Akiko Kanamori

SHAP Potentiates the CD44-mediated Leukocyte Adhesion to the Hyaluronan Substratum

CD44-hyaluronan (HA) interaction is involved in diverse physiological and pathological processes. Regulation of interacting avidity is well studied on CD44 but rarely on HA. We discovered a unique covalent modification of HA with a protein, SHAP, that corresponds to the heavy chains of inter-α-trypsin inhibitor family molecules circulating in blood. Formation of the SHAP·HA complex is often associated with inflammation, a well known process involving the CD44-HA interaction. We therefore examined the effect of SHAP on the CD44-HA interaction-mediated lymphocyte adhesion. Under both static and flowing conditions, Hut78 cells (CD44-positive) and CD44-transfected Jurkat cells (originally CD44-negative) adhered preferentially to the immobilized SHAP·HA complex than to HA. The enhanced adhesion is exclusively mediated by the CD44-HA interaction, because it was inhibited by HA, but not IαI, and was completely abolished by pretreating the cells with anti-CD44 antibodies. SHAP appears to potentiate the interaction by increasing the avidity of HA to CD44 and altering their distribution on cell surfaces. Large amounts of the SHAP·HA complex accumulate in the hyperplastic synovium of rheumatoid arthritis patients. Leukocytes infiltrated to the synovium were strongly positive for HA, SHAP, and CD44 on their surfaces, suggesting a role for the adhesion-enhancing effect of SHAP in pathogenesis.

Isolation and characterization of deaminated neuraminic acid-rich glycoprotein (KDN-gp-OF) in the ovarian fluid of rainbow trout (Salmogairdneri)

A novel highly acidic glycoprotein (deaminated neuraminic acid-rich glycoprotein; KDN-gp) was first discovered as an integral component of the vitelline envelope of rainbow trout eggs [Inoue, S., et al. (1988) Biochem. Biophys. Res. Commun. 153, 172-176]. Another member of this class of glycoprotein has now been found in the ovarian (or coelomic) fluid of ovulating rainbow trout. This ovarian fluid KDN-glycoprotein is designated as KDN-gp-OF and its amino acid and carbohydrate compositions were compared with those of the vitelline envelope KDN-gp (KDN-gp-VE). KDN-gp-OF was similar to KDN-gp-VE in the carbohydrate composition and molecular weight. However, a small but definite difference in amino acid composition and the molecular weight range was found between KDN-gp-OF and KDN-gp-VE. The results suggest that in KDN-gp-OF some peptide sequences presumably present at either the C- or N-terminus are deleted from KDN-gp-VE. Possible biological function of KDN-gp-OF is discussed.

CD43, but not P-Selectin Glycoprotein Ligand-1, Functions as an E-Selectin Counter-Receptor in Human Pre-B-Cell Leukemia NALL-1

B-cell precursor acute lymphoblastic leukemia (BCP-ALL/B-precursor ALL) is characterized by a high rate of tissue infiltration. The mechanism of BCP-ALL cell extravasation is not fully understood. In the present study, we have investigated the major carrier of carbohydrate selectin ligands in the BCP-ALL cell line NALL-1 and its possible role in the extravascular infiltration of the leukemic cells. B-precursor ALL cell lines and clinical samples from patients with BCP-ALL essentially exhibited positive flow cytometric reactivity with E-selectin, and the reactivity was significantly diminished by O-sialoglycoprotein endopeptidase treatment in NALL-1 cells. B-precursor ALL cell lines adhered well to E-selectin but only very weakly to P-selectin with low-shear-force cell adhesion assay. Although BCP-ALL cell lines did not express the well-known core protein P-selectin glycoprotein ligand-1 (PSGL-1), a major proportion of the carbohydrate selectin ligand was carried by a sialomucin, CD43, in NALL-1 cells. Most clinical samples from patients with BCP-ALL exhibited a PSGL-1(neg/low)/CD43(high) phenotype. NALL-1 cells rolled well on E-selectin, but knockdown of CD43 on NALL-1 cells resulted in reduced rolling activity on E-selectin. In addition, the CD43 knockdown NALL-1 cells showed decreased tissue engraftment compared with the control cells when introduced into gamma-irradiated immunodeficient mice. These results strongly suggest that CD43 but not PSGL-1 plays an important role in the extravascular infiltration of NALL-1 cells and that the degree of tissue engraftment of B-precursor ALL cells may be controlled by manipulating CD43 expression.

Synthesis and Antigenic Property of a Novel Sialyl 6‐O‐Sulfo Lewis X Neo‐glycolipid Containing Lactamized Neuraminic Acid

Abstract Synthesis and antigenic property of a novel 6‐O‐sulfated sLex neo‐glycolipid containing lactamized neuraminic acid are described. Coupling of methyl (methyl 4,7,8,9‐tetra‐O‐acetyl‐3,5‐dideoxy‐5‐trifluoroacetamido‐D‐glycero‐α‐D‐galacto‐2‐nonulopyranosylonate)‐(2→3)‐4,6‐di‐O‐acetyl‐2‐O‐benzoyl‐1‐thio‐β‐D‐galactopyranoside (3) with 2‐(tetradecyl)hexadecyl (2,3,4‐tri‐O‐benzyl‐α‐L‐fucopyranosyl)‐(1→3)‐2‐acetamido‐2‐deoxy‐6‐O‐4‐methoxyphenyl‐β‐D‐glucopyranoside (7) gave a protected sLex tetrasaccharide glycolipid (8). Removal of all the acyl protecting groups and subsequent lactamization afforded the lactamized sLex derivative(10), which was converted to the target compound (14) by selective removal of the 4‐methoxyphenyl group and 6‐O‐sulfation of the GlcNAc residue, and removal of all protective groups under the basic conditions furnished the target molecule. The antigenic property of the synthesized neo‐glycolipid was examined by TLC‐immunostaining with G159 monoclonal antibody. #Synthetic Studies on Sialoglycoconjugates, Part 136. For part 135, see Ref.1.

cDNA cloning of putative rat acetyl-CoA transporter and its expression pattern in brain

Rat acetyl-CoA transporter gene (Acatn) encodes a hydrophobic multi-transmembrane protein involved in the O-acetylation of gangliosides. O-acetylated gangliosides have been found to play important roles in the embryonic development of the nervous system. We have isolated rat Acatn cDNA by PCR cloning. The amino acid sequence of rat Acatn exhibited 92% and 96% homology with human and mouse sequences, respectively. The mRNA was expressed in brain at all developmental stages. Acatn expression was higher in embryonic and postnatal rats than in adult rats. Cellular localization of Acatn mRNA in adult rat brain was also analyzed by in situ hybridization. Acatn mRNA expression was detected in the neuronal cells of cerebellum, hippocampus, hypothalamus, cortex, olfactory bulb, and dorsal and ventral anterior olfactory nucleus in adult rat brain.

Cloning and characterization of a putative mouse acetyl-CoA transporter cDNA.

Abstract A mouse acetyl-CoA transporter (Acatn) cDNA was isolated by PCR cloning. Mouse Acatn exhibited 92% homology with human sequence on the basis of amino-acid sequence. The predicted gene product of Acatn is a 61kDa hydrophobic protein with six to 10 transmembrane domains. Transfection of mouse Acatn cDNA into HeLa/GT3+ cells resulted in significant increase in the amount of 9-O-acetylated gangliosides, suggesting that Acatn does play an important role in the acetylation of gangliosides. Northern blot analysis of Acatn mRNA suggested that transcript of Acatn is widely distributed in various adult tissues. Expression of Acatn was found to be developmentally regulated, with high expression levels during early embryonic stages, and then there was a subsequent decrease in expression levels in the later embryonic stages.

6-O-sulfo sialylparagloboside and sialyl Lewis X neo-glycolipids containing lactamized neuraminic acid: synthesis and antigenic reactivity against G159 monoclonal antibody.

Synthesis and antigenic reactivity of 6-O-sulfo sialylparagloboside (SPG) and sialyl Lewis X (sLeX) neo-glycolipids containing lactamized neuraminic acid are described. The suitably protected GlcNAc-β (1 → 3)-Gal-β (1 → 4)-GlcOSE derivative was glycosylated with NeuTFAc-α (2 → 3)-Gal imidate to give NeuTFAc-α (2 → 3)-Galβ (1 → 4)-GlcNAc-β (1 → 3)-Gal-β (1 → 4)-GlcOSE pentasaccharide. The partial N,O-deacylation in the NeuTFAc-α (2→3)-Gal part afforded N-deacetylated SPG derivative which was converted to the desired oligosaccharide containing lactamized neuraminic acid. Similar treatment of the sLeX hexasaccharide derivative, NeuTFAc-α (2 → 3)-Gal-β (1 → 4) [Fuc-α (1 →3)]-GlcNAc-β (1 → 3)-Gal-β (1 → 4)-GlcOSE, gave the key hexasaccharide intermediate containing lactamized neuraminic acid. These suitably protected SPG and sLex oligosaccharides were converted stepwise into the desired neo-glycolipids (GSC-551 and GSC-552) by the coupling with 2-(tetradecyl)hexadecanol, 6-O-sulfation at C-6 of the GlcNAc residure, and complete deprotection.Both lactamized-sialyl 6-O-sulfo SPG (GSC-551) and sLex (GSC-552) neo-glycolipids were clearly recognized with G159 monoclonal antibody showing that both the lactamized neuraminic acid and the 6-O-sulfate at C-6 of GlcNAc would be involved in the G159-defined determinant. However, the Fuc residue and the lipophilic (ceramide) part may not be critical for this recognition. Published in 2005

NMR structure elucidation of cyclic sialyl 6-sulfo Lewis x, a biologically dormant form of L-selectin ligand

Abstract The structure of cyclic sialyl 6-sulfo Lewis x, a new biologically dormant form of L-selectin ligand, was determined unambiguously by the accurate NMR analysis to have the lactamized 5,2 B conformation of its sialic acid. For the NMR structure elucidation were used various informations, such as chemical shifts values, appearance of amide proton, and NOE.

Genomic structure and promoter analysis of putative mouse acetyl-CoA transporter gene.

The acetyl‐CoA transporter gene (Acatn) encodes a hydrophobic, multitransmembrane protein that is involved in the process of O‐acetylation of sialic acid residues on gangliosides. O‐Acetylated gangliosides have been found to play important roles in tissue development and organization during early embryonic stages. We have cloned the gene for mouse acetyl‐CoA transporter. The gene spans approximately 20 kb and is composed of seven exons and six introns. A single transcription initiation site, 371 bp upstream of the ATG start codon, was identified. The promoter region was found to lack a TATA box. However, several potential transcription factor binding motifs such as AP1, AP2, C/EBPα, C/EBPβ, HSF, GATA2 and MZF1 were identified in the promoter region.

Assignment1 of a putative acetyl-CoA transporter gene (Acatn) to mouse chromosome band 3E1–E3 by in situ hybridization

Acetyl-CoA transporter gene (Acatn) encodes a novel protein that is involved in the process of O-acetylation of gangliosides. The human acetyl-CoA transporter gene has been cloned and sequenced (Kanamori et al., 1997). We have recently cloned and sequenced the mouse acetyl-CoA transporter gene that shows 87% homology with the human cDNA at nucleotide sequence level (unpublished data). The expression of 9-O-acetylated gangliosides is associated with neural cell differentiation and migration and it also effects virus binding, cell adhesion and the immunogenicity of sialic acid residues of gangliosides (Varki, 1992). In spite of its importance, the O-acetylation mechanism is poorly understood at the molecular and genetic level. Since acetyl-CoA transporter is involved in the process of O-acetylation, this gene will be of utmost importance to study the mechanism of O-acetylation. In this paper we report the chromosome mapping of the acetyl-CoA transporter gene. Materials and methods