Papers by Valarie Simmons
Journal of Biological Chemistry, 1999
The two hyaluronan synthases (HASs) from Streptococcus pyogenes (spHAS) and Streptococcus equisim... more 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.
Journal of Biological Chemistry, 1998
The functional sizes of the two streptococcal hyaluronan synthases (HASs) were determined by radi... more 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 HASH 6 and S. equisimilis HASH 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. Hyaluronan (HA) 1 is a linear heteropolysaccharide composed of the repeating disaccharide N-acetyl-D-glucosamine-(134)
Biochemistry, 2004
Hyaluronan synthase (HAS) utilizes UDP-GlcUA and UDP-GlcNAc in the presence of Mg 2+ to form the ... more Hyaluronan synthase (HAS) utilizes UDP-GlcUA and UDP-GlcNAc in the presence of Mg 2+ to form the GAG hyaluronan (HA). The purified HAS from Streptococcus equisimilis (seHAS) shows high fidelity in that it only polymerizes the native substrates, UDP-GlcNAc and UDP-GlcUA. However, other uridinyl nucleotides and UDP-sugars inhibited enzyme activity, including UDP-GalNAc, UDP-Glc, UDP-Gal, UDP-GalUA, UMP, UDP and UTP. Purified seHAS was ~40% more active in 25 mM, compared to 50 mM, PO 4 in the presence of either 50 mM NaCl or KCl, and displayed a slight preference for KCl over NaCl. The pH profile was surprisingly broad, with an effective range of pH 6.5-11.5 and the optimum between pH 9 and 10. SeHAS displayed two apparent pKa values at pH 6.6 and 11.8. As the pH was increased from ~6.5, both K m and V max increased until pH ~10.5, above which the kinetic constants gradually declined. Nonetheless, the overall catalytic constant (120/sec) was essentially unchanged from pH 6.5 to pH 10.5. The enzyme is temperature labile, but more stable in the presence of substrate and cardiolipin. Purified seHAS requires exogenous cardiolipin for activity and is very sensitive to the fatty acyl composition of the phospholipid. The enzyme was inactive or highly activated by synthetic cardiolipins containing, respectively, C14:0 or C18:1(Δ9) fatty acids. The apparent E a for HA synthesis is 40 kJ (9.5 kcal/mol) disaccharide. Increasing the viscosity by increasing concentrations of PEG, ethylene glycol, glycerol, or sucrose inhibited seHAS activity. For PEGs, the extent of inhibition was proportional to their molecular mass. PEGs with average masses of 2.7, 11.7, and 20 Kg/mol caused 50% inhibition of V max at 21, 6.5, and 3.5 mM, respectively. The apparent K i values for ethylene glycol, glycerol, and sucrose were, respectively, 4.5, 3.3 and 1.2 mM.
Journal of Biological Chemistry, 1994
The pH and temperature dependence of the allosteric properties of phosphofructokinase (PFK) from ... more The pH and temperature dependence of the allosteric properties of phosphofructokinase (PFK) from Bacillus stearothermophilus have been studied from 5 to 9 and 6 to 40°C, respectively. Throughout this pH and temperature range the allosteric ligands MgADP and phospho(enol)pyruvate (PEP) have no effect on k cat. The dissociation constants of the substrate, fructose 6-phosphate, and the allosteric ligands, as well as the absolute value of the coupling free energies between these ligands, all increase when the pH is raised, indicating that the inhibition by PEP and the activation by MgADP increase despite each ligand's somewhat lower affinity. However, the constituent coupling enthalpies and entropies substantially diminish in absolute value as pH is increased, suggesting that the magnitudes of molecular perturbations engendered by the binding of allosteric ligands do not correlate with the magnitudes of the functional consequences of those perturbations. Temperature and pH exert their influence on the observed allosteric behavior by changing the relative contributions made by the largely compensating ⌬H and T⌬S terms to the coupling free energy. MATERIALS AND METHODS Materials Biochemicals and buffers were purchased from Sigma Chemical Co. (St. Louis, MO). The coupling enzymes were purchased from Boehringer
Previous studies reached different conclusions about whether class I hyaluronan synthases (HASs) ... more Previous studies reached different conclusions about whether class I hyaluronan synthases (HASs) elongate hyaluronic acid (HA) by addition to the reducing or the nonreducing end. Here we used two strategies to determine the direction of HA synthesis by purified class I HASs from Streptococcus equisimilis and Streptococcus pyogenes. In the first strategy we used each of the two UDP-sugar substrates separately to pulse label either the beginning or the end of HA chains. We then quantified the relative rates of radioactive HA degradation by treatment with -glycosidases that act at the nonreducing end. The results with both purified HASs demonstrated that HA elongation occurred at the reducing end. In the second strategy, we used purified S. equisimilis HAS, UDP-glucuronic acid, and UDP[-32 P]-Glc-NAc to radiolabel nascent HA chains. Under conditions of limiting substrate, the 32 P-labeled products were separated from the substrates by paper chromatography and identified as HA-[ 32 P]UDP saccharides based on their degradation by snake venom phosphodiesterase or hyaluronidase and by their binding to a specific HAbinding protein. The 32 P radioactivity was chased (released) by incubation with unlabeled UDP-sugars, showing that the HA-UDP linkages turn over during HA biosynthesis. In contrast, HA-[ 32 P]UDP products made by the purified class II Pasteurella multocida HAS were not released by adding unlabeled UDP-sugars, consistent with growth at the nonreducing end for this enzyme. The results demonstrate that the streptococcal class I HAS enzymes polymerize HA chains at the reducing end.
The Journal of Biological Chemistry, Jan 17, 2002
Hyaluronan (HA) synthase (HAS) is a membrane-bound enzyme that utilizes UDP-glucuronic acid (GlcU... more Hyaluronan (HA) synthase (HAS) is a membrane-bound enzyme that utilizes UDP-glucuronic acid (GlcUA) and UDP-GlcNAc to synthesize HA. The HAS from Streptococcus pyogenes (spHAS, 419 amino acids) contains six Cys residues, whereas the enzyme from Streptococcus equisimilis (seHAS, 417 amino acids) contains four Cys residues. These Cys residues of seHAS are highly conserved in all Class I HAS family members. Here we investigated the structural and functional roles of these conserved cysteines in seHAS by using site-directed mutagenesis and sensitivity to sulfhydryl modifying reagents. Both seHAS and spHAS were inhibited by sulfhydryl reagents such as N-ethylmaleimide (NEM) and iodoacetamide in a dose-dependent and time-dependent manner. These inhibition curves were biphasic, indicating the presence of sensitive and insensitive components. After treatment of seHAS with NEM, the V max value was decreased ϳ50%, and the K m values changed only slightly. All the Cys-to-Ala mutants of seHAS were partially active. The least active single (C226A), double (C226A,C262A), or triple (C226A,C262A,C367A) Cys mutants retained 24, 3.2, and 1.4% activity, respectively, compared with wild-type enzyme. Surprisingly, the V max value of the seHAS cys-null mutant was ϳ17% of wild-type, although the K m values for both substrates were increased 3-6-fold. Cys residues, therefore, are not involved in a critical interaction necessary for either substrate binding or catalysis. However, the distribution of HA products was shifted to a smaller size in ϳ25% of the seHAS Cys mutants, particularly the triple mutants. Mass spectroscopic analysis of wild-type and Cys-null seHAS as well as the labeling of all double Cys-to-Ala mutants with [ 14 C]NEM demonstrated that se-HAS contains no disulfide bonds. We conclude that the four Cys residues in seHAS are not directly involved in catalysis, but that one or more of these Cys residues are located in or near substrate binding or glycosyltransferase active sites, so that their modification hinders the functions of HAS.
Biochemical Society Transactions, 1999
Journal of Biological Chemistry, Feb 12, 1999
The two hyaluronan synthases (HASs) from Streptococcus pyogenes (spHAS) and Streptococcus equisim... more 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 UDP-GlcNAc 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.
Journal of Glycomics & Lipidomics, 2012
The linear glycosaminoglycan hyaluronan (HA) is composed of unmodified disaccharide repeats: [-D-... more The linear glycosaminoglycan hyaluronan (HA) is composed of unmodified disaccharide repeats: [-D-glucuronic acid-(β1,3)-D-N-acetylglucosamine-(β-1,4)] n. HA is ubiquitous in vertebrate extracellular matrices, and it is a major component in tissues such as cartilage and dermis, and in synovial and vitreous fluids [1,2]. HA is also made by some prokaryotes and fungi [3-5]. HA plays an important role during fertilization, embryogenesis, development, wound healing, and differentiation [6,7] and in a variety of cellular functions such as migration and matrix assembly [8-10].
Glycobiology, 2001
Hyaluronan synthase (HAS), the enzyme responsible for the production of hyaluronic acid (HA), is ... more Hyaluronan synthase (HAS), the enzyme responsible for the production of hyaluronic acid (HA), is a well-conserved membrane-bound protein in both prokaryotes and eukaryotes. This enzyme performs at least six discrete functions in producing a heterodisaccharide polymer of several million molecular weight and extruding it from the cell. Among the conserved motifs and domains within the Class I HAS family are four cysteine residues. Cysteines in many proteins are important in establishing and maintaining tertiary structure or in the coordination of catalytic functions. In the present study we utilized a combination of site-directed mutagenesis, chemical labeling, and kinetic analyses to determine the importance of specific Cys residues for catalysis and structure of the HA synthase from Streptococcus pyogenes (spHAS). The enzyme activity of spHAS was partially inhibited by cysteine-reactive chemical reagents such as N-ethylmaleimide. Quantitation of the number of Cys residues modified by these reagents, using MALDI-TOF mass spectrometry, demonstrated that there are no stable disulfide bonds in spHAS. The six Cys residues of spHAS were then mutated, individually and in various combinations, to serine or alanine. The single Cys-mutants were all kinetically similar to the wild-type enzyme in terms of their V max and K m values for HA synthesis. The Cys-null mutant, in which all Cys residues were mutated to alanine, retained ∼66% of wild-type activity, demonstrating that despite their high degree of conservation within the HAS family, Cys residues are not absolutely necessary for HA biosynthesis by the spHAS enzyme.
European Journal of Biochemistry, 2002
As observed previously in cultured human skin fibroblasts, a decrease of hyaluronan production wa... more As observed previously in cultured human skin fibroblasts, a decrease of hyaluronan production was also observed in group C Streptococcus equi FM100 cells treated with 4-methylumbelliferone (MU), although there was no effect on their growth. In this study, the inhibition mechanism of hyaluronan synthesis by MU was examined using Streptococcus equi FM100, as a model. When MU was added to a reaction mixture containing the two sugar nucleotide donors and a membrane-rich fraction as an enzyme source in a cell-free hyaluronan synthesis experiment, there was no change in the production of hyaluronan. On the contrary, when MU was added to the culture medium of FM100 cells, hyaluronan production in the isolated membranes was decreased in a dose-dependent manner. However, when the effect of MU on the expression level of hyaluronan synthase was examined, MU did not decrease either the mRNA level of the has operon containing the hyaluronan synthase gene or the protein level of hyaluronan synthase. Solubilization of the enzyme from membranes of MU-treated cells and addition of the exogenous phospholipid, cardiolipin, rescued hyaluronan synthase activity. In the mass spectrometric analysis of the membrane phospholipids from FM100 cells treated with MU, changes were observed in the distribution of only cardiolipin species but not of the other major phospholipid, PtdGro. These results suggest that MU treatment may cause a decrease in hyaluronan synthase activity by altering the lipid environment of membranes, especially the distribution of different cardiolipin species, surrounding hyaluronan synthase.
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Papers by Valarie Simmons