IntroductionHyaluronan, or hyaluronic acid (HA) is a linear, highmolecular-weight (mega-Dalton) polymer comprised of repeating disaccharide units of (β1→3)D-glucuronate-(β1→4)N-acetyl-D-glucosamine. HA is synthesized by integral plasma membrane glycosyltransferases and is exported directly into the extracellular space (1, 2). Although HA is chemically homogeneous, there are three distinct mammalian HA synthases (designated Has1, Has2, and Has3), encoded by related but nonlinked genes (3-9). Each synthase has distinct catalytic properties, and the distribution and abundance of each varies during development of the mouse (6, 10). These observations suggest that the different Has enzymes play distinct roles.HA binds salt and water, expanding the extracellular space (11)(12)(13)(14). HA is especially prominent at sites where cell migration occurs, such as pathways of neural crest cell migration and in the developing cardiovascular system. In vivo, HA interacts with other extracellular matrix molecules, typically via an HA-binding domain called the link module (15). These interactions create a supramolecular architecture of the extracellular matrix, i.e., the composite matrix network of HA, link protein, and aggrecan that plays a critical role in load-bearing articular cartilage (16)(17)(18).In addition to its important physical properties, the overexpression of Has genes results in increased anchorage-independent growth and metastasis of transformed cells (19,20), suggesting a link between HA and transformation. HA is also implicated in receptor-mediated cell adhesion and intracellular signaling (21,22). Taken together, such observations suggest that HA plays a vital role in diverse cellular events, including cell migration, tissue remodeling, and metastasis. However, the near-ubiquitous distribution of HA in vivo, the biological activity of HA fragments released by degradative enzymes (23), and the inability to inhibit HA synthesis in vivo have hindered definitive analysis of the physiological roles of HA. Accordingly, we used a genetic approach to investigate the roles of HA in vivo and to identify the HA synthase that is critical during embryogenesis.Expression of Has2 appeared to correlate with expansion of cardiac cushion tissue and subsequent transformation of endocardial cells into mesenchyme. The tar- We identified hyaluronan synthase-2 (Has2) as a likely source of hyaluronan (HA) during embryonic development, and we used gene targeting to study its function in vivo. Has2 -/-embryos lack HA, exhibit severe cardiac and vascular abnormalities, and die during midgestation (E9.5-10). Heart explants from Has2 -/-embryos lack the characteristic transformation of cardiac endothelial cells into mesenchyme, an essential developmental event that depends on receptor-mediated intracellular signaling. This defect is reproduced by expression of a dominant-negative Ras in wild-type heart explants, and is reversed in Has2 -/-explants by gene rescue, by administering exogenous HA, or by expressing activated Ras. Conversely, ...

Three mammalian hyaluronan synthase genes, HAS1, HAS2, and HAS3, have recently been cloned. In this study, we characterized and compared the enzymatic properties of these three HAS proteins. Expression of any of these genes in COS-1 cells or rat 3Y1 fibroblasts yielded de novo formation of a hyaluronan coat. The pericellular coats formed by HAS1 transfectants were significantly smaller than those formed by HAS2 or HAS3 transfectants. Kinetic studies of these enzymes in the membrane fractions isolated from HAS transfectants demonstrated that HAS proteins are distinct from each other in enzyme stability, elongation rate of HA, and apparent K m values for the two substrates UDPGlcNAc and UDP-GlcUA. Analysis of the size distributions of hyaluronan generated in vitro by the recombinant proteins demonstrated that HAS3 synthesized hyaluronan with a molecular mass of 1 ؋ 10 5 to 1 ؋ 10 6 Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 ؋ 10 5 to ϳ2 ؋ 10 6 Da. Furthermore, comparisons of hyaluronan secreted into the culture media by stable HAS transfectants showed that HAS1 and HAS3 generated hyaluronan with broad size distributions (molecular masses of 2 ؋ 10 5 to ϳ2 ؋ 10 6 Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of >2 ؋ 10 6 Da). The occurrence of three HAS isoforms with such distinct enzymatic characteristics may provide the cells with flexibility in the control of hyaluronan biosynthesis and functions. Hyaluronan (HA)1 is a major component of most extracellular matrices, particularly in tissues with rapid cell proliferation and cell migration (1). The interaction of HA with various HA-binding proteins and cell-surface receptors plays important roles in regulating fundamental cell behaviors such as cell adhesion, migration, and differentiation (2, 3). Thus, HA has been greatly implicated in morphogenesis, regeneration, wound healing, tumor invasion, and cancer metastasis (4 -6). In addition, HA is an important structural molecule required for the maintenance of various aspects of tissue architecture and function. The physical and biological properties of HA appear to be affected by many factors including HA concentration and chain length. Indeed, high molecular weight HA at high concentrations suppresses endothelial cell growth, whereas low molecular weight HA stimulated cell growth leading to induction of angiogenesis (7). In addition, viscosity of the HA gel and the ability to hydrate large amounts of water were shown to be dependent on the molecular size of the HA chain.HA is a high molecular weight linear polymer composed of GlcUA ␤-1,3-GlcNAc ␤-1,4 disaccharide units and is synthesized by HA synthase at the inner face of the plasma membrane (8). Although a great deal of effort has been made to elucidate the mechanism of HA biosynthesis in mammalian cells, it has remained unclear due to difficulty in biochemical isolation of the active enzyme (9 -11). Recently, three distinct yet highly conserved genes encoding mammali...

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The last seven years have been exciting in the world of mucin biology. Molecular analyses of mucin genes and deduced protein structures have provided insight into structural features of mucins and tools with which to examine expression, secretion, and glycosylation, thereby enabling a better understanding of the role of mucins in normal physiological processes and in disease. Functional studies are in progress both in vitro using cDNAs and cell lines and in vivo utilizing mutant mice in which a particular mucin gene has been inactivated or overexpressed. These studies should help determine whether the functions of mucins are restricted to protection and lubrication, or if they are involved in the adhesion of tumor cells to other cells or tissue components or in modulation of the immune system.