The hyaluronate synthase from a eukaryotic cell line.
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ABSTRACT:
SUBMITTER: Klewes L
PROVIDER: S-EPMC1132350 | biostudies-other | 1993 Mar
REPOSITORIES: biostudies-other
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Project description:The mechanism of hyaluronate shedding from eukaryotic cell lines was analysed. All cell lines shed identical sizes of hyaluronate as were retained on the surface. They differed in the amount of hyaluronate synthesized and in the proportions of hyaluronate which were released and retained. A method was developed which could discriminate between shedding due to intramolecular degradation and that due to dissociation as intact macromolecules. This method was applied to B6 and SV3T3 cells in order to study the mechanism of hyaluronate release in more detail. The cells were pulse-labelled to form hyaluronate chains with labelled and unlabelled segments, and the sizes of labelled hyaluronate released into the medium during the pulse extension period were determined by gel filtration. B6 cells released identical sizes of hyaluronate at all labelled segment lengths, indicating that no intramolecular degradation occurred. When chain elongation was blocked by periodate-oxidized UDP-glucuronic acid, hyaluronate release was simultaneously inhibited. These results indicated that B6 cells dissociated hyaluronate as an intact macromolecule. In contrast, SV3T3 cells released hyaluronate of varying molecular mass distributions during extension of the labelled segment, suggesting partial degradation. Exogenous hyaluronate added to SV3T3 cultures was also degraded. This degradation could be prevented by the presence of radical scavengers such as superoxide dismutase and tocopherol. Degradation of endogenous hyaluronate could be inhibited by salicylate. These results led to the conclusion that SV3T3 cells released hyaluronate not only by dissociation, but also by radical-induced degradation.
| S-EPMC1131262 | biostudies-other
Project description:Hyaluronate synthase was isolated from protoblast membranes of streptococci by Triton X-114 extraction and cetylpyridinium chloride precipitation. It was identified as a 52,000-Mr protein, which bound to nascent hyaluronate and was affinity-labelled by periodate-oxidized UDP-glucuronic acid and UDP-N-acetylglucosamine. Antibodies directed against the 52,000-Mr protein inhibited hyaluronate synthesis. Mutants defective in hyaluronate synthase activity lacked the 52,000-Mr protein in membrane extracts. Synthase activity was solubilized from membranes by cholate in active form and purified by ion-exchange chromatography.
| S-EPMC1146770 | biostudies-other
Project description:Hyaluronate synthase was shed into the culture medium from growing streptococci (group C) together with nascent hyaluronate. The mechanism of solubilization was analysed using isolated protoplast membranes. Solubilization increased when membranes were suspended in larger volumes, but it was temperature-independent and was not inhibited by protease inhibitors. Increased hyaluronate chain length enhanced solubilization. The soluble synthase could re-integrate into Streptococcal membranes in a saturable manner. The soluble synthase behaved like an integral membrane protein, although it was not integrated into phospholipid vesicles. In sucrose velocity centrifugation the synthase had a higher sedimentation rate in detergent-free solution, indicating that it existed in an aggregated state.
| S-EPMC1131263 | biostudies-other
Project description:Hyaluronic acid (HA) is a crucial component of the extracellular matrix, and its level of accumulation is related to the progression of various malignant tumors. In this study, a pan-cancer analysis of the three enzymes called hyaluronan synthases (HAS1, HAS2, and HAS3) that produce HA was performed. The study comprehensively describes the characteristics of HAS1, HAS2, and HAS3 in cancers using public databases and tools, to identify the potential biological pathways involved at the molecular, protein, cellular, and clinical sample levels. The analysis showed that dysregulation of the three genes often occurs in cancer, contributing to cancer progression, metastasis, and prognosis. Overexpression of HAS2 promotes secretion of HA in GBM and enhances cell proliferation and migration. The common and specific functions of HAS in certain diseases have important research implications for the treatment and prognosis of tumors.
| S-EPMC10448108 | biostudies-literature
Project description:Differentiation of teratocarcinoma cells led to induction of hyaluronate synthesis. The synthase was recovered in the membrane fraction of cell lysates. Hyaluronate was synthesized at the membranes and was then released as a soluble product. The synthase could be stimulated by a variety of phosphate esters which prevented the degradation of the substrates UDP-GlcNAc and UDP-GlcA and the release of the growing hyaluronic acid chain from the membrane. Hyaluronidases or oligosaccharides derived from hyaluronate did not affect the synthesis. The chains grew at a rate of 60 repeating units/min. Continuous new chain initiation occurred during prolonged synthesis. Digestion of pulse-chase-labelled hyaluronate with beta-N-acetylglucosaminidase and beta-glucuronidase showed that the chains grew at the reducing end.
| S-EPMC1154342 | biostudies-other
Project description:The complete nucleotide sequence of hyaluronate synthase from Streptococcus sp. and its flanking regions is presented. The gene locus was designated has. Southern-blotting results suggested that the gene was conserved in hyaluronate-producing streptococci. A putative translation-initiation codon was identified and the open reading frame consists of 1566 bp, specifying a protein of 56 kDa. Sequences resembling the promoter and ribosome-binding site of Gram-positive organisms are found upstream of the synthase. The predicted amino-acid sequence reveals the presence of a 35-residue signal peptide. The sequence has some similarity to bacterial peptide-binding proteins.
| S-EPMC1132147 | biostudies-other
Project description:Iterative type I polyketide synthases (PKS) are megaenzymes essential to the biosynthesis of an enormously diverse array of bioactive natural products. Each PKS contains minimally three functional domains, β-ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), and a subset of reducing domains such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER). The substrate selection, condensation reactions, and β-keto processing of the polyketide growing chain are highly controlled in a programmed manner. However, the structural features and mechanistic rules that orchestrate the iterative cycles, processing domains functionality, and chain termination in this kind of megaenzymes are often poorly understood. Here, we present a biochemical and functional characterization of the KS and the AT domains of a PKS from the mallard duck Anas platyrhynchos (ApPKS). ApPKS belongs to an animal PKS family phylogenetically more related to bacterial PKS than to metazoan fatty acid synthases. Through the dissection of the ApPKS enzyme into mono- to didomain fragments and its reconstitution in vitro, we determined its substrate specificity toward different starters and extender units. ApPKS AT domain can effectively transfer acetyl-CoA and malonyl-CoA to the ApPKS ACP stand-alone domain. Furthermore, the KS and KR domains, in the presence of Escherichia coli ACP, acetyl-CoA, and malonyl-CoA, showed the ability to catalyze the chain elongation and the β-keto reduction steps necessary to yield a 3-hydroxybutyryl-ACP derivate. These results provide new insights into the catalytic efficiency and specificity of this uncharacterized family of PKSs.
| S-EPMC6334511 | biostudies-literature
Project description:Phytochelatins (PCs) are small cysteine-rich peptides involved in metal detoxification, not genetically encoded but enzymatically synthesized by phytochelatin synthases (PCSs) starting from glutathione. The constitutive PCS expression even in the absence of metal contamination, the wide phylogenetic distribution and the similarity between PCSs and the papain-type cysteine protease catalytic domain suggest a wide range of functions for PCSs. These proteins, widely studied in land plants, have not been fully analyzed in algae and cyanobacteria, although these organisms are the first to cope with heavy-metal stress in aquatic environments and can be exploited for phytoremediation. To fill this gap, we compared the features of the PCS proteins of different cyanobacterial and algal taxa by phylogenetic linkage. The analyzed sequences fall into two main, already known groups of PCS-like proteins. Contrary to previous assumptions, they are not classed as prokaryotic and eukaryotic sequences, but rather as sequences characterized by the alternative presence of asparagine and aspartic/glutamic acid residues in proximity of the catalytic cysteine. The presence of these enzymes with peculiar features suggests differences in their post-translational regulation related to cell/environmental requirements or different cell functions rather than to differences due to their belonging to different phylogenetic taxa.
| S-EPMC11314372 | biostudies-literature
Project description:IntroductionEnormous progress has been made in cardiac regeneration using human embryonic stem cell-derived cardiomyocyte (hESC-CM) grafts in pre-clinical trials. However, the rate of cell survival has remained very low due to anoikis after transplantation into the heart as single cells. Numerous solutions have been proposed to improve cell survival, and one of these strategies is to co-transplant biocompatible materials or hydrogels with the hESC-CMs.MethodsIn our study, we screened various combinations of biomaterials that could promote anoikis resistance and improve hESC-CM survival upon co-transplantation and promote cardiac functional recovery. We injected different combinations of Matrigel, alginate and hyaluronate with hESC-CM suspensions into the myocardium of rat models with myocardial infarction (MI).ResultsOur results showed that the group treated with a combination of hyaluronate and hESC-CMs had the lowest arrhythmia rates when stimulated with programmed electrical stimulation. While all three combinations of hydrogel-hESC-CM treatments improved rat cardiac function compared with the saline control group, the combination with hyaluronate most significantly reduced pathological changes from left ventricular remodelling and improved both left ventricular function and left ventricular ejection fraction by 28 days post-infarction.ConclusionHence, we concluded that hyaluronate-hESC-CM is a superior combination therapy for promoting cardiac regeneration after myocardial infarction.
| S-EPMC7705924 | biostudies-literature
Project description:Hyaluronate synthase activity is localized exclusively in plasma-membrane fractions of cultured human skin fibroblasts. The enzyme activity of plasma membranes prepared from exponential-growth-phase cells was about 6.5 times that of stationary-growth-phase cells. Hyaluronate synthase from exponential-growth-phase cells exhibited lower Km and higher Vmax. values for both UDP-N-acetylglucosamine and UDP-glucuronic acid and higher rate of elongation of hyaluronate chains compared with the enzyme from stationary-growth-phase cells. Hyaluronate synthase exhibited an extremely short half-life, 2.2 h and 3.8 h respectively when cells were treated with cycloheximide and actinomycin D. The cell-growth-phase-dependent variations in hyaluronate synthase activity appear to be due to its high turnover rate as well as due to some post-translational modification of the enzyme protein as cells progress from early exponential to stationary growth phase. The isolated plasma membranes contained a protein (Mr approx. 450,000) that was selectively autophosphorylated from [gamma-32P]ATP in vitro in the presence of hyaluronate precursors in the reaction mixture and that also exhibited some hyaluronate-synthesis-related properties. The 32P-labelled protein isolated from plasma membranes of exponentially growing cells expressed an efficient UDP-[14C]glucuronic acid- and UDP-N-acetyl[3H]glucosamine-binding activity and was able to synthesize oligosaccharides (Mr 5000) of [14C]glucuronic acid and N-acetyl[3H]glucosamine residues. The corresponding protein of stationary-growth-phase cells, which expressed much higher nucleotide-sugar-precursor-binding activity, appeared to have lost its oligosaccharide-synthesizing activity.
| S-EPMC1146992 | biostudies-other