Ribosomal protein uS7/Rps5 serine-223 in protein kinase-mediated phosphorylation and ribosomal small subunit maturation.

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.

Psychosine-triggered endomitosis is modulated by membrane sphingolipids through regulation of phosphoinositide 4,5-bisphosphate production at the cleavage furrow.

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.

Fpk1/2 kinases regulate cellular sphingoid long-chain base abundance and alter cellular resistance to LCB elevation or depletion.

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∆ 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∆ cells.

Functional evaluation of activation-dependent alterations in the sialoglycan composition of T cells.

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 α2,6 to α2,3. 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 α2,6 by α2,3 linkages may regulate immune cell interactions at several levels.

In vivo role of aldehyde reductase.

BACKGROUND: Aldehyde reductase (AKR1A; EC 1.1.1.2) catalyzes the reduction of various types of aldehydes. To ascertain the physiological role of AKR1A, we examined AKR1A knockout mice. METHODS: Ascorbic acid concentrations in AKR1A knockout mice tissues were examined, and the effects of human AKR1A transgene were analyzed. We purified AKR1A and studied the activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis. Metabolomic analysis and DNA microarray analysis were performed for a comprehensive study of AKR1A knockout mice. RESULTS: The levels of ascorbic acid in tissues of AKR1A knockout mice were significantly decreased which were completely restored by human AKR1A transgene. The activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis, were suppressed in AKR1A knockout mice. The accumulation of d-glucuronic acid and saccharate in knockout mice tissue and the expression of acute-phase proteins such as serum amyloid A2 are significantly increased in knockout mice liver. CONCLUSIONS: AKR1A plays a predominant role in the reduction of both d-glucuronic acid and d-glucurono-γ-lactone in vivo. The knockout of AKR1A in mice results in accumulation of d-glucuronic acid and saccharate as well as a deficiency of ascorbic acid, and also leads to upregulation of acute phase proteins. GENERAL SIGNIFICANCE: AKR1A is a major enzyme that catalyzes the reduction of d-glucuronic acid and d-glucurono-γ-lactone in vivo, besides acting as an aldehyde-detoxification enzyme. Suppression of AKR1A by inhibitors, which are used to prevent diabetic complications, may lead to the accumulation of d-glucuronic acid and saccharate.

Orm protein phosphoregulation mediates transient sphingolipid biosynthesis response to heat stress via the Pkh-Ypk and Cdc55-PP2A pathways.

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.

Induction of feline immunodeficiency virus from a chronically infected feline T-lymphocyte cell line.

The infection of the feline T-lymphocyte cell line FeT-J with the feline immunodeficiency virus (FIV) Petaluma strain led to the establishment of nonvirus-producing cells. One clone (C15) obtained by limiting dilution was found to express FIV in response to chemical inducers of retroviruses. The chemical treatment of C15 cells led to not only FIV protein synthesis but also an augmentation of viral production. Examination of the C15 cell derivatives obtained by recloning revealed that 10-40% of treated cells constitutively expressed FIV antigens, whereas 100% with expressed FIV antigen in response to the inducer. Chemical induction resulted in more than a 100-fold increase in infectious viral production. The results suggest that a majority of FeT-J cells that are infected with FIV exist in a non-productive state. Establishing a cell line that can be non-productively infected by FIV may help determine the mechanisms of FIV latency.

CD22 serves as a receptor for soluble IgM.

CD22 (Siglec-2) is a B-cell membrane-bound lectin that recognizes glycan ligands containing α2,6-linked sialic acid (α2,6Sia) 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 (FcγRIIB).

Quantitative transcriptomic profiling of branching in a glycosphingolipid biosynthetic pathway.

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 (LacCerSyn), 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.

CD22-antagonists with nanomolar potency: the synergistic effect of hydrophobic groups at C-2 and C-9 of sialic acid scaffold.

In earlier studies, we identified the C-9 amido derivative 1 (9-(4'-hydroxy-4-biphenyl)acetamido-9-deoxy-Neu5Gcα2-6GalOMP) and the C-9 amino derivative 2 (9-(4'-hydroxy-4-biphenyl)methylamino-9-deoxy-Neu5Gcα2-6GalOMP) 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 α 2-6Gal linkage. Both biological and computational docking simulation studies suggest that the 2-6Gal-OMP 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.

Sphingosylphosphorylcholine and lysosulfatide have inverse regulatory functions in monocytic cell differentiation into macrophages.

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.

Identification of Ypk1 as a novel selective substrate for nitrogen starvation-triggered proteolysis requiring autophagy system and endosomal sorting complex required for transport (ESCRT) machinery components.

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.

Synthesis and binding analysis of unique AG2 pentasaccharide to human Siglec-2 using NMR techniques.

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 Galfbeta(1-3)Galpalpha(1-4)Neu5Acalpha(2-3)Galpbeta(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 Galpalpha(1-4)Neu5Acalpha(2-3)Galp unit.

Anomalous expression of Thy1 (CD90) in B-cell lymphoma cells and proliferation inhibition by anti-Thy1 antibody treatment.

The anti-CD20 monoclonal antibody (Ab) rituximab is accepted to be an effective therapeutic Ab for malignant B-cell lymphoma; however, discovery of other cell surface antigens is required for the option of antibody medicine. Considering that many tumor-associated antigens are glycans, we have searched glycoconjugates for the candidate antigens that therapeutic Abs target. To this end, we first focused on the difference in the glycogenes expression in terms of Epstein-Barr virus (EBV) infection of a Burkitt's lymphoma cell line, Akata. Using DNA array, flow cytometry and Western blotting, we found that Thy1 was highly expressed in EBV-positive Akata cells. Subsequently, Thy1 was found to be expressed in other B-cell lymphoma cell lines: BJAB, MutuI, and MutuIII, irrespective of EBV infection. Treatment of these cells with an anti-Thy1 monoclonal antibody inhibited proliferation more strongly than the therapeutic Ab rituximab. The B-cell lymphoma cell lines were classified based on the extent of the proliferation inhibition, which was not correlated with the expression level of Thy1. It is suggested that stable residence of receptor tyrosine kinases in lipid rafts sustains cell growth in B-cell lymphoma cells.

Immunological property of antibodies against N-glycolylneuraminic acid epitopes in cytidine monophospho-N-acetylneuraminic acid hydroxylase-deficient mice.

The generation of pigs devoid of Galalpha1,3Galbeta1,4GlcNAc (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.

Differential CXCR4 expression and function in subpopulations of the feline lymphoma cell line 3201 susceptible to feline immunodeficiency virus.

The infection of feline thymic lymphoma 3201 cells with a cell culture-adapted Petaluma strain of feline immunodeficiency virus (FIV) led to the establishment of survivor cells designated as 3201-S after a productive infection associated with extensive cell killing. 3201-S cells were free of FIV DNA, and were found to express CXCR4, a coreceptor for infection but not CD134, a primary receptor. When 3201-S cells were reinfected with FIV, viral DNA was transiently detectable for 5 days postinfection, indicating that 3201-S cells cannot support the FIV replicative cycle. Furthermore, comparative studies found that in contrast to SDF-1alpha-responsive 3201 cells, 3201-S cells did not show a flux of Ca(2+) in response to SDF-1alpha, implying that CXCR4 is not functionally active on 3201-S cells. These results suggest that 3201 cells can be heterogeneous in the phenotype of the CXCR4 expressed, and this heterogeneity may account for the differences in susceptibility to FIV. Determining the mechanism(s) within 3201-S cells that restrict FIV could result in therapeutic strategies against FIV infection.

Protective effect of N-glycan bisecting GlcNAc residues on beta-amyloid production in Alzheimer's disease.

Alteration of glycoprotein glycans often changes various properties of the target glycoprotein and contributes to a wide variety of diseases. Here, we focused on the N-glycans of amyloid precursor protein whose cleaved fragment, beta-amyloid, is thought to cause much of the pathology of Alzheimer's disease (AD). We previously determined the N-glycan structures of normal and mutant amyloid precursor proteins (the Swedish type and the London type). In comparison with normal amyloid precursor protein, mutant amyloid precursor proteins had higher contents of bisecting GlcNAc residues. Because N-acetylglucosaminyltransferase III (GnT-III) is the glycosyltransferase responsible for synthesizing a bisecting GlcNAc residue, the current report measured GnT-III mRNA expression levels in the brains of AD patients. Interestingly, GnT-III mRNA expression was increased in AD brains. Furthermore, beta-amyloid treatment increased GnT-III mRNA expression in Neuro2a mouse neuroblastoma cells. We then examined the influence of bisecting GlcNAc on the production of beta-amyloid. Both beta-amyloid 40 and beta-amyloid 42 were significantly decreased in GnT-III-transfected cells. When secretase activities were analyzed in GnT-III transfectant cells, alpha-secretase activity was increased. Taken together, these results suggest that upregulation of GnT-III in AD brains may represent an adaptive response to protect them from additional beta-amyloid production.

Potent small molecule mouse CD22-inhibitors: exploring the interaction of the residue at C-2 of sialic acid scaffold.

Our previous study revealed that compound 1 (9-(4'-hydroxy-4-biphenyl)acetamido-9-deoxy-Neu5Gcalpha2-6GalOMP) 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.

Tropoelastin regulates chemokine expression in fibroblasts in Costello syndrome.

Costello syndrome is a multiple congenital anomaly associated with growth and mental retardation, cardiac and skeletal anomalies, and a predisposition to develop neoplasia. Comprehensive expression analysis revealed remarkable up-regulation of several cytokines and chemokines including Gro family proteins, interleukin-1beta (IL-1beta), IL-8 and MCP-1 but down-regulation of extracellular matrix components including collagens and proteoglycans of skin fibroblasts derived from a Japanese Costello syndrome patient characterized by significantly reduced tropoelastin mRNA, impaired elastogenesis and enhanced cell proliferation. In contrast, decreases in these chemokines and IL-1beta expression were observed in Costello fibroblastic cell lines stably expressing the bovine tropoelastin (btEln) gene and in restored elastic fibers. These results strongly suggest that the human TE gene (ELN) transfer could be applicable for the gene therapy of a group of Costello syndrome patients with reduced ELN gene expression.