The two hyaluronan synthases (HASs) from Streptococcus pyogenes (spHAS) and Streptococcus equisimilis (seHAS) were expressed in Escherichia coli as recombinant proteins containing His 6 tails. Both enzymes were expressed as major membrane proteins, accounting for ϳ5-8% of the total membrane protein. Using nickel chelate affinity chromatography, the HASs were purified to homogeneity from n-dodecyl ␤-D-maltoside extracts. High levels of HAS activity could be achieved only if the purified enzymes were supplemented with either bovine or E. coli cardiolipin (CL), although bovine CL gave consistently greater activity. Mass spectroscopic analysis revealed that the fatty acid compositions of these two CL preparations did not overlap. The two HAS enzymes showed similar but distinct activation profiles with the 10 other lipids tested. For example, phosphatidic acid and phosphatidylethanolamine stimulated seHAS, but not spHAS. Phosphatidylserine stimulated both enzymes. spHAS appears to be more CL-specific than se-HAS, although both purified enzymes still contain endogenous CL that can not easily be removed. Both seHAS and spHAS were inhibited by phosphatidylcholine, sphingomyelin, and sulfatides and were not substantially stimulated by cerebrosides, phosphatidylglycerol, or phosphatidylinositol. With both HASs, CL increased the K m for UDP-GlcUA, but decreased the K m for UDP-GlcNAc and gave an overall stimulation of V max . A kinetic characterization of the two membrane-bound and purified HASs is presented in the accompanying paper (Tlapak-Simmons, V. L., Baggenstoss, B. A., Kumari, K., Heldermon, C., and Weigel, P. H. (1999) J. Biol. Chem. 274, 4246 -4253). Both purified HASs became inactive after storage for ϳ5 days at 4°C. Both purified enzymes also lost activity over 4 -5 days when stored at -80°C in the presence of CL, but reached a level of activity that then slowly decreased over a period of months. Although the purified enzymes stored in the absence of CL at ؊80°C were much less active, the enzymes retained this same low level of activity for at least 5 weeks. When both spHAS and seHAS were stored without CL at ؊80°C, even after 2 months, they could be stimulated by the addition of bovine CL to ϳ60% of the initial activity of the freshly purified enzyme.Since the discovery of HA 1 over 60 years ago (1), this saccharide polymer, which contains repeating disaccharide units of GlcUA␤(1,3)GlcNAc␤(1,4), has been shown to have numerous biological functions. For example, HA provides the viscous lubrication of synovial fluid in joints and provides cartilage with its viscoelastic properties. HA is involved in a wide variety of cellular functions and behaviors, including cell migration (2, 3) development (4 -6), differentiation (7-9), phagocytosis (6), and proteoglycan synthesis (2, 4). As well as being a major structural component of the matrix, HA has wound healing, pharmaceutical, and analgesic effects (10 -14) and is also being used as a vehicle for drug delivery (15,16).Although cell-free HA biosynthesis was achieved ...

OBJECTIVE Oligodendrocyte progenitor cells (OPCs) recruited to demyelinating lesions often fail to mature into oligodendrocytes (OLs) that remyelinate spared axons. The glycosaminoglycan hyaluronan (HA) accumulates in demyelinating lesions and has been implicated in the failure of OPC maturation and remyelination. We tested the hypothesis that OPCs in demyelinating lesions express a specific hyaluronidase, and that digestion products of this enzyme inhibit OPC maturation. METHODS Mouse OPCs grown in vitro were analyzed for hyaluronidase expression and activity. Gain of function studies were used to define the hyaluronidases that blocked OPC maturation. Mouse and human demyelinating lesions were assessed for hyaluronidase expression. Digestion products from different hyaluronidases and a hyaluronidase inhibitor were tested for their effects on OPC maturation and functional remyelination in vivo. RESULTS OPCs demonstrated hyaluronidase activity in vitro and expressed multiple hyaluronidases including HYAL1, HYAL2, and PH20. HA digestion by PH20 but not other hyaluronidases inhibited OPC maturation into OLs. In contrast, inhibiting HA synthesis did not influence OPC maturation. PH20 expression was elevated in OPCs and reactive astrocytes in both rodent and human demyelinating lesions. HA-digestion products generated by the PH20 hyaluronidase but not another hyaluronidase inhibited remyelination following lysolecithin-induced demyelination. Inhibition of hyaluronidase activity lead to increased OPC maturation and promoted increased conduction velocities through lesions. INTERPRETATION We determined that PH20 is elevated in demyelinating lesions and that increased PH20 expression is sufficient to inhibit OPC maturation and remyelination. Pharmacological inhibition of PH20 may therefore be an effective way to promote remyelination in multiple sclerosis and related conditions.

Background: HARE mediates systemic clearance of hyaluronan (HA), which turns over continuously in tissues. Results: HARE uptake of 40 -400-kDa, but not larger or smaller, HA stimulated NF-B activation. Conclusion: HA-HARE signal complexes activate NF-B and gene transcription only with optimally sized HA. Significance: HARE responsiveness to a narrow size range of HA degradation products may be a sensing system to detect tissue ECM stress.

The functional sizes of the two streptococcal hyaluronan synthases (HASs) were determined by radiation inactivation analysis of isolated membranes. The native enzymes in membranes from Group A Streptococcus pyogenes HAS and Group C Streptococcus equisimilis HAS were compared with the recombinant proteins expressed in Escherichia coli membranes. Based on their amino acid sequences, the masses of these four proteins as monomers are ϳ48 kDa. In all cases, loss of enzyme activity was a simple single exponential function with increasing radiation dose. The functional sizes calculated from these data were identical for the four HASs at ϳ64 kDa. In contrast, the sizes of the proteins estimated by the loss of antibody reactivity on Western blots were essentially identical at 41 kDa for the four HAS species, consistently lower than the functional size by ϳ23 kDa. Matrix-assisted laser desorption time of flight mass spectrometry analysis of purified S. pyogenes HAS-H 6 and S. equisimilis HAS-H 6 gave masses that differed by <0.07% from the predicted monomer sizes, which confirms that neither protein is posttranslationally modified or covalently attached to another protein. Ongoing studies indicate that the purified HAS enzymes require cardiolipin (CL) for maximal activity and stability. When irradiated membranes were detergent solubilized and the extracts were incubated with exogenous CL, the residual level of HAS activity increased. Consequently, the calculated functional size decreased by ϳ23 kDa to the expected size of the HAS monomer. The ϳ23-kDa larger size of the functional HAS enzyme, compared with the HAS monomer, is due, therefore, to CL molecules. We propose that the active streptococcal HA synthases are monomers in complex with ϳ16 CL molecules.

The two hyaluronan synthases (HASs) from Streptococcus pyogenes (spHAS) and Streptococcus equisimilis (seHAS) were expressed in Escherichia coli as recombinant proteins containing His 6 tails. The accompanying paper has described the purification and lipid dependence of both HASs, their preference for cardiolipin, and their stability during storage (Tlapak-Simmons, V. L., Baggenstoss, B. A., Clyne, T., and Weigel, P. H. (1999) J. Biol. Chem. 274, 4239 -4245). Kinetic characterization of the enzymes in isolated membranes gave K m values for UDP-GlcUA of 40 ؎ 4 M for spHAS and 51 ؎ 5 M for seHAS. In both cases, the V max profiles at various concentrations of UDP-GlcNAc were hyperbolic, with no evidence of cooperativity. In contrast, membrane-bound spHAS, but not seHAS, showed sigmoidal behavior as the UDP-GlcNAc concentration was increased, with a Hill number of ϳ2, indicating significant cooperativity. The Hill number for UDP-GlcNAc utilization by seHAS was 1, confirming the lack of cooperativity for UDPGlcNAc in this enzyme. The K m values for UDP-GlcNAc were 60 ؎ 7 M for seHAS and 149 ؎ 3 M for spHAS in the isolated membranes. The kinetic characteristics of the two affinity-purified HAS enzymes were assessed in the presence of cardiolipin after 8 -9 days of storage at -80°C without cardiolipin. With increasing storage time, the enzymes showed a gradual increase in their K m values for both substrates and a decrease in V max . Even in the presence of cardiolipin, the detergent-solubilized, purified HASs had substantially higher K m values for both substrates than the membrane-bound enzymes. The K UDP-GlcUA for purified spHAS and seHAS increased 2-4-fold. The K UDP-GlcNAc for spHAS and seHAS increased 4-and 5-fold, respectively. Despite the higher K m values, the V max values for the purified HASs were only ϳ50% lower than those for the membrane-bound enzymes. Significantly, purified spHAS displayed the same cooperative interaction with UDP-GlcNAc (n H ϳ 2), whereas purified seHAS showed no cooperativity. HA1 is a polysaccharide composed of two alternating sugars, ␤1,3-linked glucuronic acid and ␤1,4-linked N-acetylglucosamine (1). Although the structure of HA seems quite simple, the molecule, nonetheless, has unusual physical properties that are important for its numerous biological functions (2-5). For example, HA forms very viscous solutions and gels due to its high molecular mass and its ability to bind cations and to hydrate large amounts of water. This characteristic of HA provides the viscous lubrication of synovial fluid and helps provide cartilage with its viscoelasticity. These characteristics are also ideal for the role HA has in the extracellular matrices (4, 5) of the skin and virtually every vertebrate tissue as well as in the fluid of the vitreous humor of the eye. HA also plays an important role in morphogenesis, wound healing (6 -9), and angiogenesis (10, 11). HA receptors and HA-binding proteins, particularly CD44 (12) and the receptor for hyaluronic acidmediated mobility (RHAMM; Ref. 13), modul...