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...

No abstract

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.

The three mammalian hyaluronan synthase (HAS) genes and the related Xenopus laevis gene, DG42, belong to a larger evolutionarily conserved vertebrate HAS gene family. We have characterized additional vertebrate HAS genes from chicken (chas2 and chas3) and Xenopus (xhas2, xhas3, and a unique Xenopus HAS-related sequence, xHAS-rs). Genomic structure analyses demonstrated that all vertebrate HAS genes share at least one exon-intron boundary, suggesting that they evolved from a common ancestral gene. Furthermore, the Has2 and Has3 genes are identical in structure, suggesting that they arose by a gene duplication event early in vertebrate evolution. Significantly, similarities in the genomic structures of the mouse Has1 and Xenopus DG42 genes strongly suggest that they are orthologues. Northern analyses revealed a similar temporal expression pattern of HAS genes in developing mouse and Xenopus embryos. Expression of mouse Has2, Has3, and Xenopus Has1 (DG42) led to hyaluronan biosynthesis in transfected mammalian cells. However, only mouse Has2 and Has3 expressing cells formed significant hyaluronan-dependent pericellular coats in culture, implying both functional similarities and differences among vertebrate HAS enzymes. We propose that vertebrate hyaluronan biosynthesis is regulated by a comparatively ancient gene family that has arisen by sequential gene duplication and divergence.Simple linear carbohydrate polymers make up the largest biomass on the Earth. These polymers include cellulose, chitin, and arguably their vertebrate equivalent, hyaluronan (HA).

No abstract

We describe a vertebrate hyaluronan and proteoglycan binding link protein gene family (HAPLN), consisting of four members including cartilage link protein. The encoded proteins share 45-52% overall amino acid identity. In contrast to the average sequence identity between family members, the sequence conservation between vertebrate species was very high. Human and mouse link proteins share 81-96% amino acid sequence identity. Two of the four link protein genes (HAPLN2 and HAPLN4) were restricted in expression to the brain/central nervous system, while one of the four genes (HAPLN3) was widely expressed. Genomic structures revealed that all four HAPLN genes were similar in exon-intron organization and were also similar in genomic organization to the 5 exons for the CSPG core protein genes. Strikingly, all four HAPLN genes were located immediately adjacent to the four CSPG core protein genes creating four pairs of CSPG-HAPLN genes within the mammalian genome. Furthermore, the two brain-specific HAPLN genes (HAPLN2 and HAPLN4) were physically linked to the brain-specific CSPG genes encoding brevican and neurocan, respectively. The tight physical association of the HAPLN and CSPG genes supports a hypothesis that the first HAPLN gene arose as a partial gene duplication event from an ancestral CSPG gene. There is some degree of coordinated expression of each gene pair. Collectively, the four HAPLN genes are expressed by most tissue types, reflecting the fundamental importance of the hyaluronan-dependent extracellular matrix to tissue architecture and function in vertebrate species. Comparison of the genomic structures for the HAPLN, CSPG genes and other members of the link module superfamily provide strong support for a common evolutionary origin from an ancestral gene containing one link module encoding exon.

Hyaluronan synthases (HASs) are plasma membrane enzymes that simultaneously elongate, bind, and extrude the growing hyaluronan chain directly into extracellular space. In cells transfected with green fluorescent protein (GFP)-tagged Has3, the dorsal surface was decorated by up to 150 slender, 3-20-m-long microvillus-type plasma membrane protrusions, which also contained filamentous actin, the hyaluronan receptor CD44, and lipid raft microdomains. Enzymatic activity of HAS was required for the growth of the microvilli, which were not present in cells transfected with other GFP proteins or inactive GFP-Has3 mutants or in cells incubated with exogenous soluble hyaluronan. The microvilli induced by HAS3 were gradually withered by introduction of an inhibitor of hyaluronan synthesis and rapidly retracted by hyaluronidase digestion, whereas they were not affected by competition with hyaluronan oligosaccharides and disruption of the CD44 gene, suggesting independence of hyaluronan receptors. The data bring out the novel concept that the glycocalyx created by dense arrays of hyaluronan chains, tethered to HAS during biosynthesis, can induce and maintain prominent microvilli.Vertebrate cells display slender plasma membrane protrusions like filopodia, microspikes, and microvilli, the formation and maintenance of which are thought to depend on the dynamics of a bundle of actin filaments in the core of these extensions (1). In selected environments, most of the components in the actin polymerization apparatus, when overexpressed or experimentally activated, have been reported to increase the growth of filopodia or microvilli (2-7). This suggests that there is a strong intrinsic potential in cells to display these extensions, ready for implementation by factors even outside the effector and signaling chains directly related to actin. Indeed, at the molecular level, mechanisms that induce the growth of microvilli in vivo are still somewhat obscure (8). We present a novel factor, active hyaluronan synthesis, apparently unrelated to any of the previously described signaling or actin polymerization processes, that triggers extensive microvillus formation in several cell types. Interestingly, the process is primarily driven by the cell surface glycocalyx rather than intracellular systems.Hyaluronan is an ubiquitous pericellular and extracellular matrix glycosaminoglycan important in embryonic development (9), wound healing (10), inflammation (11, 12), mammalian fertilization (13), and cancer (14). It is involved in cell adhesion (15), migration (16 -19), proliferation and epithelial-mesenchymal transition (9), resistance to apoptosis, and multidrug resistance (14). Some of its effects are thought to be mediated by an ability to form a hydrated pericellular matrix, a coat that can modify cellular interactions with other matrix components and neighboring cells, directly regulating processes such as differentiation and migration (20). Some of the biological signals elicited by hyaluronan are dependent on its CD44 receptor on cell su...