The functional molecular mass of the Pasteurella hyaluronan synthase is a monomer

Hyaluronan (HA), a linear polysaccharide composed of beta1,3-GlcNAc-beta1,4-GlcUA repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. All known HA synthases (HASs) are dual-action glycosyltransferases that catalyze the addition of two different sugars from UDP-linked precursors to the growing HA chain. The bacterial hyaluronan synthase, PmHAS from Gram-negative Pasteurella multocida, is a 972-residue membrane-associated protein. Previously, the Gram-positive Streptococcus pyogenes enzyme, SpHAS (419 residues), and the vertebrate enzyme, XlHAS1 (588 residues), were found to function as monomers of protein, but the PmHAS is not similar at the protein sequence level and has quite different enzymological properties. We have utilized radiation inactivation to measure the target size of recombinant full-length and truncated PmHAS. The target size of HAS activity was confirmed using internal enzyme standards of known molecular weight. We found that the Pasteurella HA synthase protein functions catalytically as a monomer. Functional truncated soluble PmHAS also behaves as a polypeptide monomer as assessed by gel filtration chromatography and light scattering.     

The functional molecular mass of the Pasteurella hyaluronan synthase is a monomer

Hyaluronan (HA), a linear polysaccharide composed of β1,3-GlcNAc-β1,4-GlcUA repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. All known HA synthases (HASs) are dual-action glycosyltransferases that catalyze the addition of two different sugars from UDP-linked precursors to the growing HA chain. The bacterial hyaluronan synthase, PmHAS from Gram-negative Pasteurella multocida, is a 972-residue membrane-associated protein. Previously, the Gram-positive Streptococcus pyogenes enzyme, SpHAS (419 residues), and the vertebrate enzyme, XlHAS1 (588 residues), were found to function as monomers of protein, but the PmHAS is not similar at the protein sequence level and has quite different enzymological properties. We have utilized radiation inactivation to measure the target size of recombinant full-length and truncated PmHAS. The target size of HAS activity was confirmed using internal enzyme standards of known molecular weight. We found that the Pasteurella HA synthase protein functions catalytically as a monomer. Functional truncated soluble PmHAS also behaves as a polypeptide monomer as assessed by gel filtration chromatography and light scattering.     

Molecular cloning, genomic organization and developmental expression of the Xenopus laevis hyaluronan synthase 3.

The content of hyaluronan (HA), a polymer of the extracellular matrix involved in a variety of physiological and pathological processes, depends on the activity of synthetic (HAS) and degrading enzymes. Since HA is also involved in embryogenesis, we have used Xenopus as a model organism because information is available for HAS1 and HAS2, but not for HAS3. We report the sequence of xlHAS3 mRNA, its genomic organization and its expression in adult tissues as well as during embryonic development. Interestingly, evidence from in situ hybridization indicates that xlHAS3 expression is restricted to the developing inner ear and cement gland. In addition, we have correlated the expression pattern of the enzymes involved in HA metabolism with the HA content during development.     

Biosynthesis of hyaluronan: direction of chain elongation

Hyaluronan (HA), a functionally essential glycosaminoglycan in vertebrate tissues and a putative virulence factor in certain pathogenic bacteria, is an extended linear polymer composed of alternating units of glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc). Uncertainty regarding the mechanism of HA biosynthesis has included the directionality of chain elongation, i.e. whether addition of monosaccharide units occurs at the reducing or non-reducing terminus of nascent chains. We have investigated this problem using yeast-derived recombinant HA synthases from Xenopus laevis (xlHAS1) and from Streptococcus pyogenes (spHAS). The enzymes were incubated with UDP-[3H]GlcUA and UDP-[14C]GlcNAc, under experimental conditions designed to yield HA chains with differentially labeled reducing-terminal and non-reducing terminal domains. Digestion of the products with a mixture of beta-glucuronidase and beta-N-acetylglucosaminidase exoenzymes resulted in truncation of the HA chain strictly from the non-reducing end and release of labeled monosaccharides. The change in 3H/14C ratio of the monosaccharide fraction, during the course of exoglycosidase digestion, was interpreted to indicate whether sugar units had been added at the reducing or non-reducing end. The results demonstrate that the vertebrate xlHAS1 and the bacterial spHAS extend HA in opposite directions. Chain elongation catalyzed by xlHAS1 occurs at the non-reducing end of the HA chain, whereas elongation catalyzed by spHAS occurs at the reducing end. The spHAS is the first glycosyltransferase that has been unanimously demonstrated to function at the reducing end of a growing glycosaminoglycan chain.     

Radiation inactivation analysis of influenza virus reveals different target sizes for fusion, leakage, and neuraminidase activities

The size of the functional units responsible for several activities carried out by the influenza virus envelope glycoproteins was determined by radiation inactivation analysis. Neuraminidase activity, which resides in the glycoprotein NA, was inactivated exponentially with an increasing radiation dose, yielding a target size of 94 +/- 5 kilodaltons (kDa), in reasonable agreement with that of the disulfide-bonded dimer (120 kDa). All the other activities studied are properties of the HA glycoprotein and were normalized to the known molecular weight of the neuraminidase dimer. Virus-induced fusion activity was measured by two phospholipid dilution assays: relief of energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dipalmitoyl-L-alpha- phosphatidylethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)-dioleoyl-L-alpha-phosphatidylethanolamine (N-Rh-PE) in target liposomes and relief of self-quenching of N-Rh-PE in target liposomes. Radiation inactivation of fusion activity proceeded exponentially with radiation dose, yielding normalized target sizes of 68 +/- 6 kDa by assay i and 70 +/- 4 kDa by assay ii. These values are close to the molecular weight of a single disulfide-bonded (HA1 + HA2) unit (75 kDa), the "monomer" of the HA trimer. A single monomer is thus inactivated by each radiation event, and each monomer (or some part of it) constitutes a minimal functional unit capable of mediating fusion. Virus-induced leakage of calcein from target liposomes and virus-induced leakage of hemoglobin from erythrocytes (hemolysis) both showed more complex inactivation behavior: a pronounced shoulder was present in both inactivation curves, followed by a steep drop in activity at higher radiation levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of subunit interactions on enzymatic activity of glutathione S-transferases: a radiation inactivation study.

The glutathione S-transferases are a family of dimeric enzymes. Three isozymes from the alpha family, termed YaYa, YaYc, and YcYc, and three from the mu family, termed Yb1Yb1, Yb1Yb2, and Yb2Yb2, were purified from rat liver. Binding studies were performed by equilibrium dialysis using a radiolabeled product, S(-)[14C](dinitrophenyl)glutathione. Each isozyme contained two independent binding sites which had equal affinity for the ligand. The presence of two independent active sites per enzyme dimer suggests that each subunit contains a complete active site. This conclusion was examined further using radiation inactivation which also allowed for assessment of the importance of subunit interactions in catalytic activity. The activity target size of YaYa (47 kDa) was significantly larger than the protein monomer target size (31 kDa); similarly the activity target size of YaYc was that of the dimer (54 kDa). In contrast, the activity target sizes of Yb1Yb1 and Yb2Yb2 were the same, being 35 and 29 kDa, respectively, and the protein monomer target size of Yb1Yb1 also was similar, being 32 kDa. These data indicate that interactions between subunits are critical for the maintenance of enzymatic activity of alpha class enzymes whereas each subunit of the two mu class proteins is capable of independent catalytic activity.     

Differential effect of growth factors on hyaluronan synthase gene expression in fibroblasts exposed to oxidative stress

The aim of this study was to evaluate how growth factors (PDGF-BB, EGF, and TGF-1beta) modulate hyaluronan synthase (HAS) activities in normal or stressed cultured human skin fibroblasts. The effects of concomitant treatment with cytokines and FeSO4 plus ascorbate on HAS mRNA expression, protein synthesis, and hyaluronic acid (HA) concentrations were also studied. Treatment of fibroblasts with growth factors up-regulated HAS gene expression and increased HAS enzymes and HA production. PDGF-BB induced HAS mRNA expression, protein synthesis, and HA production more efficiently than EGF and TGF-1beta. EGF was less effective than TGF-1beta. In addition, TGF-1beta reduced the expression and synthesis of HAS3, while PDGF-BB and EGF had the opposite effect. Concomitant treatment with growth factors and the oxidant was able to further increase HAS mRNA expression, once again with the exception of HAS3 with TGF-1beta. HAS protein synthesis was reduced, while HA levels were unaffected in comparison to those obtained from exposure to FeSO4 plus ascorbate alone. In conclusion, although growth factors plus the oxidant synergistically induced HAS mRNA expression in part, enzyme production was not correlated with this increase. Moreover, the increase in HAS mRNA levels was not translated into a consequent rise in HA concentration.     

Engineering of cell membrane to enhance heterologous production of hyaluronic acid in Bacillus subtilis

Hyaluronic acid (HA) is a high‐value biopolymer used in the biomedical, pharmaceutical, cosmetic, and food industries. Current methods of HA production, including extraction from animal sources and streptococcal cultivations, are associated with high costs and health risks. Accordingly, the development of bioprocesses for HA production centered on robust “Generally Recognized as Safe (GRAS)” organisms such as Bacillus subtilis is highly attractive. Here, we report the development of novel strains of B. subtilis in which the membrane cardiolipin (CL) content and distribution has been engineered to enhance the functional expression of heterologously expressed hyaluronan synthase (HAS) of Streptococcus equisimilis (SeHAS), in turn, improving the culture performance for HA production. Elevation of membrane CL levels via overexpressing components involved in the CL biosynthesis pathway, and redistribution of CL along the lateral membrane via repression of the cell division initiator protein FtsZ resulted in increases to the HA titer of up to 204% and peak molecular weight of up to 2.2 MDa. Moreover, removal of phosphatidylethanolamine and neutral glycolipids from the membrane of HA‐producing B. subtilis via inactivation of pssA and ugtP, respectively, has suggested the lipid dependence for functional expression of SeHAS. Our study demonstrates successful application of membrane engineering strategies to develop an effective platform for biomanufacturing of HA with B. subtilis strains expressing Class I streptococcal HAS.     

Target size analysis by radiation inactivation: the use of free radical scavengers

Several model systems were employed to assess indirect effects that occur in the process of using radiation inactivation analysis to determine protein target sizes. In the absence of free radical scavengers, such as mannitol and benzoic acid, protein functional unit sizes can be drastically overestimated. In the case of glutamate dehydrogenase, inclusion of free radical scavengers reduced the apparent target size from that of a hexamer to that of a trimer based on enzyme activity determinations. For glucose-6-phosphate dehydrogenase, the apparent target size was reduced from a dimer to a monomer. The target sizes for both glutamate dehydrogenase and glucose-6-phosphate dehydrogenase in the presence of free radical scavengers corresponded to subunit sizes when determinations of protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or immunoblotting were done rather than enzyme activity. The free radical scavengers appear to compete with proteins for damage by secondary radiation products, since irradiation of these compounds can result in production of inhibitory species. Addition of benzoic acid/mannitol to samples undergoing irradiation was more effective in eliminating secondary damage than were 11 other potential free radical scavenging systems. Addition of a free radical scavenging system enables more accurate functional unit size determinations to be made using radiation inactivation analysis.     

An engineered hyaluronan synthase: characterization for recombinant human hyaluronan synthase 2 Escherichia coli

The Class I hyaluronan synthase (HAS) is a unique glycosyltransferase synthesizing hyaluronan (HA), a polysaccharide composed of GlcUA and GlcNAc, by using one catalytic domain that elongates two different monosaccharides. As for the synthetic mechanism, there are two alternative manners for the sugar elongation process. Some bacterial HASs add new sugars to the non-reducing end of the acceptor to grow polymers. On the other hand, some vertebrate enzymes seem to transfer sugars to the reducing end. Expression of vertebrate HASs as active and soluble proteins will accelerate further precise insight into mechanisms of sugar elongation reactions by natural HASs. Since large scale production of HA polymers and oligomers would become powerful tools both for basic studies and new biotechnology to create functional carbohydrates in medicinal purposes, advent of an efficient method for the expression of HASs in Escherichia coli is strongly expected. Here we communicate the first success of the production of recombinant human HAS2 proteins composed of only the catalytic region in E. coli as the active form. It was demonstrated that an engineered HAS2 expressed in E. coli exhibited significant activity to synthesize a mixture of HAS oligomers from 8-mer (HA8) to 16-mer (HA16). Engineered HAS2 prepared herein elongated sugars from exogenous tetrasaccharide to form polymers with a direction to the non-reducing end. According to the present results, large scale production of engineered recombinant HASs is to be performed using E. coli that will provide practical and economic advantages in manufacturing enzymes for use in the synthesis of various oligomeric HA molecules and their industrial applications.     

Evaluation of critical structural elements of UDP-sugar substrates and certain cysteine residues of a vertebrate hyaluronan synthase

The hyaluronan (HA) synthases catalyze the addition of two different monosaccharides from UDP-sugar substrates to the linear heteropolysaccharide chain. To accomplish this task, the HA synthases must be able to bind and to transfer from both UDP-sugar substrates. Until now, it has been impossible to distinguish between these two abilities. We have created a mutant of xlHAS1, a HA synthase from Xenopus laevis, that allows for the examination of the enzyme's ability to bind substrate only. The ability of different compounds to protect the xlHAS1(C337S) mutant enzyme from loss of activity due to treatment with N-ethylmaleimide, a cysteine-modifying reagent, yields information on the relative affinity of a variety of nucleotides and nucleotide-sugars. We have observed that the substrate binding selectivity is more relaxed than the specificity of catalytic transfer. The only attribute that appears to be absolutely required for binding is a nucleotide containing two phosphates complexed with magnesium ion. The role of certain cysteine residues in catalysis was also evaluated. Cys307 of xlHAS1 may play a role in catalysis or in maintaining structure. Mutation of Cys337 raises the UDP-GlcUA Michaelis constant (K(m)), suggesting that this residue participates in UDP-GlcUA substrate binding or in catalytic complex formation.     

Radiation inactivation analysis of kidney microvillar peptidases

Five membrane peptidases were studied by radiation inactivation analysis of pig kidney microvillar membranes. One heterodimeric enzyme, gamma-glutamyl transferase, presented a target size corresponding to the dimeric Mr. The other enzymes are known to be homodimers. Three of these, aminopeptidase A. aminopeptidase N and dipeptidyl peptidase IV, gave results clearly indicating the monomer to be the target and, hence, in this group the association of the subunits was not essential for activity. The target size for endopeptidase-24.11 was intermediate between those for monomer and dimer and its functional state was not resolved by the experiments.     

Yeast and horse liver alcohol dehydrogenases: potential problems in target size analysis and evidence for a monomer active unit

Yeast and horse alcohol dehydrogenases are commonly used as standards for radiation inactivation analysis of proteins, usually assuming that the minimal functional unit corresponds to the physical size in solution, a tetramer (Mr = 148,000) and a dimer (Mr = 80,000), respectively. Results described in this paper demonstrate that molecular weight overestimates may be obtained for the yeast protein as a result of its unusual sensitivity to secondary radiation products. Irradiation in the presence of sulfhydryl reagents results in a smaller functional size estimate (67,000 +/- 3000) than that obtained in their absence (128,000 +/- 5000), indicating that some sulfhydryl groups in the enzyme may be particularly susceptible to attack by radiolytic species. Analysis of the horse liver enzyme reveals that although it has structural and functional similarities to the yeast protein, it is not as prone to secondary radiation damage and gives a minimal functional size estimate (33,000 +/- 1000) that most closely corresponds to a monomer. Quantitation of disappearance of the protein from a sodium dodecyl sulfate gel as a function of radiation dose also gives a target size (48,000 +/- 3000) in reasonable agreement with the monomer molecular weight. These results indicate that the individual subunits of horse liver alcohol dehydrogenase have independent catalytic capacity and imply that the same may be true for the yeast enzyme.     

The human hyaluronan synthase genes: genomic structures,proximal promoters and polymorphic microsatellite markers

The glycosaminoglycan (GAG) hyaluronan (HA) is a key component of the vertebrate extracellular matrix (ECM) and is synthesised by the HA synthase (HAS) enzymes HAS1, HAS2 and HAS3 at the plasma membrane. Accumulating evidence emphasises the relevance of HA metabolism in an increasing number of processes of clinical interest including renal fibrosis and peritoneal mesothelial wound healing. In the present study, the genomic sequences and organisation of the genes encoding the human HAS isoforms were deduced, in silico, from reference cDNA and genomic sequence data. These data were confirmed in vitro by sequencing of PCR-amplified HAS exons and flanking genomic sequences, comparison with sequence data for the corresponding murine Has orthologues, rapid amplification of 5' cDNA ends analysis and luciferase reporter assays on putative proximal promoter sequences. The HAS1 gene comprised five exons, with the translation start site situated 9bp from the 3' end of exon 1. In contrast, the genomic structures for HAS2 and both HAS3 variants spanned four exons, exon 1 forming a discrete 5'-untranslated region (5'-UTR) and the translation start site lying at nucleotide 1 of exon 2. Dinucleotide microsatellite loci were identified in intron 1 of HAS1 and HAS2, and immediately upstream of the HAS3 gene and their utility as linkage markers demonstrated in genomic DNA (gDNA) studies. We thus present a comprehensive resource for mutation detection screening of all HAS exons and/or linkage analysis of each HAS gene in a variety of disorders for which they are attractive candidates.     

Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties.

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 UDP-GlcNAc 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 x 10(5) to 1 x 10(6) Da, shorter than those synthesized by HAS1 and HAS2 which have molecular masses of 2 x 10(5) to approximately 2 x 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 x 10(5) to approximately 2 x 10(6) Da), whereas HAS2 generated hyaluronan with a broad but extremely large size (average molecular mass of >2 x 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.     

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is a reliable internal standard for radiation-inactivation studies of membranes in the frozen state

The target size of four soluble enzymes (beta-galactosidase, pyruvate kinase, alcohol dehydrogenase, and glucose-6-phosphate dehydrogenase) in the presence or absence of subcellular membrane fractions has been determined by the radiation-inactivation method using samples in the frozen state. For each of the four enzymes, full activity was recovered after freezing and thawing in the absence of radiation. We found minimal (less than 20%) binding of the enzymes to either submitochondrial vesicles or sarcoplasmic reticulum vesicles. Under the conditions tested, beta-galactosidase, pyruvate kinase, and alcohol dehydrogenase exhibited target sizes which varied according to the experimental conditions, i.e., the buffer selected and also the presence or absence of membrane preparations. For these tetrameric enzymes, the target sizes were generally comparable to either a monomer or a dimer. By contrast, the target size of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was found to be essentially invariant when frozen in a variety of buffers and in the presence or absence of either cryoprotectant (sucrose or glycerol) or different membrane preparations. The target size from 19 separate determinations gave an average value of 104 +/- 16 kDa, which is comparable to the molecular weight of the enzyme (104 kDa). We conclude that glucose-6-phosphate dehydrogenase from L. mesenteroides is a reliable internal standard for radiation-inactivation studies of membrane preparations in the frozen state.     

Evidence for the Heparin-Binding Ability of the Ascidian Xlink Domain and Insight into the Evolution of the Xlink Domain in Chordates

The vertebrate Xlink domain is found in two types of genes: lecticans and their associated hyaluronan-and-proteoglycan-binding-link-proteins (HAPLNs), which are components of the extracellular matrix, and those represented by CD44 and stabilins, which are expressed on the surface of lymphocytes. In both types of genes, Xlink functions as a hyaluronan binding domain. We have already reported that protochordate ascidians possess only the latter type of gene. The present analysis of the expression of ascidian Xlink domain genes revealed that these genes function in blood cell migration and apoptosis. While the Xlink domain is found in various metazoans, including ascidians and nematodes, hyaluronan is believed to be specific for vertebrates. In comprehensive genome surveys for hyaluronan synthase (HAS), we found no HAS gene in ascidians. We also established that hyaluronan is absent from the ascidian body biochemically. Therefore, ascidians possess the Xlink domain, but they lack HA. We recovered one ascidian Xlink domain gene that encoded a heparin-binding protein, although it shows no affinity for hyaluronan. Based on these findings, we conclude that in invertebrates, the Xlink domain serves as heparin-binding protein domain and functions in blood cell migration and apoptosis. Its binding affinity for HA might have been acquired in the vertebrate lineage.     

Calmodulin-activated cyclic nucleotide phosphodiesterase from brain. Relationship of subunit structure to activity assessed by radiation inactivation

The apparent target sizes of the basal and calmodulin-dependent activities of calmodulin-activated phosphodiesterase from bovine brain were estimated using target theory analysis of data from radiation inactivation experiments. Whether crude or highly purified samples were irradiated, the following results were obtained. Low doses of radiation caused a 10 to 15% increase in basal activity, which, with further irradiation, decayed with an apparent target size of approximately 60,000 daltons. Calmodulin-dependent activity decayed with an apparent target size of approximately 105,000 daltons. The percentage stimulation of enzyme activity by calmodulin decreased markedly as a function of radiation dosage. These observations are consistent with results predicted by computer-assisted modeling based on the assumptions that: 1) the calmodulin-activated phosphodiesterase exists as a mixture of monomers which are fully active in the absence of calmodulin and dimers which are inactive in the absence of calmodulin; 2) in the presence of calmodulin, a dimer exhibits activity equal to that of two monomers; 3) on radiations destruction of a dimer, an active monomer is generated. This monomer-dimer hypothesis provides a plausible explanation for and definition of basal and calmodulin-dependent phosphodiesterase activity.