, but not by GSC718 treatment, suggesting that CD22 restored BCR ligation-induced signaling in Cd45-/- mature B cells by altering cellular phenotypes during development. CD22 was required for the increase in the surface amount of IgM-BCR on Cd45-/- B cells, which augmented signaling. Because B cell survival depends on steady-state BCR signaling, IgM-BCR abundance was likely increased by the selective survival of IgM-BCRhi Cd45-/- B cells because of CD22-mediated signaling under conditions of substantially reduced steady-state signaling. Because the amount of surface IgM-BCR is increased on B cells from patients with other BCR signaling deficiencies, including X-linked agammaglobulinemia, our findings suggest that CD22 may contribute to the partial restoration of B cell function in these patients.

CD22 is an inhibitory B cell coreceptor that regulates B cell development and activation by downregulating BCR signaling through activation of SH2-containing protein tyrosine phosphatase-1 (SHP-1). CD22 recognizes a2,6 sialic acid as a specific ligand and interacts with a2,6 sialic acid-containing membrane molecules, such as CD45, IgM, and CD22, expressed on the same cell. Functional regulation of CD22 by these endogenous ligands enhances BCR ligation-induced signaling and is essential for normal B cell responses to Ags. In this study, we demonstrate that CD45 plays a crucial role in CD22-mediated inhibition of BCR ligationinduced signaling. However, disruption of ligand binding of CD22 enhances CD22 phosphorylation, a process required for CD22- mediated signal inhibition, upon BCR ligation in CD452/2 as well as wild-type mouse B cells but not in mouse B cells expressing a loss-of-function mutant of SHP-1. This result indicates that SHP-1 but not CD45 is required for ligand-mediated regulation of CD22. We further demonstrate that CD22 is a substrate of SHP-1, suggesting that SHP-1 recruited to CD22 dephosphorylates nearby CD22 as well as other substrates. CD22 dephosphorylation by SHP-1 appears to be augmented by homotypic CD22 clustering mediated by recognition of CD22 as a ligand of CD22 because CD22 clustering increases the number of nearby CD22. Our results suggest that CD22 but not CD45 is an endogenous ligand of CD22 that enhances BCR ligation-induced signaling through SHP- 1_mediated dephosphorylation of CD22 in CD22 clusters.

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells.   Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. The importance of reactive oxygen species (ROS) production for the toxicity of each agent was investigated by using an inhibitor of ROS production, N-acetyl-L-cysteine (NAC).    Results:  Andro reduced the viability of THP-1 and H929 in a dose-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, ROS production, and mitochondrial membrane depolarization. Whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. The apoptotic activities of Andro were largely suppressed by NAC, an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR.  Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. The high toxicity for (thus forming: The high toxicity for H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.) H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.

Guillain-Barré syndrome (GBS), including its variant Miller Fisher syndrome (MFS), is an acute peripheral neuropathy that involves autoimmune mechanisms leading to the production of autoantibodies to gangliosides; sialic acid-containing glycosphingolipids. Although association with various genetic polymorphisms in the major histocompatibility complex (MHC) is shown in other autoimmune diseases, GBS is an exception, showing no such link. No significant association was found by genome wide association studies, suggesting that GBS is not associated with common variants. To address the involvement of rare variants in GBS, we analyzed Siglec-10, a sialic acid-recognizing inhibitory receptor expressed on B cells. Here we demonstrate that two rare variants encoding R47Q and A108V substitutions in the ligand-binding domain are significantly accumulated in patients with GBS. Because of strong linkage disequilibrium, there was no patient carrying only one of them. Recombinant Siglec-10 protein containing R47Q but not A108V shows impaired binding to gangliosides. Homology modeling revealed that the R47Q substitution causes marked alteration in the ligand-binding site. Thus, GBS is associated with a rare variant of the SIGLEC10 gene that impairs ligand binding of Siglec-10. Because Siglec-10 regulates antibody production to sialylated antigens, our finding suggests that Siglec-10 regulates development of GBS by suppressing antibody production to gangliosides, with defects in its function predisposing to disease.

The AMPA-type glutamate receptor (AMPAR) is a homotetrameric or heterotetrameric ion channel composed of various combinations of four subunits (GluA1-4), and its abundance in the synapse determines the strength of synaptic activity. The formation of oligomers in the endoplasmatic reticulum (ER) is crucial for AMPAR subunits' ER-exit and translocation to the cell membrane. Although N-glycosylation on different AMPAR subunits has been shown to regulate the ER-exit of hetero-oligomers, its role in the ER-exit of homo-oligomers remains unclear. In this study, we investigated the role of N-glycans at GluA1N63/N363 and GluA2N370 in ER-exit under the homo-oligomeric expression conditions, whose mutants are known to show low cell surface expressions. In contrast to the N-glycosylation site mutant GluA1N63Q, the cell surface expression levels of GluA1N363Q and GluA2N370Q increased in a time-dependent manner. Unlike wild-type (WT) GluA1, GluA2WT rescued surface GluA2N370Q expression. Additionally, the expression of GluA1N63Q reduced the cell surface expression level of GluA1WT. In conclusion, our findings suggest that these N-glycans have distinct roles in the ER-exit of GluA1 and GluA2 homo-oligomers; N-glycan at GluA1N63 is a prerequisite for GluA1 ER-exit, whereas N-glycans at GluA1N363 and GluA2N370 control the ER-exit rate.

The number and subunit compositions of AMPA receptors (AMPARs), hetero- or homotetramers composed of four subunits GluA1-4, in the synapse is carefully tuned to sustain basic synaptic activity. This enables stimulation-induced synaptic plasticity, which is central to learning and memory. The AMPAR tetramers have been widely believed to be stable from their formation in the endoplasmic reticulum until their proteolytic decomposition. However, by observing GluA1 and GluA2 at the level of single molecules, we find that the homo- and heterotetramers are metastable, instantaneously falling apart into monomers, dimers, or trimers (in 100 and 200 ms, respectively), which readily form tetramers again. In the dendritic plasma membrane, GluA1 and GluA2 monomers and dimers are far more mobile than tetramers and enter and exit from the synaptic regions. We conclude that AMPAR turnover by lateral diffusion, essential for sustaining synaptic function, is largely done by monomers of AMPAR subunits, rather than preformed tetramers.

Glucocorticoids (GCs) potently induce T-cell apoptosis in a GC receptor (GR)-dependent manner and are used to control lymphocyte function in clinical practice. However, its downstream pathways remain controversial. Here, we showed that GC-induced transcript 1 (GLCCI1) is a novel downstream molecule of the GC-GR cascade that acts as an antiapoptotic mediator in thymic T cells. GLCCI1 was highly phosphorylated and colocalized with microtubules in GLCCI1-transfected human embryonic kidney QBI293A cells. GR-dependent up-regulation of GLCCI1 was associated with GC-induced proapoptotic events in a cultured thymocyte cell line. However, GLCCI1 knockdown in a thymocyte cell line led to apoptosis. Consistently, transgenic mice overexpressing human GLCCI1 displayed enlarged thymi that consisted of larger numbers of thymocytes. Further molecular characterization showed that GLCCI1 bound to both dynein light chain LC8-type 1 (LC8) and its functional kinase, p21-protein activated kinase 1 (PAK1), thereby inhibiting the kinase activity of PAK1 toward LC8 phosphorylation, a crucial event in apoptotic signaling. GLCCI1 induction facilitated LC8 dimer formation and reduced Bim expression. Thus, GLCCI1 is a candidate factor involved in apoptosis regulation of thymic T cells.-Kiuchi, Z., Nishibori, Y., Kutsuna, S., Kotani, M., Hada, I., Kimura, T., Fukutomi, T., Fukuhara, D., Ito-Nitta, N., Kudo, A., Takata, T., Ishigaki, Y., Tomosugi, N., Tanaka, H., Matsushima, S., Ogasawara, S., Hirayama, Y., Takematsu, H., Yan, K. GLCCI1 is a novel protector against glucocorticoid-induced apoptosis in T cells.

The human natural killer-1 (HNK-1) carbohydrate epitope, composed of a unique sulfated trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-R), is highly expressed during brain development and regulates higher brain function. However, it remains unclear which glycoprotein carries the HNK-1 epitope in the embryonic brain and the functional role it plays. Here, we showed that one of the major HNK-1 carrier proteins in the embryonic brain is tenascin-C (TNC), an extracellular matrix protein that regulates neurite outgrowth by interacting with the GPI-anchored protein contactin-1 (CNTN). Because the alternatively spliced fibronectin type-III (FNIII) repeats in TNC give rise to many isoforms and affect neuronal function, we evaluated neurite outgrowth of primary hippocampal neurons on purified recombinant FNIII repeats with or without the HNK-1 epitope as a substrate. We found that the presence of the HNK-1 epitope on the C domain of TNC promoted neurite outgrowth, and that this signal was mediated by CNTN, which is an HNK-1-expressing neuronal receptor. The neurite-promoting activity of the HNK-1 epitope on TNC required neuronal HNK-1 expression, which was defective in neurons lacking the glucuronyltransferases GlcAT-P and GlcAT-S. These results suggest that the HNK-1 epitope is a key modifier of TNC and CNTN in the regulation of embryonic brain development.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are expressed in various immune cells and most of them carry signaling functions. High-affinity synthetic sialoside ligands have been developed for various Siglecs. Therapeutic potentials of the nanoparticles and compounds that contain multiple numbers of these sialosides and other reagents such as toxins and antigens have been demonstrated. However, whether immune responses can be regulated by monomeric sialoside ligands has not yet been known. CD22 (also known as Siglec-2) is an inhibitory molecule preferentially expressed in B lymphocytes (B cells) and is constitutively bound and functionally regulated by a2,6 sialic acids expressed on the same cell (cis-ligands). Here, we developed synthetic sialosides GSC718 and GSC839 that bind to CD22 with high affinity (IC50 ~100 nM), and inhibit ligand binding of CD22. When B cells are activated by B cell antigen receptor (BCR) ligation, both GSC718 and GSC839 downregulate proliferation of B cells, and this regulation requires both CD22 and α2,6 sialic acids. This result suggests that these sialosides regulate BCR ligation-induced B cell activation by reversing endogenous ligand-mediated regulation of CD22. By contrast, GSC718 and GSC839 augment B cell proliferation induced by TLR ligands or CD40 ligation, and this augmentation requires CD22 but not a2,6 sialic acids. Thus, these sialosides appear to enhance B cell activation by directly suppressing the inhibitory function of CD22 independently of endogenous ligand-mediated regulation. Moreover, GSC839 augments B cell proliferation that depends on both BCR ligation and CD40 ligation as is the case for in vivo B cell responses to antigens, and enhanced antibody production to the extent comparable to CpG oligonuleotides or a small amount of alum. Although these known adjuvants induce production of the inflammatory cytokines or accumulation of inflammatory cells, CD22-binding sialosides do not. Thus, synthetic sialosides that bind to CD22 with high-affinity modulate B cell activation through endogenous ligand-dependent and independent pathways, and carry an adjuvant activity without inducing inflammation.

Cellular translation should be precisely controlled in response to extracellular cues. However, knowledge is limited concerning signal transduction-regulated translation. In the present study, phosphorylation was identified in the 40S small subunit ribosomal protein uS7 (Yjr123w/previously called as Rps5) by Ypk1 and Pkc1, AGC family protein kinases in yeast Saccharomyces cerevisiae. Serine residue 223 (Ser223) of uS7 in the conserved C-terminal region was crucial for this phosphorylation event. S223A mutant uS7 caused severe reduction of small ribosomal subunit production, likely due to compromised interaction with Rio2, resulting in both reduced translation and reduced cellular proliferation. Contrary to optimal culture conditions, heat stressed S223A mutant cells exhibited increased heat resistance and induced heat shock proteins. Taken together, an intracellular signal transduction pathway involving Ypk1/Pkc1 seemed to play an important role in ribosome biogenesis and subsequent cellular translation, utilizing uS7 as a substrate.

Glucose uptake is crucial for providing both an energy source and a signal that regulates cell proliferation. Therefore, it is important to clarify the mechanisms underlying glucose uptake and its transmission to intracellular signaling pathways. In this study, we searched for a novel regulatory factor involved in glucose-induced signaling by using Saccharomyces cerevisiae as a eukaryotic model. Requirement of the extracellular protein Ecm33 in efficient glucose uptake and full activation of the nutrient-responsive TOR kinase complex 1 (TORC1) signaling pathway is shown. Cells lacking Ecm33 elicit a series of starvation-induced pathways even in the presence of extracellular high glucose concentration. This results in delayed cell proliferation, reduced ATP, induction of autophagy, and dephosphorylation of the TORC1 substrates Atg13 and Sch9.

Unbiased transcriptome profiling and functional genomics approaches have identified ubiquitin-specific protease 40 (USP40) as a highly specific glomerular transcript. This gene product remains uncharacterized, and its biological function is completely unknown. Here, we showed that mouse and rat glomeruli exhibit specific expression of the USP40 protein, which migrated at 150 kDa and was exclusively localized in the podocyte cytoplasm of the adult kidney. Double-labeling immunofluorescence staining and confocal microscopy analysis of fetal and neonate kidney samples revealed that USP40 was also expressed in the vasculature, including in glomerular endothelial cells at the premature stage. USP40 in cultured glomerular endothelial cells and podocytes was specifically localized to the intermediate filament protein nestin. In glomerular endothelial cells, immunoprecipitation confirmed actual protein-protein binding of USP40 with nestin, and USP40-small-interfering RNA transfection revealed significant reduction of nestin. In a rat model of minimal-change nephrotic syndrome, USP40 expression was apparently reduced, which was also associated with the reduction of nestin. Zebrafish morphants lacking Usp40 exhibited disorganized glomeruli with the reduction of the cell junction in the endothelium and foot process effacement in the podocytes. Permeability studies in these zebrafish morphants demonstrated a disruption of the selective glomerular permeability filter. These data indicate that USP40/Usp40 is a novel protein that might play a crucial role in glomerulogenesis and the glomerular integrity after birth through the modulation of intermediate filament protein homeostasis.

Unbiased transcriptome profiling and functional genomics approaches have identified ubiquitin-specific protease 40 (USP40) as a highly specific glomerular transcript. This gene product remains uncharacterized, and its biological function is completely unknown. Here, we showed that mouse and rat glomeruli exhibit specific expression of the USP40 protein, which migrated at 150 kDa and was exclusively localized in the podocyte cytoplasm of the adult kidney. Double-labeling immunofluorescence staining and confocal microscopy analysis of fetal and neonate kidney samples revealed that USP40 was also expressed in the vasculature, including in glomerular endothelial cells at the premature stage. USP40 in cultured glomerular endothelial cells and podocytes was specifically localized to the intermediate filament protein nestin. In glomerular endothelial cells, immunoprecipitation confirmed actual protein-protein binding of USP40 with nestin, and USP40-small-interfering RNA transfection revealed significant reduction of nestin. In a rat model of minimal-change nephrotic syndrome, USP40 expression was apparently reduced, which was also associated with the reduction of nestin. Zebrafish morphants lacking Usp40 exhibited disorganized glomeruli with the reduction of the cell junction in the endothelium and foot process effacement in the podocytes. Permeability studies in these zebrafish morphants demonstrated a disruption of the selective glomerular permeability filter. These data indicate that USP40/Usp40 is a novel protein that might play a crucial role in glomerulogenesis and the glomerular integrity after birth through the modulation of intermediate filament protein homeostasis.

All vertebrate cell surfaces display a dense glycan layer often terminated with sialic acids, which have multiple functions due to their location and diverse modifications. The major sialic acids in most mammalian tissues are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), the latter being derived from Neu5Ac via addition of one oxygen atom at the sugar nucleotide level by CMP-Neu5Ac hydroxylase (Cmah). Contrasting with other organs that express various ratios of Neu5Ac and Neu5Gc depending on the variable expression of Cmah, Neu5Gc expression in the brain is extremely low in all vertebrates studied to date, suggesting that neural expression is detrimental to animals. However, physiological exploration of the reasons for this long term evolutionary selection has been lacking. To explore the consequences of forced expression of Neu5Gc in the brain, we have established brain-specific Cmah transgenic mice. Such Neu5Gc overexpression in the brain resulted in abnormal locomotor activity, impaired object recognition memory, and abnormal axon myelination. Brain-specific Cmah transgenic mice were also lethally sensitive to a Neu5Gc-preferring bacterial toxin, even though Neu5Gc was overexpressed only in the brain and other organs maintained endogenous Neu5Gc expression, as in wild-type mice. Therefore, the unusually strict evolutionary suppression of Neu5Gc expression in the vertebrate brain may be explained by evasion of negative effects on neural functions and by selection against pathogens.

Endomitosis is a special type of mitosis in which only cytokinesis-the final step of the cell division cycle-is defective, resulting in polyploid cells. Although endomitosis is biologically important, its regulatory aspects remain elusive. Psychosine, a lysogalactosylceramide, prevents proper cytokinesis when supplemented to proliferating cells. Cytokinetic inhibition by psychosine does not inhibit genome duplication. Consequently cells undergo multiple rounds of endomitotic cell cycles, resulting in the formation of giant multiploid cells. Here we successfully quantified psychosine-triggered multiploid cell formation, showing that membrane sphingolipids ratios modulate psychosine-triggered polyploidy in Namalwa cells. Among enzymes that experimentally remodel cellular sphingolipids, overexpression of glucosylceramide synthase to biosynthesize glycosylsphingolipids (GSLs) and neutral sphingomyelinase 2 to hydrolyze sphingomyelin (SM) additively enhanced psychosine-triggered multiploidy; almost all of the cells became polyploid. In the presence of psychosine, Namalwa cells showed attenuated cell surface SM clustering and suppression of phosphatidylinositol 4,5-bisphosphate production at the cleavage furrow, both important processes for cytokinesis. Depending on the sphingolipid balance between GSLs and SM, Namalwa cells could be effectively converted to viable multiploid cells with psychosine.

Background: Cells have evolved the mechanisms to survive nutritional shortages in the environment. In Saccharomyces cerevisiae, alpha-mannosidase Ams1 is known to play a role in catabolism of N-linked free oligosaccharides in the cytosol. Although, this enzyme is also known to be transported selectively from the cytosol to the vacuoles by autophagy, the physiological significance of this transport has not been clarified. Methods: To elucidate the regulatory mechanism of the activity of Ams1, we assessed the enzymatic activity of the cell free extract of the wild-type and various gene disruptants under different nutritional conditions. In addition, the regulation of Ams1 at both transcription and post-translation was examined. Results: The activity of Ams1 was significantly increased upon the depletion of glucose in the medium. Interestingly, the activity of the enzyme was also stimulated by nitrogen starvation. Our data showed that the activity of Ams1 is regulated by the stress responsive transcriptional factors Msn2/4 through the protein kinase A and the target of rapamycin complex 1 pathways. In addition, Ams1 is post-translationally activated by Pep4-dependent processing in the vacuoles. Conclusion: Yeast cells monitor extracellular nutrients to regulate mannoside catabolism via the cellular signaling pathway. General significance: This study revealed that intracellular Ams1 activity is exquisitely upregulated in response to nutrient starvation by induced expression as well as by Pep4-dependent enhanced activity in the vacuoles. The signaling molecules responsible for regulation of Ams1 were also clarified. (C) 2016 Elsevier B.V. All rights reserved.

Human natural killer-1 (HNK-1) carbohydrate (HSO3-3GlcA beta 1-3Gal beta 1-4GlcNAc-R) is highly expressed in the brain and required for learning and neural plasticity. We previously demonstrated that expression of the HNK-1 epitope is mostly abolished in knockout mice for GlcAT-P (B3gat1), a major glucuronyltransferase required for HNK-1 biosynthesis, but remained in specific regions such as perineuronal nets (PNNs) in these mutant mice. Considering PNNs are mainly composed of chondroitin sulfate proteoglycans (CSPGs) and regulate neural plasticity, GlcAT-P-independent expression of HNK-1 in PNNs is suggested to play a role in neural plasticity. However, the function, structure, carrier glycoprotein and biosynthetic pathway for GlcAT-P-irrelevant HNK-1 epitope remain unclear. In this study, we identified a unique HNK-1 structure on aggrecan in PNNs. To determine the biosynthetic pathway for the novel HNK-1, we generated knockout mice for GlcAT-S (B3gat2), the other glucuronyltransferase required for HNK-1 biosynthesis. However, GlcAT-P and GlcAT-S double-knockout mice did not exhibit reduced HNK-1 expression compared with single GlcAT-P-knockout mice, indicating an unusual biosynthetic pathway for the HNK-1 epitope in PNNs. Aggrecan was purified from cultured cells in which GlcAT-P and -S are not expressed and we determined the structure of the novel HNK-1 epitope using liquid chromatography/mass spectrometry (LC/MS) as a sulfated linkage region of glycosaminoglycans (GAGs), HSO3-GlcA-Gal-Gal-Xyl-R. Taken together, we propose a hypothetical model where GlcAT-I, the sole glucuronyltransferase required for synthesis of the GAG linkage, is also responsible for biosynthesis of the novel HNK-1 on aggrecan. These results could lead to discovery of new roles of the HNK-1 epitope in neural plasticity.

The AMPA-type glutamate receptor (AMPAR), which is a tetrameric complex composed of four subunits (GluA1-4) with several combinations, mediates the majority of rapid excitatory synaptic transmissions in the nervous system. Cell surface expression levels of AMPAR modulate synaptic plasticity, which is considered one of the molecular bases for learning and memory formation. To date, a unique trisaccharide (HSO3-3GlcA beta 1-3Gal beta 1-4GlcNAc), human natural killer-1 (HNK-1) carbohydrate, was found expressed specifically on N-linked glycans of GluA2 and regulated the cell surface expression of AMPAR and the spine maturation process. However, evidence that the HNK-1 epitope on N-glycans of GluA2 directly affects these phenomena is lacking. Moreover, it is thought that other N-glycans on GluA2 also have potential roles in the regulation of AMPAR functions. In the present study, using a series of mutants lacking potential N-glycosylation sites (N256, N370, N406, and N413) within GluA2, we demonstrated that the mutant lacking the N-glycan at N370 strongly suppressed the intracellular trafficking of GluA2 from the endoplasmic reticulum (ER) in HEK293 cells. Cell surface expression of GluA1, which is a major subunit of AMPAR in neurons, was also suppressed by co-expression of the GluA2 N370S mutant. The N370S mutant and wild-type GluA2 were co-immunoprecipitated with GluA1, suggesting that N370S was properly associated with GluA1. Moreover, we found that N413 was the main potential site of the HNK-1 epitope that promoted the interaction of GluA2 with N-cadherin, resulting in enhanced cell surface expression of GluA2. The HNK-1 epitope on N-glycan at the N413 of GluA2 was also involved in the cell surface expression of GluA1. Thus, our data suggested that site-specific N-glycans on GluA2 regulate the intracellular trafficking and cell surface expression of AMPAR.

Autophagy occurs in human neutrophils after the phagocytosis of multidrug-resistant bacteria and drug-sensitive strains, including Escherichia coli and Pseudomonas aeruginosa. The present study detected autophagy by immunoblot analysis of LC3B conversion, by confocal scanning microscopic examination of LC3B aggregate formation and by transmission electron microscopic examination of bacteria-containing autophagosomes. Patients with severe bacterial infections are often treated with IVIG alongside antimicrobial agents. Here, we showed that IVIG induced neutrophil-mediated phagocytosis of multidrug-resistant strains. Compared with untreated neutrophils, neutrophils exposed to IVIG showed increased levels of bacterial cell killing, phagocytosis, O-2(-) release, MPO release, and NET formation. IVIG also increased autophagy in these cells. Inhibiting the late phase of autophagy (fusion of lysosomes with autophagosomes) with bafilomycin A1-reduced, neutrophil-mediated bactericidal activity. These findings indicate that autophagy plays a critical role in the bactericidal activity mediated by human neutrophils. Furthermore, the autophagosomes within the neutrophils contained bacteria only and their organelles only, or both bacteria and their organelles, a previously undocumented observation. Taken together, these results suggest that the contents of neutrophil autophagosomes may be derived from specific autophagic systems, which provide the neutrophil with an advantage. Thus, IVIG promotes the neutrophil-mediated killing of multidrug-resistant bacteria as well as drug-sensitive strains.

Aberrant glycosylation of dystroglycan causes congenital muscular dystrophies associated with cobblestone lissencephaly, classified as dystroglycanopathy. However, pathological features in the onset of brain malformations, including the precise timing and primary cause of the pial basement membrane disruption and abnormalities in the migration of pyramidal neurons, remain unexplored. Using the Pomgnt2-knockout (KO) mouse as a dystroglycanopathy model, we show that breaches of the pial basement membrane appeared at embryonic day 11.5, coinciding with the ectopic clustering of Cajal-Retzius cells and subplate neurons and prior to the migration onset of pyramidal neurons. Furthermore, in the Pomgnt2-KO cerebral cortex, preplate splitting failure likely occurred due to the aggregation of Cajal-Retzius and subplate cells, and migrating pyramidal neurons lost polarity and radial orientation. Our findings demonstrate the initial pathological events in dystroglycanopathy mice and contribute to our understanding of how dystroglycan dysfunction affects brain development and progresses to cobblestone lissencephaly.

Cells manage the expression of their genomic contents to cope with their needs. In multicellular organisms, different cell types result from differential usage of the genome. In addition, cell surface glycans are often cell type specific and the mechanisms regulating glycan expression are controlled by gene expression, just like other cellular phenotypes. Here, a recently developed method for assessing the glycan-related genes that regulate cell surface glycans will be described. This method, called correlation index-based responsible enzyme gene screening (CIRES), uses statistical analysis of quantitative transcriptome data and glycan expression. Not only is this method useful for identifying the genes that positively regulate glycan expression, but CIRES can also systematically identify genes that negatively regulate glycan expression. CIRES involves three steps: (1) obtaining a glycan expression profile in several cell lines and gene expression profiles of glycan-related genes in the same set of cells, (2) correlating glycan and gene expression, and (3) evaluating the correlations in a gene perturbation study. Using this technique, one can determine the genes that quantitatively regulate the expression of cell surface glycans. This method complements conventional loss-of-function experiments, which can identify the exclusiveness of the gene for glycan expression.

Sphingolipids are a family of eukaryotic lipids biosynthesized from sphingoid long-chain bases (LCBs). Sphingolipids are an essential class of lipids, as their depletion results in cell death. However, acute LCB supplementation is also toxic; thus, proper cellular LCB levels should be maintained. To characterize the "sphingolipid-signaling intercross," we performed a kinome screening assay in which budding yeast protein kinase-knockout strains were screened for resistance to ISP-1, a potent inhibitor of LCB biosynthesis. Here, one pair of such DIR (deletion-mediated ISP-1 resistance) genes, FPK1 and FPK2, was further characterized. Cellular LCB levels increased in the fpk1/2 Delta strain, which was hypersensitive to phytosphingosine (PHS), a major LCB species of yeast cells. Concomitantly, this strain acquired resistance to ISP-1. Fpk1 and Fpk2 were involved in two downstream events; that is, ISP-1 uptake due to aminophospholipid flippase and LCB degradation due to LCB4 expression. RSK3, which belongs to the p90-S6K subfamily, was identified as a functional counterpart of Fpk1/2 in mammalian cells as the RSK3 gene functionally complemented the ISP-1-resistant phenotype of fpk1/2 Delta cells.

The human natural killer-1 (HNK-1) carbohydrate comprising a sulfated trisaccharide (HSO3-3GlcA beta 1-3Gal beta 1-4GlcNAc-) is expressed on N-linked and O-mannose-linked glycans in the nervous system and involved in learning and memory functions. Although whole/core glycan structures and carrier glycoproteins for the N-linked HNK-1 epitope have been studied, carrier glycoproteins and the biosynthetic pathway of the O-mannose-linked HNK-1 epitope have not been fully characterized. Here, using mass spectrometric analyses, we identified the major carrier glycoprotein of the O-linked HNK-1 as phosphacan in developing mouse brains and determined the major O-glycan structures having the terminal HNK-1 epitope from partially purified phosphacan. The O-linked HNK-1 epitope on phosphacan almost disappeared due to the knockout of protein O-mannose beta 1,2-N-acetylglucosaminyltransferase 1, an N-acetylglucosaminyltransferase essential for O-mannose-linked glycan synthesis, indicating that the reducing terminal of the O-linked HNK-1 is mannose. We also showed that glucuronyltransferase-P (GlcAT-P) was involved in the biosynthesis of O-mannose-linked HNK-1 using the gene-deficient mice of GlcAT-P, one of the glucuronyltransferases for HNK-1 synthesis. Consistent with this result, we revealed that GlcAT-P specifically synthesized O-linked HNK-1 onto phosphacan using cultured cells. Furthermore, we characterized the as-yet-unknown epitope of the 6B4 monoclonal antibody (mAb), which was thought to recognize a unique phosphacan glycoform. The reactivity of the 6B4 mAb almost completely disappeared in GlcAT-P-deficient mice, and exogenously expressed phosphacan was selectively recognized by the 6B4 mAb when co-expressed with GlcAT-P, suggesting that the 6B4 mAb preferentially recognizes O-mannose-linked HNK-1 on phosphacan. This is the first study to show that 6B4 mAb-reactive O-mannose-linked HNK-1 in the brain is mainly carried by phosphacan.

Background: Sialic acids play key roles in molecular recognition. Results: T-cell activation alters the principal sialic acid species profile, regulating expression of siglec ligands, T-cell activation per se, and T cell-B cell interactions. Conclusion: This activation-dependent change in the sialoglycan profile modulates immune responses. Significance: Pronounced changes in the sialoglycan profile not only serve as cellular markers but also reflect cellular functionality. Sialic acids (Sias) are often conjugated to the termini of cellular glycans and are key mediators of cellular recognition. Sias are nine-carbon acidic sugars, and, in vertebrates, the major species are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), differing in structure at the C5 position. Previously, we described a positive feedback loop involving regulation of Neu5Gc expression in mouse B cells. In this context, Neu5Gc negatively regulated B-cell proliferation, and Neu5Gc expression was suppressed upon activation. Similarly, resting mouse T cells expressed principally Neu5Gc, and Neu5Ac was induced upon activation. In the present work, we used various probes to examine sialoglycan expression by activated T cells in terms of the Sia species expressed and the linkages of Sias to glycans. Upon T-cell activation, sialoglycan expression shifted from Neu5Gc to Neu5Ac, and the linkage shifted from 2,6 to 2,3. These changes altered the expression levels of sialic acid-binding immunoglobulin-like lectin (siglec) ligands. Expression of sialoadhesin and Siglec-F ligands increased, and that of CD22 ligands decreased. Neu5Gc exerted a negative effect on T-cell activation, both in terms of the proliferative response and in the context of activation marker expression. Suppression of Neu5Gc expression in mouse T and B cells prevented the development of nonspecific CD22-mediated T cell-B cell interactions. Our results suggest that an activation-dependent shift from Neu5Gc to Neu5Ac and replacement of 2,6 by 2,3 linkages may regulate immune cell interactions at several levels.

Sphingoid intermediates accumulate in response to a variety of stresses, including heat, and trigger cellular responses. However, the mechanism by which stress affects sphingolipid biosynthesis has yet to be identified. Recent studies in yeast suggest that sphingolipid biosynthesis is regulated through phosphorylation of the Orm proteins, which in humans are potential risk factors for childhood asthma. Here we demonstrate that Orm phosphorylation status is highly responsive to sphingoid bases. We also demonstrate, by monitoring temporal changes in Orm phosphorylation and sphingoid base production in cells inhibited for yeast protein kinase 1 (Ypk1) activity, that Ypk1 transmits heat stress signals to the sphingolipid biosynthesis pathway via Orm phosphorylation. Our data indicate that heat-induced sphingolipid biosynthesis in turn triggers Orm protein dephosphorylation, making the induction transient. We identified Cdc55-protein phosphatase 2A (PP2A) as a key phosphatase that counteracts Ypk1 activity in Orm-mediated sphingolipid biosynthesis regulation. In total, our study reveals a mechanism through which the conserved Pkh-Ypk kinase cascade and Cdc55-PP2A facilitate rapid, transient sphingolipid production in response to heat stress through Orm protein phosphoregulation. We propose that this mechanism serves as the basis for how Orm phosphoregulation controls sphingolipid biosynthesis in response to stress in a kinetically coupled manner.

CD22 (Siglec-2) is a B-cell membrane-bound lectin that recognizes glycan ligands containing a2,6-linked sialic acid (a2,6Sia) and negatively regulates signaling through the B-cell Ag receptor (BCR). Although CD22 has been investigated extensively, its precise function remains unclear due to acting multiple phases. Here, we demonstrate that CD22 is efficiently activated in trans by complexes of Ag and soluble IgM (sIgM) due to the presence of glycan ligands on sIgM. This result strongly suggests sIgM as a natural trans ligand for CD22. Also, CD22 appears to serve as a receptor for sIgM, which induces a negative feedback loop for B-cell activation similar to the Fc receptor for IgG (Fc?RIIB).

Cellular biosynthesis of macromolecules often involves highly branched enzyme pathways, thus cellular regulation of such pathways could be rather difficult. To understand the regulatory mechanism, a systematic approach could be useful. We genetically analyzed a branched biosynthetic pathway for glycosphingolipid (GSL) GM1 using correlation index-based responsible enzyme gene screening (CIRES), a novel quantitative phenotype-genotype correlation analysis. CIRES utilizes transcriptomic profiles obtained from multiple cells. Among a panel of B cell lines, expression of GM1 was negatively correlated with and suppressed by gene expression of CD77 synthase (CD77Syn), whereas no significant positive correlation was found for enzymes actually biosynthesizing GM1. Unexpectedly, a GM1-suppressive phenotype was also observed in the expression of catalytically inactive CD77Syn, ruling out catalytic consumption of lactosylceramide (LacCer) as the main cause for such negative regulation. Rather, CD77Syn seemed to limit other branching reaction(s) by targeting LacCer synthase (LacCer-Syn), a proximal enzyme in the pathway, because they were closely localized in the Golgi apparatus and formed a complex. Moreover, turnover of LacCerSyn was accelerated upon CD77Syn expression to globally change the GSL species expressed. Collectively, these data suggest that transcriptomic assessment of macromolecule biosynthetic pathways can disclose a global regulatory mechanism(s) even when unexpected.

「冬虫夏草」由来の免疫抑制物質ISP-1をリード化合物として合成されたFTY720は，昨年米国やロシアで自己免疫疾患の一種である多発性硬化症の治療薬「Gilenya」として承認された．ISP-1やFTY720の免疫抑制作用にはスフィンゴ脂質が深く関与していることが知られている．ここでは，ISP-1やFTY720の作用機構について，スフィンゴ脂質の関与を中心に解説する．

In earlier studies, we identified the C-9 amido derivative 1 (9-(4'-hydroxy-4-biphenyl)acetamido-9-deoxy-Neu5Gc alpha 2-6GalOMP) and the C-9 amino derivative 2 (9-(4'-hydroxy-4-biphenyl)methylamino-9-deoxy-Neu5Gc alpha 2-6GalOMP) have the most promising affinity for mouse CD22 and human CD22, respectively. Replacing the subterminal galactose residue (2-6Gal-OMP) of 1 with benzyl (5) or biphenylmethyl (6) as aglycone led to even higher potency for mCD22. In this study, both compounds showed improved potency and selectivity for CD22 (IC(50) 70 nM) and 712-fold more selective for CD22 than for MAG. The corresponding derivatives of 2, compounds 8 and 9, showed comparable activity to 2 but lower potency and selectivity than 5 and 6. Although compounds 5-9 are simple and small molecular weight antagonists, they showed much high potency and selectivity than the corresponding compounds having a 2-6Gal linkage. Both biological and computational docking simulation studies suggest that the 2-6GalOMP residues of 1 and 2 are not critical for binding process and could be replaced with hydrophobic non-carbohydrate moieties. The data presented herein has significant implications for the design and discovery of next-generation CD22-antagonists. (C) 2011 Elsevier Ltd. All rights reserved.

The discovery of novel bioactive molecules advances our systems-level understanding of biological processes and is crucial for innovation in drug development. For this purpose, the emerging field of chemical genomics is currently focused on accumulating large assay data sets describing compound-protein interactions (CPIs). Although new target proteins for known drugs have recently been identified through mining of CPI databases, using these resources to identify novel ligands remains unexplored. Herein, we demonstrate that machine learning of multiple CPIs can not only assess drug polypharmacology but can also efficiently identify novel bioactive scaffold-hopping compounds. Through a machine-learning technique that uses multiple CPIs, we have successfully identified novel lead compounds for two pharmaceutically important protein families, G-protein-coupled receptors and protein kinases. These novel compounds were not identified by existing computational ligand-screening methods in comparative studies. The results of this study indicate that data derived from chemical genomics can be highly useful for exploring chemical space, and this systems biology perspective could accelerate drug discovery processes. Molecular Systems Biology 7: 472; published online 1 March 2011; doi:10.1038/msb.2011.5

Sphingolipids act as signaling mediators that regulate a diverse range of cellular events. Although numerous sphingolipid functions have been studied, little is known about the effect of sphingolipids on monocyte differentiation into macrophages. Here, we report that two lysosphingolipids, sphingosylphosphorylcholine (SPC) and lysosulfatide (LSF), inversely affect macrophagic differentiation of monocytic cell lines, U937 and THP-1. Molecular analyses revealed that SPC enhances, whereas LSF suppresses, phorbol ester-induced classical (M1-polarized) differentiation to macrophages. The expression of CD11b, a macrophage marker, was induced in accordance with the activation status of the Raf/MEK/ERK signaling pathway in which SPC and LSF had opposite effects. Pharmacological inhibition of this pathway aborted the differentiation, indicating that this signaling pathway is required. Consistently, SPC promoted, while LSF inhibited, monocyte adhesion to fibronectin, through the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway. The effects of SPC on Raf/MEK/ERK and PI3K/Akt signaling were dependent on G(i/o), whereas the SPC-induced calcium influx was dependent on G(q). Thus SPC utilizes G-protein coupled receptor. In contrast, the effects of LSF were independent of G(i/o) and G(q). These results suggest that SPC enhances, whereas LSF suppresses, monocyte differentiation into macrophages through regulating the Raf/MEK/ERK and PI3K/Akt signaling pathways via distinct mechanisms. (C) 2010 Elsevier Inc. All rights reserved.

Nitrogen starvation-mediated reduction of Ypk1 is suggested to suppress translational initiation, possibly in parallel with the target of rapamycin complex 1 (TORC1) signaling. However, the molecular mechanism that regulates Ypk1 in nitrogen-starved cells is poorly understood. Here we report that Ypk1 is a novel selective substrate for nitrogen starvation-triggered proteolysis requiring autophagy system. Among various nutrient starvation methods used to elicit autophagy, rapid Ypk1 degradation was specific to nitrogen starvation. In screening genes required for such nitrogen starvation-specific vacuolar proteolysis, we found that autophagy-related degradation of Ypk1 depended on the endosomal sorting complex required for transport (ESCRT) machinery, which is conventionally thought to function in endosomal trafficking. In microscopic analyses, the disruption of ESCRT subunits resulted in the accumulation of both Ypk1 and autophagosomal Atg8 at a perivacuolar site that was distinct from conventional endosomes. ESCRT machinery was not involved in autophagic flux induced by the TORC1 inhibitor rapamycin, thus suggesting that ESCRT represents an exclusive mechanism of nitrogen starvation-specific proteolysis of Ypk1. Overall, we propose a novel regulation of Ypk1 that is specific to nitrogen limitation.

Siglec-2 is a mammalian sialic acid binding protein expressed on B-cell surfaces and is involved in the modulation of B-cell mediated immune response. We synthesized a unique starfish ganglioside, AG2 pentasaccharide Galf beta(1-3)Galp alpha(1-4)Neu5Ac alpha(2-3)Galp beta(1-4)Glcp, and found that the synthetic pentasaccharide binds to human Siglec-2 by performing (1)H NMR experiments. Saturation transfer difference NMR experiments indicated that the C7-C9 side-chain and the acetamide moiety of the central sialic acid residue were located in the binding face of human Siglec-2. We determined the binding epitope of AG2 pentasaccharide to human Siglec-2, as the Galp alpha(1-4)Neu5Ac alpha(2-3)Galp unit. (C) 2010 Elsevier Ltd. All rights reserved.

By use of a mouse mucosal immunization model, LT-IIb(T13I), a nontoxic mutant type II heat-labile enterotoxin, was shown to have potent mucosal and systemic adjuvant properties. In contrast to LT-IIb, which binds strongly to ganglioside receptors decorated with either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc), LT-IIb(T13I) binds NeuAc gangliosides much less well. Rather, LT-IIb(T13I) binds preferentially to NeuGc gangliosides. To determine if the adjuvant properties of LT-IIb(T13I) are altered in the absence of NeuGc ganglioside receptors, experiments were conducted using a Cmah-null mouse line which is deficient in the synthesis of NeuGc gangliosides. Several immunomodulatory properties of LT-IIb(T13I) were shown to be dependent on NeuGc gangliosides. LT-IIb(T13I) had reduced binding activity for NeuGc-deficient B cells and macrophages; binding to NeuGc-deficient T cells and dendritic cells (DC) was essentially undetectable. Treatment of Cmah-null macrophages with LT-IIb(T13I), however, upregulated the transcription of interleukin-4 (IL-4), IL-6, IL-17, and gamma interferon (IFN-gamma), four cytokines important for promoting immune responses. The production of mucosal IgA and serum IgG against an immunizing antigen was augmented in NeuGc-deficient mice administered LT-IIb(T13I) as a mucosal adjuvant. Notably, NeuGc gangliosides are not expressed in humans. Still, treatment of human monocytes with LT-IIb(T13I) induced the secretion of IL-6, an inflammatory cytokine that mediates differential control of leukocyte activation. These results suggested that NeuAc gangliosides are sufficient to mediate the immunomodulatory properties of LT-IIb(T13I) in mice and in human cells. The nontoxic mutant enterotoxin LT-IIb(T13I), therefore, is potentially a new and safe human mucosal adjuvant.

The generation of pigs devoid of Gal alpha 1,3Gal beta 1,4GlcNAc (Gal) residues has stimulated interest in non-Gal Ags as potentially important targets for Ab binding leading to rejection of pig organ xenografts in humans. Although N-glycolylneuraminic acid (NeuGc) epitopes, which are widely expressed on the endothelial cells of all mammals except humans, are likely targets of anti-non-Gal Abs, this aspect has not been investigated intensively owing to the absence of an appropriate animal model. In this study, we used CMAH(-/-) mice, which are completely deficient in NeuGc and thus produce anti-NeuGc Abs. Sera obtained from CMAH(-/-) mice and healthy human volunteers having anti-NeuGc Abs initiated complement-mediated lysis against CMAH(+/+) cells in vitro. The cytotoxic activity of anti-NeuGc Abs was also determined in vivo (i.e., NeuGc-expressing CMAH(+/+) mouse splenocytes that had been i.v. injected were completely eliminated in syngeneic CMAH(-/-) mice). CMAH(-/-) mice rejected the islets transplanted from syngeneic CMAH(+/+) mice. Thus, the anti-NeuGc Ab-mediated response may be crucially involved in xenograft loss. This is the first direct demonstration of the immunogenic property of NeuGc determinants as targets of the corresponding Abs in CMAH(+/+)-to-CMAH(-/-) transplantation setting. The Journal of Immunology, 2010, 184: 3269-3275.

Our previous study revealed that compound 1 (9-(4'-hydroxy-4-biphenyl)acetamido-9-deoxy-Neu5Gc alpha 2-6GalOMP) has the most promising affinity for mCD22. Replacing the subterminal galactose residue of 1 with benzyl or biphenylmethyl as aglycone led to 38- and 20-fold higher potency, respectively. This discovery represents a new direction in inhibitor design suitable for pharmaceutical development. (C) 2009 Elsevier Ltd. All rights reserved.

Biotin conjugate of the most potent ligand for mouse CD22 was designed and synthesized. The key synthetic step involved the dual capability of Hanessian reaction for debenzylation and simultaneous conversion of the anomeric p-methoxyphenyl group into the corresponding phenylthioglycoside in one step. Competition enzyme-linked immunosorbent assay (ELISA) for testing the binding affinity of synthetic sialosides was developed based on this biotinylated ligand. (C) 2009 Elsevier Ltd. All rights reserved.