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Biomedical subjects

Reiji Kannagi

Publications and source records attributed to Reiji Kannagi.

45 records · Page 3Linked to original sources
PubMed2002

Distinct sulfation requirements of selectins disclosed using cells that support rolling mediated by all three selectins under shear flow. L-selectin prefers carbohydrate 6-sulfation totyrosine sulfation, whereas p-selectin does not.

l- and P-selectin are known to require sulfation in their ligand molecules. We investigated the significance of carbohydrate 6-sulfation and tyrosine sulfation in selectin-mediated cell adhesion. COS-7 cells were genetically engineered to express P-selectin glycoprotein ligand-1 (PSGL-1) or its mutant in various combinations with 6-O-sulfotransferase (6-Sul-T) and/or alpha1-->3fucosyltransferase VII (Fuc-T VII). The cells transfected with PSGL-1, 6-Sul-T, and Fuc-T VII cDNAs supported rolling mediated by all three selectins and provided the best experimental system so far to estimate kinetic parameters in selectin-mediated cell adhesion for all three selectins using the identical rolling substrate and to compare the ligand specificity of each selectin. L-selectin-mediated rolling was drastically impaired if the cells lacked carbohydrate 6-sulfation elaborated by 6-Sul-T, but not affected when PSGL-1 was replaced with a mutant lacking three tyrosine residues at its NH(2) terminus. L-selectin-mediated adhesion was also hardly affected by mocarhagin treatment of the cells, which cleaved a short peptide containing sulfated tyrosine residues from PSGL-1. In contrast, P-selectin-mediated rolling was abolished when PSGL-1 was either mutated or cleaved by mocarhagin at its NH(2) terminus, whereas the cells expressing PSGL-1 and Fuc-T VII but not 6-Sul-T showed only a modest decrease in P-selectin-mediated adhesion. These results indicate that L-selectin prefers carbohydrate 6-sulfation much more than tyrosine sulfation, whereas P-selectin favors tyrosine sulfation in the PSGL-1 molecule far more than carbohydrate 6-sulfation. E-selectin-mediated adhesion was sulfation-independent requiring only Fuc-T VII, and thus the three members of the selectin family have distinct requirements for ligand sulfation.

Animals↗
PubMed2002

Reduced sialidase expression in highly metastatic variants of mouse colon adenocarcinoma 26 and retardation of their metastatic ability by sialidase overexpression.

Sialidase expression levels are inversely correlated with the metastatic potential of mouse colon adenocarcinoma 26 sublines, as assessed by activity assays and RT-PCR, irrespective of total and cell surface sialic acid contents. Compared with low metastatic NL4 and NL44 cell lines, the highly metastatic NL17 and NL22 cells exhibit low expression of sialidases, accompanied with higher levels of sialylLe(x) and GM3. To investigate whether these properties of NL17 cells can be altered by sialidase overexpression, we transfected a cytosolic sialidase gene into NL17 cells. The result was markedly inhibited lung metastasis, invasion and cell motility with a concomitant decrease in sialylLe(x) and GM3 levels, in line with the case of spontaneously low metastatic sublines having relatively high endogenous sialidase levels, implying that sialidase level is a determining factor affecting metastatic ability. Treatment of the cells with antibodies against sialylLe(x) and GM3 affected cell adhesion and/or cell motility, providing evidence that desialylation of these molecules, as targets of sialidase, is involved in the suppression of metastasis.

Adenocarcinoma↗
PubMed2002

Regulatory roles of carbohydrate ligands for selectins in the homing of lymphocytes.

Cell surface carbohydrate determinants figure heavily in the regulation of lymphocyte homing and the inflammatory recruitment of leukocytes. The recently described sulfated carbohydrate ligand for selectins, sialyl 6-sulfo Lewis x, is mainly involved in the routine homing of various subsets of lymphocytes, such as nai;ve helper T cells, and skin- and gut-homing helper memory T cells. The homing of lymphocytes is regulated by a unique post-translational modification of sialic acid moieties that occurs specifically in sulfated selectin ligands, whereby the sialic acid loses its N-acetyl group and is converted into a 1-5 cyclic derivative. By contrast, nonsulfated carbohydrate ligands of the selectins, such as sialyl Lewis x, are mainly involved in the recruitment of leukocytes during inflammation. The increment of sialyl Lewis x expression upon inflammatory stimuli is mediated by transcriptional induction of fucosyltransferase genes.

Animals↗
PubMed2002

6-Sulfo LacNAc, a novel carbohydrate modification of PSGL-1, defines an inflammatory type of human dendritic cells.

The monoclonal antibody M-DC8 defines a major subset of human blood dendritic cells (DCs). Here we identify the M-DC8 structure as 6-sulfo LacNAc, a novel carbohydrate modification of the P selectin glycoprotein ligand 1 (PSGL-1). In contrast to previously described blood DCs, M-DC8+ DCs lack the cutaneous lymphocyte antigen (CLA) on PSGL-1 and fail to bind P and E selectin. Yet they express anaphylatoxin receptors (C5aR and C3aR) and the Fcgamma receptor III (CD16), which recruit cells to inflammatory sites. While sharing with DC1 the expression of myeloid markers and a potent capacity to prime T cells in vitro, M-DC8+ DCs produce far more TNF-alpha in response to the bacterial endotoxin lipopolysaccharide (LPS). Thus, 6-sulfo LacNAc-expressing DCs appear as a novel proinflammatory DC subset.

Adult↗
PubMed2002

P-selectin-dependent macrophage migration into the tubulointerstitium in unilateral ureteral obstruction.

BACKGROUND: Interstitial infiltration of macrophages (Mø) is one of the main causal factors for the tubulointerstitial injury. However, precise mechanisms of Mø infiltration into tubulointerstitium have not been fully explored. The purposes of this study were to assess the role of selectins in the acute infiltration of Mø in rats with unilateral ureteral obstruction (UUO) and to evaluate the role of vasa recta, that is, whether they facilitate massive influx of Mø into the interstitium by functioning as specialized vessels. METHODS: To evaluate the role of selectins in Mø infiltration into tubulointerstitium, the expression of selectins and L-selectin ligands was examined by immunohistochemistry and immunoelectron microscopy. The functional role of P-selectin in vasa recta was studied by Stamper-Woodruff assay, in vivo p-Mø migration assay and in vivo blocking experiments with the monoclonal antibody (mAb) ARP2-4. RESULTS: Selective expression of P-selectin was detected in vasa recta as early as one hour after UUO, and the expression increased thereafter for 96 hours. In contrast, endothelial expression of L-selectin ligands and E-selectin were not detectable. In the Stamper-Woodruff assay on kidney sections of rats with UUO, the adhesion of isolated rat peritoneal Mø (p-Mø) to vasa recta was significantly inhibited by the mAb ARP2-4 (P-selectin blocker; P < 0.01), but not by mAb ARE-5 (E-selectin blocker) or rLECIg (rat L-selectin chimeric protein). In the in vivo transfer experiments with fluorescein-labeled p-Mø into rats 48 hours after UUO, labeled p-Mø had accumulated around vasa recta at three minutes and had infiltrated predominantly into the outer medulla at 180 minutes. The number of labeled p-Mø was reduced when the rats were pretreated with ARP2-4 (P < 0.01). Finally, ARP2-4 (10 mg/kg), injected 15 minutes before UUO, reduced the number of infiltrated Mø (P < 0.01). CONCLUSION: The results suggest that vasa recta, which express P-selectin, contribute to massive infiltration of Mø into the interstitium by functioning as specialized post-capillary venules.

Animals↗
PubMed2002

TNF-alpha increases the carbohydrate sulfation of CD44: induction of 6-sulfo N-acetyl lactosamine on N- and O-linked glycans.

CD44 and sulfation have both been implicated in leukocyte adhesion. In monocytes, the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) stimulates CD44 sulfation, and this correlates with the induction of CD44-mediated adhesion events. However, little is known about the sulfation of CD44 or its induction by inflammatory cytokines. We determined that TNF-alpha induces the carbohydrate sulfation of CD44. CD44 was established as a major sulfated cell surface protein on myeloid cells. In the SR91 myeloid cell line, the majority of CD44 sulfation was attributed to the glycosaminoglycan chondroitin sulfate. However, TNF-alpha stimulation increased CD44 sulfation two- to threefold, largely attributed to the increased sulfation of N- and O-linked glycans on CD44. Therefore, TNF-alpha induced a decrease in the percentage of CD44 sulfation due to chondroitin sulfate and an increase due to N- and O-linked sulfation. Furthermore, TNF-alpha induced the expression of 6-sulfo N-acetyl lactosamine (LacNAc)/Lewis x on these cells, which was detected by a monoclonal antibody after neuraminidase treatment. This 6-sulfo LacNAc/Lewis x epitope was induced on N-linked and (to a lesser extent) on O-linked glycans present on CD44. This demonstrates that CD44 is modified by sulfated carbohydrates in myeloid cells and that TNF-alpha modifies both the type and amount of carbohydrate sulfation occurring on CD44. In addition, it demonstrates that TNF-alpha can induce the expression of 6-sulfo N-acetyl glucosamine on both N- and O-linked glycans of CD44 in myeloid cells.

Amino Sugars↗
PubMed2001

Molecular basis of evolutionary loss of the alpha 1,3-galactosyltransferase gene in higher primates.

Galactose-alpha1,3-galactose (alphaGal) epitopes, the synthesis of which requires the enzyme product of alpha1,3-galactosyltransferase (alpha1,3GT), are sugar chains on the cell surface of most mammalian species. Notable exceptions are higher primates including Old World monkeys, apes, and humans. The alphaGal-negative species as well as mice with deletion of the alpha1,3GT gene produce abundant anti-alphaGal antibodies. The evolutionary loss of alphaGal epitopes has been attributed to point mutations in the coding region of the gene. Because no transcripts could be found in the higher primate species with Northern blot analysis, a potential alternative explanation has been loss of upstream regulation of the gene. Here, we have demonstrated that the rhesus promoter is functional. More importantly, a variety of full-length transcripts were detected with sensitive PCR-based methods in the tissues of rhesus monkeys, orangutans, and humans. Five crucial mutations were delineated in the coding region of the human and rhesus and three in the orangutan, any one of which could be responsible for inactivation of the alpha1,3GT gene. Two of the mutations were shared by all three higher primates. These findings, which elucidate the molecular basis for the evolutionary loss of alphaGal expression, may have implications in medical research.

Amino Acid Sequence↗
PubMed2001

Specificities of N-acetylglucosamine-6-O-sulfotransferases in relation to L-selectin ligand synthesis and tumor-associated enzyme expression.

N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) catalyzes the transfer of sulfate from adenosine 3'-phosphate,5'-phosphosulfate to the C-6 position of the non-reducing GlcNAc. Three human GlcNAc6STs, namely GlcNAc6ST-1, GlcNAc6ST-2 (HEC-GlcNAc6ST), and GlcNAc6ST-3 (I-GlcNAc6ST), were produced as fusion proteins to protein A, and their substrate specificities as well as their enzymological properties were determined. Both GlcNAc6ST-1 and GlcNAc6ST-2 efficiently utilized the following oligosaccharide structures as acceptors: GlcNAcbeta1-6[Galbeta1-3]GalNAc-pNP (core 2), GlcNAcbeta1-6ManOMe, and GlcNAcbeta1-2Man. The ratios of activities to these substrates were not significantly different between the two enzymes. However, GlcNAc6ST-2 but not GlcNAc6ST-1 acted on core 3 of GlcNAcbeta1-3GalNAc-pNP. GlcNAc6ST-3 used only the core 2 structure among the above mentioned oligosaccharide structures. The ability of GlcNAc6ST-1 to sulfate core 2 structure as efficiently as GlcNAc6ST-2 is consistent with the view that GlcNAc6ST-1 is also involved in the synthesis of l-selectin ligand. Indeed, cells doubly transfected with GlcNAc6ST-1 and fucosyltransferase VII cDNAs supported the rolling of L-selectin-expressing cells. The activity of GlcNAc6ST-2 on core 3 and its expression in mucinous adenocarcinoma suggested that this enzyme corresponds to the sulfotransferase, which is specifically expressed in mucinous adenocarcinoma (Seko, A., Sumiya, J., Yonezawa, S., Nagata, K., and Yamashita, K. (2000) Glycobiology 10, 919-929).

Base Sequence↗