Circulating leukocytes transmigrate into the tissue through a multistep process involving rolling, adhesion, and extravasation. This process is relatively rapid and takes about several minutes. Molecules involved in this process have recently been extensively clarified. It has been known that leukocytes that have transmigrated into the tissue migrate through the tissue by interacting with the extracellular matrix components in a span of several hours or longer, whereas the molecular mechanisms underlying this process are almost unknown (1). We have recently found that a chondroitin sulfate proteoglycan versican, an extracellular matrix component, binds adhesion molecules, such as L-selectin, P-selectin and CD44, and chemokines. We are speculating that these interactions may be involved in leukocyte migration in the tissue. In this article, we will focus on the interaction of chondroitin sulfate proteoglycans with selectins, CD44, and chemokines, and will review recent progress in this field including our own work.

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Glycosaminoglycan (GAG) has many kinds of structural domains, and is known to participate in physiological functions such as anticoagulation and antithrombotic activities. It is important to prepare GAG oligosaccharides as investigation models in order to analyze their relationship between structure and function. In this review, I describe the methods of synthesis for the reconstruction of chondroitin sulfate oligosaccharides using testicular hyaluronidase, which is an endo-β-N-acetylhexosaminidase. The reaction occurs with changing combinations of chondroitin (Ch), chondroitin 4-sulfate (Ch4S), chondroitin 6-sulfate (Ch6S) and the other GAGs as an acceptor and a donor of transglycosylation, which reconstructs the sugar chain. Thus, it is possible to custom-synthesize oligosaccharides by controlling their combinations (Fig. 1). Therefore, it was possible to prepare many libraries of chondroitin sulfate oligosaccharides including chimeric unnatural GAGs. I expect that these libraries will contribute to new developments in glycotechnology in the future.

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Sulfotransferases so far cloned which are involved in the synthesis of chondroitin sulfate are classified into three gene families: chondroitin 6-sulfotransferase (C6ST) family, chondroitin 4-sulfotransferase (C4ST) family and uronosyl 2-O-sulfotransferase (UA2OST) family. The C6ST family includes sulfotransferases which catalyze the transfer of sulfate to position 6 of Gal residue of keratan sulfate and sialyllactosamine oligosaccharide, and to position 6 of nonreducing terminal GlcNAc residue. In the C4ST family, sulfotransferases which catalyze the transfer of sulfate to position 4 of nonreducing terminal GalNAc residue and to position 3 of nonreducing terminal GlcA residue are present. UA2OST was cloned as a cDNA showing homology with heparan sulfate 2-sulfotransferase that transfers sulfate to position 2 of Ido A residue in heparan sulfate. It became evident that, in some cases, sulfotransferases involved in the sulfation of glycosaminoglycans and sulfotransferases involved in the sulfation of oligosaccharides of glycoproteins are included in a common gene family. A sulfotransferase with a novel specificity may be found in these families, or a new sulfotransferase other than known families may be revealed in the future. Findings of a new sulfotransferase will offer important information about the biological function of sulfated sugar chains.

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Oversulfated chondroitin sulfate (CS) variant chains, CS-D, CS-E, CS-H and CS-K, all of which are characterized by di-or trisulfated disaccharide units, were originally discovered in tissues of lower marine organisms: shark cartilage, squid cartilage, hagfish notochord and king crab cartilage, respectively. Our studies and others have demonstrated oversulfated structures of CS in various vertebrate tissues including mammalian brains. Our studies in collaboration with A. Faissner have recently demonstrated neurite outgrowth promoting activities towards embryonic rat hippocampal neurons for shark cartilage CS-D and squid cartilage CS-E. We have also demonstrated that cortical neuronal cell adhesion, mediated by heparin (Hep)-binding neuroregulatory factor midkine, is specifically inhibited by squid cartilage CS-E as well as Hep. Furthermore we have shown direct molecular interactions of CS-E with midkine. Recent studies by others have also demonstrated the specific binding of oversulfated CS chains to another Hep-binding growth factor pleiotrophin, which forms a unique gene family with midkine. A systematic structural analysis of various oligosaccharides isolated from the oversulfated CS variants with such intriguing biological activities has revealed various characteristic sulfation profiles. Highly heterogenous sulfated patterns found in these oligosaccharides and the specific molecular interactions of the CS chains with Hep-binding growth factors may indicate the occurrence of analogous structures in higher organisms and are involved in the regulation of various biological processes such as neuronal cell adhesion, migration and neurite outgrowth promotion through specific interactions with the corresponding proteins including some Hep-binding growth factors.

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Transcription factors regulate gene expression and are responsible for various biological processes such as cell proliferation, cell differentiation, and apoptosis. Recently, analysis of genomes in various organisms is advancing rapidly, and technology for the analysis of multiple gene expression such as DNA chip technology is developing rapidly. Therefore, the necessity has arisen for many researchers in various fields to obtain information on transcription factors, which regulate gene expression. “TRANSFAC” is a database of transcription factors. In this paper, I will explain the practical and elementary operations not only of the TRANSFAC database to search for information on the transcriptional regulatory mechanism of transcription factors and genes, but also of a program, MatInspector, to predict the potential binding sites of the gene that interests you.

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