Hiromu Takematsu

N-Glycolylneuraminic Acid Deficiency in Mice: Implications for Human Biology and Evolution

ABSTRACT Humans and chimpanzees share >99% identity in most proteins. One rare difference is a human-specific inactivating deletion in the CMAH gene, which determines biosynthesis of the sialic acid N-glycolylneuraminic acid (Neu5Gc). Since Neu5Gc is prominent on most chimpanzee cell surfaces, this mutation could have affected multiple systems. However, Neu5Gc is found in human cancers and fetuses and in trace amounts in normal human tissues, suggesting an alternate biosynthetic pathway. We inactivated the mouse Cmah gene and studied the in vivo consequences. There was no evidence for an alternate pathway in normal, fetal, or malignant tissue. Rather, null fetuses accumulated Neu5Gc from heterozygous mothers and dietary Neu5Gc was incorporated into oncogene-induced tumors. As with humans, there were accumulation of the precursor N-acetylneuraminic acid and increases in sialic acid O acetylation. Null mice showed other abnormalities reminiscent of the human condition. Adult mice showed a diminished acoustic startle response and required higher acoustic stimuli to increase responses above the baseline level. In this regard, histological abnormalities of the inner ear occurred in older mice, which had impaired hearing. Adult animals also showed delayed skin wound healing. Loss of Neu5Gc in hominid ancestors ∼2 to 3 million years ago likely had immediate and long-term consequences for human biology.

Germinal Center Marker GL7 Probes Activation-Dependent Repression of N-Glycolylneuraminic Acid, a Sialic Acid Species Involved in the Negative Modulation of B-Cell Activation

ABSTRACT Sialic acid (Sia) is a family of acidic nine-carbon sugars that occupies the nonreducing terminus of glycan chains. Diversity of Sia is achieved by variation in the linkage to the underlying sugar and modification of the Sia molecule. Here we identified Sia-dependent epitope specificity for GL7, a rat monoclonal antibody, to probe germinal centers upon T cell-dependent immunity. GL7 recognizes sialylated glycan(s), the α2,6-linked N-acetylneuraminic acid (Neu5Ac) on a lactosamine glycan chain(s), in both Sia modification- and Sia linkage-dependent manners. In mouse germinal center B cells, the expression of the GL7 epitope was upregulated due to the in situ repression of CMP-Neu5Ac hydroxylase (Cmah), the enzyme responsible for Sia modification of Neu5Ac to Neu5Gc. Such Cmah repression caused activation-dependent dynamic reduction of CD22 ligand expression without losing α2,6-linked sialylation in germinal centers. The in vivo function of Cmah was analyzed using gene-disrupted mice. Phenotypic analyses showed that Neu5Gc glycan functions as a negative regulator for B-cell activation in assays of T-cell-independent immunization response and splenic B-cell proliferation. Thus, Neu5Gc is required for optimal negative regulation, and the reaction is specifically suppressed in activated B cells, i.e., germinal center B cells.

Hypoxic culture induces expression of sialin, a sialic acid transporter, and cancer-associated gangliosides containing non-human sialic acid on human cancer cells.

Tumor hypoxia figures heavily in malignant progression by altering the intracellular glucose metabolism and inducing angiogenic factor production, thus, selecting and expanding more aggressive cancer cell clones. Little is known, however, regarding hypoxia-induced antigenic changes in cancers. We investigated the expression of N-glycolyl sialic acid (NeuGc)-G(M2), a cancer-associated ganglioside containing non-human sialic acid, NeuGc, in human cancers. Cancer tissues prepared from patients with colon cancers frequently expressed NeuGc-G(M2), whereas it was virtually absent in nonmalignant colonic epithelia. Studies on cultured cancer cells indicated that the non-human sialic acid was incorporated from culture medium. Hypoxic culture markedly induced mRNA for a sialic acid transporter, sialin, and this accompanied enhanced incorporation of NeuGc as well as N-acetyl sialic acid. Transfection of cells with sialin gene conferred accelerated sialic acid transport and induced cell surface expression of NeuGc-G(M2). We propose that the preferential expression of NeuGc-G(M2) in cancers is closely associated with tumor hypoxia. Hypoxic culture of tumor cells induces expression of the sialic acid transporter, and enhances the incorporation of non-human sialic acid from the external milieu. A consequence of this is the acquisition of cancer-associated cell surface gangliosides, typically G(M2), containing non-human sialic acid (NeuGc), which is not endogenously synthesized through CMP-N-acetyl sialic acid hydroxylase because humans lack the gene for the synthetic enzyme. As hypoxia is associated with diminished response to radiotherapy and chemotherapy, NeuGc-G(M2) is a potential therapeutic target for hypoxic cancer cells.

Sli2 (Ypk1), a Homologue of Mammalian Protein Kinase SGK, Is a Downstream Kinase in the Sphingolipid-Mediated Signaling Pathway of Yeast

ABSTRACT ISP-1 is a new type of immunosuppressant, the structure of which is homologous to that of sphingosine. In a previous study, ISP-1 was found to inhibit mammalian serine palmitoyltransferase, the primary enzyme involved in sphingolipid biosynthesis, and to reduce the intracellular pool of sphingolipids. ISP-1 induces the apoptosis of cytotoxic T cells, which is triggered by decreases in the intracellular levels of sphingolipids. In this study, the inhibition of yeast (Saccharomyces cerevisiae) proliferation by ISP-1 was observed. This ISP-1-induced growth inhibition was also triggered by decreases in the intracellular levels of sphingolipids. In addition, DNA duplication without cytokinesis was detected in ISP-1-treated yeast cells on flow cytometry analysis. We have cloned multicopy suppressor genes of yeast which overcome the lethal sphingolipid depletion induced by ISP-1. One of these genes, SLI2, is synonymous withYPK1, which encodes a serine/threonine kinase. Kinase-dead mutants of YPK1 did not show any resistance to ISP-1, leading us to predict that the kinase activity of the Ypk1 protein should be essential for this resistance to ISP-1. Ypk1 protein overexpression had no effect on sphingolipid biosynthesis by the yeast. Furthermore, both the phosphorylation and intracellular localization of the Ypk1 protein were regulated by the intracellular sphingolipid levels. These data suggest that the Ypk1 protein is a downstream kinase in the sphingolipid-mediated signaling pathway of yeast. The Ypk1 protein was reported to be a functional homologue of the mammalian protein kinase SGK, which is a downstream kinase of 3-phosphoinositide-dependent kinase 1 (PDK1). PDK1 phosphotidylinositol (PI) is regulated by PI-3,4,5-triphosphate and PI-3,4-bisphosphate through the pleckstrin homology (PH) domain. Overexpression of mammalian SGK also overcomes the sphingolipid depletion in yeast. Taking both the inability to produce PI-3,4,5-triphosphate and PI-3,4-bisphosphate and the lack of a PH domain in the yeast homologue of PDK1, the Pkh1 protein, into account, these findings further suggest that yeast may use sphingolipids instead of inositol phospholipids as lipid mediators.

B Cell Antigen Receptor Signal Strength and Peripheral B Cell Development are Regulated by a 9-O-Acetyl Sialic Acid Esterase

We show that the enzymatic acetylation and deacetylation of a cell surface carbohydrate controls B cell development, signaling, and immunological tolerance. Mice with a mutation in sialate:O-acetyl esterase, an enzyme that specifically removes acetyl moieties from the 9-OH position of α2–6-linked sialic acid, exhibit enhanced B cell receptor (BCR) activation, defects in peripheral B cell development, and spontaneously develop antichromatin autoantibodies and glomerular immune complex deposits. The 9-O-acetylation state of sialic acid regulates the function of CD22, a Siglec that functions in vivo as an inhibitor of BCR signaling. These results describe a novel catalytic regulator of B cell signaling and underscore the crucial role of inhibitory signaling in the maintenance of immunological tolerance in the B lineage.

Analysis of multiple compound–protein interactions reveals novel bioactive molecules

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.

Human B-lymphocytes Express α2-6-Sialylated 6-Sulfo-N-acetyllactosamine Serving as a Preferred Ligand for CD22/Siglec-2

CD22/Siglec-2, an important inhibitory co-receptor on B-lymphocytes, is known to recognize α2-6-sialylated glycan as a specific ligand. Here we propose that the α2-6-sialylated and 6-GlcNAc-sulfated determinant serves as a preferred ligand for CD22 because the binding of a human B-cell line to CD22 was almost completely abrogated after incubating the cells with NaClO3, an inhibitor of cellular sulfate metabolism, and was also significantly inhibited by a newly generated monoclonal antibody specific to the α2-6-sialylated 6-sulfo-N-acetyllactosamine (LacNAc) determinant (KN343, murine IgM). The α2-6-sialylated 6-sulfo-LacNAc determinant defined by the antibody was significantly expressed on a majority of normal human peripheral B-lymphocytes as well as follicular B-lymphocytes in peripheral lymph nodes. The determinant was also expressed in endothelial cells of high endothelial venules of secondary lymphoid tissues, including lymph nodes, tonsils, and intestine-associated lymphoid tissues, more strongly than on B-lymphocytes, suggesting a role for CD22 in B-cell interaction with blood vessels and trafficking. These results indicate that the α2-6-sialylated 6-sulfo-LacNAc determinant serves as an endogenous ligand for human CD22 and suggest the possibility that 6-GlcNAc sulfation as well as α2-6-sialylation may regulate CD22/Siglec-2 functions in humans.

Physical and Functional Association of Glucuronyltransferases and Sulfotransferase Involved in HNK-1 Biosynthesis

HNK-1 carbohydrate expressed predominantly in the nervous system is considered to be involved in cell migration, recognition, adhesion, and synaptic plasticity. Human natural killer-1 (HNK-1) carbohydrate has a unique structure consisting of a sulfated trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-) and is sequentially biosynthesized by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) and a sulfotransferase (HNK-1ST). Considering that almost all the HNK-1 carbohydrate structures so far determined in the nervous system are sulfated, we hypothesized that GlcAT-P or GlcAT-S functionally associates with HNK-1ST, which results in efficient sequential biosynthesis of HNK-1 carbohydrate. In this study, we demonstrated that both GlcAT-P and GlcAT-S were co-immunoprecipitated with HNK-1ST with a transient expression system in Chinese hamster ovary cells. Immunofluorescence staining revealed that these enzymes are mainly co-localized in the Golgi apparatus. To determine which domain is involved in this interaction, we prepared the C-terminal catalytic domains of GlcAT-P, GlcAT-S, and HNK-1ST, and we then performed pulldown assays with the purified enzymes. As a result, we obtained evidence that mutual catalytic domains of GlcAT-P or GlcAT-S and HNK-1ST are important and sufficient for formation of an enzyme complex. With an in vitro assay system, the activity of HNK-1ST increased about 2-fold in the presence of GlcAT-P or GlcAT-S compared with that in its absence. These results suggest that the function of this enzyme complex is relevant to the efficient sequential biosynthesis of the HNK-1 carbohydrate.

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).

Regulation of biosynthesis ofN-glycolylneuraminic acid-containing glycoconjugates: characterization of factors required for NADH-dependent cytidine 5′monophosphate-N-acetylneuraminic acid hydroxylation

The hydroxylation of CMP-NeuAc has been demonstrated to be carried out by several factors including the soluble form of cytochromeb5. In the present study, mouse liver cytosol was subjected to ammonium sulfate fractionation and cellulose phosphate column chromatography for the separation of two other essential fractions participating in the hydroxylation. One of the fractions, which bound to a cellulose phosphate column, was able to reduce the soluble cytochromeb5, using NADH as an electron donor. The other fraction, which flowed through the column, was assumed to contain the terminal enzyme which accepts electrons from cytochromeb5, activates oxygen, and catalyses the hydroxylation of CMP-NeuAc. Assay conditions for the quantitative determination of the terminal enzyme were established, and the activity of the enzyme in several tissues of mouse and rat was measured. The level of the terminal enzyme activity is associated with the expression ofN-glycolylneuraminic acid in these tissues, indicating that the expression of the terminal enzyme possibly regulates the overall velocity of CMP-NeuAc hydroxylation.