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Heparan sulfate chains are initially synthesized on core proteins as linear polysaccharides composed of glucuronic acid-N-acetylglucosamine repeating units and subjected to marked structural modification by sulfation at various places and epimerization of hexuronic acid residues (C5-epimerase) at the Golgi lumen and further by 6-O-desulfation at the cell surface, which generates their characteristic divergent fine structures. This chapter focuses on the biological and physiological functions of 6-O-sulfation in HS and the characterization of the enzymes catalyzing 6-O-sulfation (HS6ST). HS6STs in mammals such as humans and mice comprise of three isoforms (HS6ST-1, -2, and -3) and one alternatively spliced form of HS6ST-2 (HS6ST-2S). Each of these isoforms has distinct substrate preferences, albeit overlapping each other. These HS6ST isoforms are expressed in a spatiotemporally regulated manner in most organs. HS6ST-1-deficient mice are lethal mostly at later embryonic stages and exhibit abnormal angiogenesis in labyrinthine zone of placenta and aberrant lung morphology similar to pulmonary emphysema. These knockout mice also exhibit retinal axon guidance abnormality at the optic chiasm. Other HS6ST-deficient animals reveal various malformations in muscle development and branching morphology of the caudal vein of zebrafish, in tracheal formation of Drosophila, and in axon guidance of ventral nerve cord interneurons of Caenorhabditis elegans. Mouse embryonic fibroblasts prepared from HS6ST-1/HS6ST-2 double knockout mice did produce HS lacking 6-O-sulfation and responded differently to various FGFs dependent signaling.

Publisher Summary Hyaluronan synthase (HAS) expression provides powerful tools for understanding the in vivo function of hyaluronan (HA), particularly in connection with cancer cell behavior. The central aim of the present review is to highlight the role of HA in cancer progression with respect to abnormal HA biosynthesis. A wealth of data is accumulated on HA function in the promotion of malignancies, which shows that enhanced cancer invasion and dissemination may be partly dependent on the mesenchymal conversion of cancer cells by HA overexpression. Furthermore, recent studies have enabled postulation of reliable mechanisms by which HA influences tumor growth and invasion by modulating the tumor microenvironment to recruit stromal cells and vasculature. Although the angiogenic function of HA oligosaccharides is well-established, the anti-angiogenicity of high molecular weight HA being modulated by HA-binding molecules needs further clarification and study. The roles of HA in cancer progression may differ according to the HAS isoforms expressed, meaning cancer cells at different stages may differentially utilize the three HAS isoforms to maximize their survival. Studies are now in progress to identify exactly which HAS proteins are associated with cancer progression. This will provide an opportunity to develop new strategies for cancer therapy targeting specific cancer-associated HAS species.

Proteoglycans have sulfated linear polysaccharide chains, that is, heparan sulfate, heparin, chondroitin sulfates, dermatan sulfate, and keratan sulfate. Many glycosyltransferases and sulfotransferases are involved in biosynthesis of the polysaccharides. Specificities of these enzymes have been mainly determined by evaluating their activities to various acceptor carbohydrates and by analyzing the structure of the products. For the latter purpose, enzymatic hydrolysis using heparitinases, heparinase, and chondroitinases or chemical degradation employing nitrous acid deamination has been effectively used in combination with high‐performance liquid chromatography (HPLC) of the degraded products. As examples, we describe methods for assays and product characterization of sulfotransferases involved in biosynthesis of these polysaccharides, namely heparan sulfate 2‐sulfotransferase, heparan sulfate 6‐sulfotransferases, chondroitin 4‐sulfotransferases, chondroitin 6‐sulfotransferase, N‐acetylgalactosamine 4‐sulfate 6‐sulfotransferase, and N‐acetylglucosamine 6‐sulfotransferases.

This chapter presents an introduction to the formation of the serum-derived hyaluronan-associated protein (SHAP)–hyaluronan complex and the related physiological significance. This chapter discusses its possible roles in pathological processes. The main point is that the association of SHAP changes the properties of hyaluronan. Mass spectrometry revealed a unique protein–glycosaminoglycan linkage in both IαI family molecules and the SHAP–hyaluronan complex, where an ester bond is formed between the carboxyl group of the asparate residue at the C-terminus of the heavy chains/SHAPs, and the C-6 hydroxyl group of an internal N-acetylgalactosamine residue of the bikunin chondroitin sulfate or an N-acetylglucosamine residue of hyaluronan. Based on the similarity of the protein–glycosaminoglycan linkage and the homology of both the N- and C-terminus between the heavy chain and corresponding SHAP, it is concluded that the SHAP–hyaluronan complex is formed via a trans-esterification reaction in which hyaluronan substitutes for bikunin chondroitin sulfate to form the same type of ester bond with the heavy chains, accompanied by the release of the bikunin proteoglycan.

Publisher Summary This chapter summarizes general aspects of proteoglycans/glycosaminoglycan, and focuses on chondroitin sulfate/dermatan sulfate proteoglycans and hyaluronan in normal and cirrhotic liver. Proteoglycans/glycosaminoglycans consist of a collection of macromolecules that surround the plasma membrane of cell and constitute part of the substrate upon which cells are attached and perform their major functions. The solid frameworks needed for the structural support and functional organizations of these assemblies are made primarily of cross-linked fibers of collagen embedded in a fine meshwork of proteoglycans/glycosaminoglycans, and sometimes reinforced by deposited minerals. Proteoglycans/glycosaminoglycans are involved in maintaining the transparency of the cornea, the tensile strength of the skin and tendon, the viscoelasticity of blood vessels, the compressive properties of cartilage, and the mineralized matrix of bones. In addition, proteoglycans play a key role as storage depots for growth factors and cytokines, and by virtue of their multifunctional properties, they alter the biology of these factors. The extracellular matrix and its components are used as culture substrata to improve the attachment and survival of the cultured hepatocytes; materials used as substrata include a biomatrix composed of fibers isolated from liver tissue, collagens, laminin, and fibronectin.

Hyaluronan (HA) is a widely distributed nonsulfated glycosaminogtycan with many physiological and biological functions including promotion of cell proliferation and migration. Accumulation of HA in extracellular matrix is an early event in tissue repair, and also the appearance and removal of HA at specific loci are precisely controlled during embryogencsis. Liver regeneration following partial hepatectomy (PH) is well established in vivo system to study cell proliferation and tissue reorganization. Therefore we studied tissue distribution of HA, its serum level, and the expression patterns of the genes of HA synthases (HAS1, HAS2, HAS3) in regenerating rat liver after PH. In the regenerating liver, HA were transiently accumulated along sinusoid from 24 hour to 48 hour after PH. Normally, low concentrations of HA circulate in the blood, but, during the liver regeneration serum concentration of HA was transiently elevated with the same temporal pattern as the tissue accumulation of HA. Semi-quantitative RT-PCR analyses showed expression level of HAS1 in regenerating liver was elevated immediately after PH and was kept at high level for 1 week. Transient accumulation and synthesis of HA may play important roles in liver regeneration by remodeling the microenvironment.

Hyaluronan (HA) was shown not only to be associated noncovalently with some extracellular molecules such as HA-binding proteoglycans but also to be covalently linked to SHAP (Serum-derived Hyaluronan-Associated Protein), the heavy chains of inter-alpha-trypsin inhibitor (ITI) through a unique ester linkage. Such non-covalent and covalent associations may play important roles in the formation and maintenance of HA-rich matrix. In the present study, we examined the binding activity of ITI to various extracellular matrix molecules by enzyme-linked immunosorbent assay, and found that the heavy chains but not the bikunin of ITI bound to PG-M/versican, to link protein, and to the hyaluronan-binding region of aggrecan from various sources, which suggests specific interactions between the conserved regions of the heavy chains and the conserved HA-binding regions of these matrix molecules. In the further binding experiments using recombinant proteins for various portions of the heavy chains of human ITI, we found that the polypeptide near the C-terminus of the heavy chain and the HA-binding region of aggrecan were involved in binding to each other. Such a noncovalent protein-protein interaction may be important as an initial step for the formation of the HA-SHAP complex as well as for the formation and stabilization of HA-rich matrix.

Publisher Summary Adhesion of cells to extracellular substrate molecules is one of the fundamental processes for formation and maintenance of animal tissues and bodies. The core protein moiety generally has the ability to bind to other extracellular matrix molecules. Aggrecan, for example, is bound at the amino-terminal globular domains to hyaluronan and link proteins and also at the carboxyl-terminal domains with C-type lectin like activity to some ligand molecules in the matrix. Proteoglycans are considered, therefore, as molecules which enable GAGs to be immobilized in extracellular and/ or cell surface proteins by such core protein-mediated interactions.