Bruce A. Baggenstoss

The Active Streptococcal Hyaluronan Synthases (HASs) Contain a Single HAS Monomer and Multiple Cardiolipin Molecules

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 CStreptococcus equisimilis HAS were compared with the recombinant proteins expressed in Escherichia colimembranes. Based on their amino acid sequences, the masses of these four proteins as monomers are ∼48 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 ∼64 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 ∼23 kDa. Matrix-assisted laser desorption time of flight mass spectrometry analysis of purified S. pyogenes HAS-H6 andS. equisimilis HAS-H6 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 ∼23 kDa to the expected size of the HAS monomer. The ∼23-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 ∼16 CL molecules.

The hyaluronan receptor for endocytosis (HARE) activates NF-κB-mediated gene expression in response to 40-400-kDa, but not smaller or larger, hyaluronans.

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-κB activation. Conclusion: HA-HARE signal complexes activate NF-κB 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 hyaluronan (HA) receptor for endocytosis (HARE; Stabilin-2) binds and clears 14 different ligands, including HA and heparin, via clathrin-mediated endocytosis. HA binding to HARE stimulates ERK1/2 activation (Kyosseva, S. V., Harris, E. N., and Weigel, P. H. (2008) J. Biol. Chem. 283, 15047–15055). To assess a possible HA size dependence for signaling, we tested purified HA fractions of different weight-average molar mass and with narrow size distributions and Select-HATM for stimulation of HARE-mediated gene expression using an NF-κB promoter-driven luciferase reporter system. Human HARE-mediated gene expression was stimulated in a dose-dependent manner with small HA (sHA) >40 kDa and intermediate HA (iHA) <400 kDa. The hyperbolic dose response saturated at 20–50 nm with an apparent Km ∼10 nm, identical to the Kd for HA-HARE binding. Activation was not detected with oligomeric HA (oHA), sHA <40 kDa, iHA >400 kDa, or large HA (lHA). Similar responses occurred with rat HARE. Activation by sHA-iHA was blocked by excess nonsignaling sHA, iHA, or lHA, deletion of the HA-binding LINK domain, or HA-blocking antibody. Endogenous NF-κB activation also occurred in the absence of luciferase plasmids, as assessed by degradation of IκB-α. ERK1/2 activation was also HA size-dependent. The results show that HA-HARE interactions stimulate NF-κB-activated gene expression and that HARE senses a narrow size range of HA degradation products. We propose a model in which optimal length HA binds multiple HARE proteins to allow cytoplasmic domain interactions that stimulate intracellular signaling. This HARE signaling system during continuous HA clearance could monitor the homeostasis of tissue biomatrix turnover throughout the body.

Mutation of Two Intramembrane Polar Residues Conserved within the Hyaluronan Synthase Family Alters Hyaluronan Product Size

We identified two conserved polar amino acids within different membrane domains (MD) of Streptococcus equisimilis hyaluronan synthase (seHAS), Lys48 in MD2 and Glu327 in MD4. In eukaryotic HASs, the position of the Glu is very similar and the Lys is replaced by a conserved polar Gln. To assess whether Lys48 and Glu327 interact or influence seHAS activity, we investigated the effects of changing Lys48 to Arg or Glu and Glu327 to Lys, Asp, or Gln. Mutants, including a double switch variant with Lys48 and Glu327 exchanged, were expressed and assayed in Escherichia coli membranes. SeHAS(E327Q) and seHAS(E327K) were expressed at low levels, whereas seHAS(E327D) and the Lys48 mutants were expressed well. The specific enzyme activities (relative to wild type) were 17 and 7% for the K48R and K48E mutants and 26 and 38% for the E327Q and E327D mutants, respectively. In contrast, seHAS(E327K) showed only 0.16% of wild-type activity but was rescued over 46-fold by changing Lys48 to Glu. Expression of the seHAS(E327K,K48E) protein was also rescued to near wild-type levels. Based on size exclusion chromatography coupled to multiangle laser light scattering analysis, all the variants synthesized hyaluronan (HA) of smaller weight-average molar mass than wild-type enzyme (3.6 MDa); the smallest HA (∼0.6 MDa) was made by seHAS(E327K,K48E) and seHAS(K48E). The results indicate that Glu327 within MD4 is a critical residue for the stability of seHAS, that it may interact with Lys48 within MD2, and that these residues are involved in the ability of HAS to synthesize very large HA.

Increased Hyaluronan Synthase-2 mRNA Expression and Hyaluronan Accumulation with Choroidal Thickening: Response during Recovery from Induced Myopia

PURPOSE Several studies have convincingly shown that in chicks, compensation for imposed focus involves immediate changes in choroid thickness. The molecular events associated with choroidal thickening and the regulation of the choroidal response are largely unknown. METHODS Form-deprivation myopia was induced in the right eyes of 2-day-old chicks by the application of translucent occluders for 10 days and was followed by unrestricted vision for an additional 1 to 20 days (recovery). Individual choroids were isolated from treated and control eyes and used for reverse transcription-quantitative PCR, hyaluronan (HA) localization with biotinylated hyaluronic acid binding protein (b-HABP), and analyses of HA size and concentration by size exclusion chromatography-multiangle laser light scattering (SEC-MALLS). RESULTS HAS2 gene expression increased significantly after 6 hours of unrestricted vision (>7-fold) and peaked at 24 hours (>9-fold). In untreated eyes, HA was localized to perivascular sheaths of larger choroidal blood vessels; however, after 4 to 15 days of recovery, intense labeling for HA was detected throughout the thickened choroidal stroma. Analyses of choroidal HA by SEC-MALLS indicated that HA concentration was significantly increased in recovering choroids compared with controls after 4 to 8 days of recovery (≈3.5-fold). CONCLUSIONS Newly synthesized HA accumulates in the choroidal stroma of recovering eyes and is most likely responsible for the stromal swelling observed during recovery from myopia. This HA accumulation is initiated by a rapid increase in choroidal expression of the HAS2 gene in response to myopic defocus.

Hyaluronan synthase polymerizing activity and control of product size are discrete enzyme functions that can be uncoupled by mutagenesis of conserved cysteines

Streptococcus equisimilis hyaluronan (HA) synthase (SeHAS) contains four cysteines (C226, C262, C281 and C367) that are conserved in the mammalian HAS family. Previous studies of single Cys-to-Ser and all possible Cys-to-Ala mutants of SeHAS found that: the Cys-null mutant is active, Cys modification inhibits HAS activity and the conserved cysteines are clustered at the membrane-enzyme interface in substrate-binding sites (Kumari K, Weigel PH. 2005. Identification of a membrane-localized cysteine cluster near the substrate binding sites of the Streptococcus equisimilis hyaluronan synthase. Glycobiology. 15:529-539). We re-examined these Cys mutants using a single technique (size exclusion chromatography-multi-angle laser light scattering) that allows simultaneous assays on the same sample for both HA synthesis activity and HA product size. Among 18 mutants compared with wild type, 4 showed no change in either function and 3 showed changes in both (decreased activity and HA size). Only one of the two functions was altered in 11 other mutants, which showed either decreased polymerizing activity or product size. No mutants made larger HA, 8 made smaller HA and 10 showed no change in HA size. Nine mutants showed no change in activity and nine were less active. The mutants fell into four of nine possible groups in terms of changes in HA size or synthesis rate (i.e. none, increased or decreased). Specific Cys residues were associated with each mutant group and the pattern of effects on both functions. Thus, the four conserved Cys residues, individually and in specific combinations, influence the rate of sugar assembly by HAS and HA product size, but their participation in one function is independent of the other.

Hyaluronan synthase assembles chitin oligomers with -GlcNAc(α1→)UDP at the reducing end

Class I hyaluronan synthases (HASs) assemble a polysaccharide containing the repeating disaccharide [GlcNAc(β1,4)GlcUA(β1,3)]n-UDP and vertebrate HASs also assemble (GlcNAc-β1,4)n homo-oligomers (chitin) in the absence of GlcUA-UDP. This multi-membrane domain CAZy GT2 family glycosyltransferase, which couples HA synthesis and translocation across the cell membrane, is atypical in that monosaccharides are incrementally assembled at the reducing, rather than the non-reducing, end of the growing polymer. Using Escherichia coli membranes containing recombinant Streptococcus equisimilis HAS, we demonstrate that a prokaryotic Class I HAS also synthesizes chitin oligomers (up to 15-mers, based on MS and MS/MS analyses of permethylated products). Furthermore, chitin oligomers were found attached at their reducing end to -4GlcNAc(α1→)UDP [i.e. (GlcNAcβ1,4)nGlcNAc(α1→)UDP]. These oligomers, which contained up to at least seven HexNAc residues, consisted of β4-linked GlcNAc residues, based on the sensitivity of the native products to jack bean β-N-acetylhexosaminidase. Interestingly, these oligomers exhibited mass defects of -2, or -4 for longer oligomers, that strictly depended on conjugation to UDP, but MS/MS analyses indicate that these species result from chemical dehydrogenations occurring in the gas phase. Identification of (GlcNAc-β1,4)n-GlcNAc(α1→)UDP as HAS reaction products, made in the presence of GlcNAc(α1→)UDP only, provides strong independent confirmation for the reducing terminal addition mechanism. We conclude that chitin oligomer products made by HAS are derived from the cleavage of these novel activated oligo-chitosyl-UDP oligomers. Furthermore, it is possible that these UDP-activated chitin oligomers could serve as self-assembled primers for initiating HA synthesis and ultimately modify the non-reducing terminus of HA with a chitin cap.

What is special about 200 kDa hyaluronan that activates hyaluronan receptor signaling

The polydispersity of hyaluronan (HA) presents challenges for analyzing its solution properties, such as the relationship between mass and particle size. The broad mass range of natural HA (≤50-fold) makes molecular characterization difficult and ambiguous compared to molecules with known molecular weights (e.g., proteins). Biophysical studies show that large >MDa HA behaves like a random coil, whereas very small (e.g., 10 kDa) HA behaves like a rod. However, the mass range for this conformational transition is not easily determined in natural polydisperse HA. Some HA receptors (e.g., CD44 and HARE) initiate signaling responses upon binding HA in the 100-300 kDa range, but not larger MDa HA. Size-dependent responses are studied using nonnatural HA: purified narrow-size range HA [Pandey MS, Baggenstoss BA, Washburn J, Harris EN, Weigel PH. 2013. The hyaluronan receptor for endocytosis (HARE) activates NF-κB-mediated gene expression in response to 40-400 kDa, but not smaller or sarger, hyaluronans. J Biol Chem. 288:14068-14079] and very narrow size range Select-HA made chemo-enzymatically [Jing W, DeAngelis PL. 2004. Synchronized chemoenzymatic synthesis of monodisperse hyaluronan polymers. J Biol Chem. 279:42345-42349]. Here, we used size exclusion chromatography and multiangle light scattering to determine the weight-average molar mass and diameter of ~60 very narrow size preparations from 29 to 1650 kDa. The ratio of HA mass to HA diameter showed a transition in the 150-250 kDa size range (~65 nm). The HA rod-to-coil transition occurs within the size range that specifically activates cell signaling by some receptors. Thus, size-specific signaling could be due to unique external receptor•HA conformation changes that enable transmembrane-mediated activation of cytoplasmic domains. Alternatively and more likely, transition-size HA may enable multiple receptors to bind the same HA, creating new internal signal-competent cytoplasmic domain complexes.

Hyaluronan synthase assembles hyaluronan on a [GlcNAc(β1,4)]n-GlcNAc(α1→)UDP primer and hyaluronan retains this residual chitin oligomer as a cap at the nonreducing end

Class I hyaluronan synthases (HAS) assemble [GlcNAc(β1,4)GlcUA(β1,3)]n-UDP at the reducing end and also make chitin. Streptococcus equisimilis HAS (SeHAS) also synthesizes chitin-UDP oligosaccharides, (GlcNAc-β1,4)n-GlcNAc(α1→)UDP (Weigel et al. 2015). Here we determined if HAS uses chitin-UDPs as primers to initiate HA synthesis, leaving the non-HA primer at the nonreducing (NR) end. HA made by SeHAS membranes was purified, digested with streptomyces lyase, and hydrophobic oligomers were enriched by solid phase extraction and analyzed by MALDI-TOF MS. Jack bean hexosaminidase (JBH) and MS/MS were used to analyze 19 m/z species of possible GnHn ions with clustered GlcNAc (G) residues attached to disaccharide units (H): (GlcNAcβ1,4)2-5[GlcUA(β1,3)GlcNAc]2-6. JBH digestion sequentially removed GlcNAc from the NR-end of GnHn oligomers, producing successively smaller GnH2-3 series members. Since lyase releases dehydro-oligos (dHn; M-18), only the unique NR-end oligo lacks dehydro-GlcUA. Hn oligomers were undetectable in lyase digests, whereas JBH treatment created new H2-6m/z peaks (i.e. HA tetra- through dodeca-oligomers). MS/MS of larger GnHn species produced chitin (2-5 GlcNAcs), HA oligomers and multiple smaller series members with fewer GlcNAcs. All NR-ends (97%) started with GlcNAc, as a chitin trimer (three GlcNAcs), indicating that GlcNAc(β1,4)2GlcNAc(α1→)-UDP may be optimal for initiation of HA synthesis. Also, HA made by live S. pyogenes cells had G4Hn chitin-oligo NR-ends. We conclude that chitin-UDP functions in vitro and in live cells as a primer to initiate synthesis of all HA chains and these primers remain at the NR-ends of HA chains as residual chitin caps [(GlcNAc-β1,4)3-4].

Methods for Measuring Class I Membrane-Bound Hyaluronan Synthase Activity

Detecting and quantifying hyaluronan (HA) made by Class I HA synthase (HAS) and determining the level of activity of these membrane-bound enzymes is critical in studies to understand the normal biology of HA and how changes in HAS activity and HA levels or size are important in inflammatory and other diseases, tumorigenesis, and metastasis. Unlike the products made by the vast majority of glycosyltransferases, HA products are more complicated since they are made as a heterogeneous population of sizes spanning a broad mass range. Three radioactive and nonradioactive assay methods are described that can give the amount of HA made with or without information about the distribution of product sizes.

Clustered Conserved Cysteines in Hyaluronan Synthase Mediate Cooperative Activation by Mg(2+) Ions and Severe Inhibitory Effects of Divalent Cations.

Hyaluronan synthase (HAS) uses UDP-GlcUA and UDP-GlcNAc to make hyaluronan (HA). Streptococcus equisimilis HAS (SeHAS) contains four conserved cysteines clustered near the membrane, and requires phospholipids and Mg2+ for activity. Activity of membrane-bound or purified enzyme displayed a sigmoidal saturation profile for Mg2+ with a Hill coefficient of 2. To assess if Cys residues are important for cooperativity we examined the Mg2+ dependence of mutants with various combinations of Cys-to-Ala mutations. All Cys-mutants lost the cooperative response to Mg2+. In the presence of Mg2+, other divalent cations inhibited SeHAS with different potencies (Cu2+~Zn2+ >Co2+ >Ni2+ >Mn2+ >Ba2+ Sr2+ Ca2+). Some divalent metal ions likely inhibit by displacement of Mg2+-UDP-Sugar complexes (e.g. Ca2+, Sr2+ and Ba2+ had apparent Ki values of 2-5 mM). In contrast, Zn2+ and Cu2+ inhibited more potently (apparent Ki ≤ 0.2 mM). Inhibition of Cys-null SeHAS by Cu2+, but not Zn2+, was greatly attenuated compared to wildtype. Double and triple Cys-mutants showed differing sensitivities to Zn2+ or Cu2+. Wildtype SeHAS allowed to make HA prior to exposure to Zn2+ or Cu2+ was protected from inhibition, indicating that access of metal ions to sensitive functional groups was hindered in processively acting HA•HAS complexes. We conclude that clustered Cys residues mediate cooperative interactions with Mg2+ and that transition metal ions inhibit SeHAS very potently by interacting with one or more of these -SH groups.