Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.36 (hyaluronidase)
4,606 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hyaluronan is a constituent of the extracellular matrix of connective tissue and is actively synthesized during wound healing and tissue repair to provide a framework for ingrowth of blood vessels and fibroblasts. Changes in the serum concentration of hyaluronan are associated with inflammatory and degenerative arthropathies such as rheumatoid arthritis. In addition, hyaluronan has been implicated as an important substrate for migration of adhesion of leukocytes during inflammation. A human hyaluronan synthase (HuHAS1) cDNA was isolated by a functional expression cloning approach. Transfection of CHO cells conferred hyaluronidase-sensitive adhesiveness of a mucosal T cell line via the lymphocyte hyaluronan receptor, CD44, as well as increased hyaluronan levels in the cultures of transfected cells. The HuHAS1 amino acid sequence shows considerable homology to the hasA gene product of Streptococcus pyogenes, a glycosaminoglycan synthetase from Xenopus laevis (DG42), and is the human homolog of a recently described murine hyaluronan synthase.
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PMID:Functional cloning of the cDNA for a human hyaluronan synthase. 879 44

We previously reported the first cloning of a functional glycosaminoglycan synthase, the hyaluronan synthase (HAS) from Group A Streptococcus pyogenes (spHAS) (DeAngelis, P. L., Papaconstantinou, J., and Weigel, P. H. (1993) J. Biol. Chem. 268, 19181-19184). Group A spHAS was unrelated to a putative Group C HA synthase reported by others (Lansing, M., Lellig, S., Mausolf, A., Martini, I. , Crescenzi, F., Oregon, M., and Prehm, P. (1993) Biochem. J. 289, 179-184). Here we report the isolation of a bona fide HA synthase gene from a highly encapsulated strain of Group C Streptococcus equisimilis. The encoded protein, designated seHAS, is 417 amino acids long (calculated molecular weight, 47,778; calculated pI, 9.1) and is the smallest member of the HAS family identified thus far. The enzyme migrates anomalously fast in SDS-polyacrylamide gel electrophoresis (approximately 42,000 Da). The seHAS protein shows no similarity (<2% identity) to the previously reported Group C gene, which is not an HA synthase. The seHAS and spHAS protein and coding sequences are 72 and 70% identical, respectively. seHAS is also similar to eukaryotic HAS1 (approximately 31% identical), HAS2 (approximately 28% identical), and HAS3 (28% identical). The deduced protein sequence of seHAS was confirmed by reactivity with a synthetic peptide antibody. Recombinant seHAS expressed in Escherichia coli was recovered in membranes as a major protein (approximately 10% of the total protein) and synthesized very large HA (Mr >7 x 10(6)) in the presence of UDP-GlcNAc and UDP-GlcA. The product contained equimolar amounts of both sugars and was degraded by the specific Streptomyces hyaluronidase. Comparison of the two recombinant streptococcal enzymes in isolated membranes showed that seHAS and spHAS are essentially identical in the steady-state size distribution of HA chains they synthesize, but seHAS has an intrinsic 2-fold faster rate of chain elongation (Vmax) than spHAS. seHAS is the most active HA synthase identified thus far; it polymerizes HA at an average rate of 160 monosaccharides/s. The two bacterial HA synthase genes may have arisen from a common ancient gene shared with the early evolving vertebrates.
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PMID:Molecular cloning, expression, and characterization of the authentic hyaluronan synthase from group C Streptococcus equisimilis. 940 67

In the present study we describe a method to prepare membranes with high hyaluronan synthase activity from human glioma cells by pretreatment of the cells with both testicular hyaluronidase and 4-phorbol 12-myristate 13-acetate (PMA). A 23-fold increase in hyaluronan synthase activity was detected in comparison to untreated cells. Using isolated membranes as a source of hyaluronan synthase activity we demonstrate that chain elongation occurs at the reducing end of the hyaluronan molecule. We also present a method to solubilize hyaluronan synthase in active form with 1% digitonin. The solubilized synthase synthesized shorter hyaluronan chains than the membrane bound enzyme. Partial purification of the solubilized enzyme on a Superdex-200 column revealed a 12-fold increase in specific activity. Affinity purified polyclonal antibodies, raised against a synthetic peptide corresponding to the carboxy-terminus of the deduced protein sequence of human hyaluronan synthase recognized a 66 kDa component in the purified preparations. The elution position of the solubilized hyaluronan synthesizing activity immediately after V0 corresponding to a molecular mass of about 600 kDa, suggested that the 66 kDa enzyme forms a complex with other components which may have accessory or regulatory roles during hyaluronan synthesis.
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PMID:Characterization of hyaluronan synthase from a human glioma cell line. 955 98

The regulation of hyaluronan synthesis and shedding was analysed in human fibroblasts and in two melanoma cells that differed in the metastatic potential and proteolysis of the hyaluronan receptor CD44. Dissociation of nascent hyaluronan from plasma membranes isolated from fibroblasts by high salt concentrations led to activation of hyaluronan synthase. Hyaluronan synthesis was also enhanced in plasma membranes from fibroblasts that had been treated with hyaluronidase or trypsin. Hyaluronan oligosaccharides stimulated hyaluronan production in fibroblast cultures. These results indicated that nascent high-molecular-mass hyaluronan inhibited its own chain elongation, if it was retained in the vicinity of the synthase by cell-surface receptors. The results also indicated that increased hyaluronan synthesis and shedding correlated with proteolysis of CD44 on the melanoma cell lines, which has been observed by others.
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PMID:Synthesis and shedding of hyaluronan from plasma membranes of human fibroblasts and metastatic and non-metastatic melanoma cells. 1049 13

Hyaluronan is a major molecule in joint fluid and plays a crucial role in joint motion and the maintenance of joint homeostasis. The concentration and average molecular weight of hyaluronan in the joint fluids are reduced in osteoarthritis and rheumatoid arthritis. To elucidate the underlying mechanism, we analyzed the message expression of three isoforms of hyaluronan synthase and hyaluronidase from knee synovium, using real-time reverse transcriptase polymerase chain reaction. Synovia were obtained from 17 patients with osteoarthritis, 14 patients with rheumatoid arthritis, and 20 healthy control donors. The message expression of hyaluronan synthase-1 and -2 in the synovium of both types of arthritis was significantly less than in the control synovium, whereas that of hyaluronidase-2 in the synovium of both arthritides was significantly greater than in the control synovium. The decreased expression of the messages for hyaluronan synthase-1 and -2 and/or the increased expression of the message for hyaluronidase-2 may be reflected in the reduced concentration and decreased average molecular weight of hyaluronan in the joint fluids of patients with osteoarthritis and rheumatoid arthritis.
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PMID:Expression analysis of three isoforms of hyaluronan synthase and hyaluronidase in the synovium of knees in osteoarthritis and rheumatoid arthritis by quantitative real-time reverse transcriptase polymerase chain reaction. 1553 29

Basic fibroblast growth factor (FGF-2) can enhance biological potentials of periodontal ligament cells and its topical application induces considerable periodontal tissue regeneration in vivo. In this study, we examined the effect of FGF-2 on the production of hyaluronan (HA), an extracellular matrix playing important roles in homeostasis and inflammatory/wound healing responses, by human periodontal ligament (HPDL) cells. An inhibition binding-protein assay revealed that FGF-2 significantly increased HA production by HPDL cells in a dose dependent manner. Analysis by HPLC revealed that in conditioned medium of FGF-2-treated HPDL cells HA had a higher molecular mass, compared to that of untreated HPDL cells. RT-PCR analysis revealed the enhancement of mRNA expression of hyaluronan synthase (HAS) 1 and HAS 2, both of which contribute to the production of HA with a high molecular mass, but not HAS 3 in the FGF-2-treated HPDL cells. In contrast, three isoforms of hyaluronidase (HYAL) transcript were unchanged in the FGF-2-treated HPDL cells. These results provide new evidence for the possible involvement of FGF-2 in the regulation of HA production and its appreciable roles in not only homeostasis but also regeneration of periodontal tissues.
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PMID:Fibroblast growth factor-2 regulates the synthesis of hyaluronan by human periodontal ligament cells. 1557 76

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 beta-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[beta-32P]-Glc-NAc to radiolabel nascent HA chains. Under conditions of limiting substrate, the 32P-labeled products were separated from the substrates by paper chromatography and identified as HA-[32P]UDP saccharides based on their degradation by snake venom phosphodiesterase or hyaluronidase and by their binding to a specific HA-binding protein. The 32P radioactivity was chased (released) by incubation with unlabeled UDP-sugars, showing that the HA-UDP linkages turn over during HA biosynthesis. In contrast, HA-[32P]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.
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PMID:Hyaluronan biosynthesis by class I streptococcal hyaluronan synthases occurs at the reducing end. 1566 42

Within tumors there appears to be an intricate balance between hyaluronan (HA) synthesis and degradation where the invading edges display increased HA metabolism. The metabolism of HA has not been characterized in breast cancer cell lines; therefore, this study quantitatively identifies and characterizes the enzymes responsible for the synthesis and degradation of HA while correlating gene expression to cancer cell invasiveness and HA receptor status. In ten well-established breast cancer cell lines, the expression of the genes for each hyaluronan synthase (HAS) and hyaluronidase (Hyal) isoform was quantitated using real-time and reverse transcriptase polymerase chain reaction (PCR). The synthesis and degradation rates of hyaluronan were determined by ELISA, while quantitation of HA receptors, CD44 and RHAMM was performed by comparative Western blotting. The molecular weight of HA synthesized by each HAS isoform and the degradation products of each hyaluronidase were characterized by size exclusion chromatography. It was demonstrated that highly invasive cell lines preferentially expressed the HAS2 and Hyal-2 isoforms, while less invasive cells expressed HAS3 and Hyal-3. There was a correlation between elevated levels of HA synthesis, CD44 expression and cancer cell migration thereby highlighting the pivotal role that HA metabolism plays in the aggressive breast cancer phenotype.
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PMID:The over-expression of HAS2, Hyal-2 and CD44 is implicated in the invasiveness of breast cancer. 1612

The glycosaminoglycan hyaluronan is important in many tissuerepair processes. We have investigated the synthesis of hyaluronan in a panel of cell lines of fibroblastic and epithelial origin in response to PDGF (platelet-derived growth factor)-BB and other growth factors. Human dermal fibroblasts exhibited the highest hyaluronan-synthesizing activity in response to PDGF-BB. Analysis of HAS (hyaluronan synthase) and HYAL (hyaluronidase) mRNA expression showed that PDGF-BB treatment induced a 3-fold increase in the already high level of HAS2 mRNA, and increases in HAS1 and HYAL1 mRNA, whereas the levels of HAS3 and HYAL2 mRNA were not affected. Furthermore, PDGF-BB also increased the amount and activity of HAS2 protein, but not of HYAL1 and HYAL2 proteins. Using inhibitors for MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1/2] (U0126) and for PI3K (phosphoinositide 3-kinase) (LY294002), as well as the SN50 inhibitor, which prevents translocation of the active NF-kappaB (nuclear factor kappaB) to the nucleus, we observed a complete inhibition of both HAS2 transcriptional activity and hyaluronan synthesis, whereas inhibitors of other signalling pathways were without any significant effect. TGF-beta1 (transforming growth factor-beta1) did not increase the activity of hyaluronan synthesis in dermal fibroblasts, but increased the activity of HYALs. Importantly, inhibition of hyaluronan binding to its receptor CD44 by the monoclonal antibody Hermes-1, inhibited PDGF-BB-stimulated [3H]thymidine incorporation of dermal fibroblasts. We conclude that the ERK MAPK and PI3K signalling pathways are necessary for the regulation of hyaluronan synthesis by PDGF-BB, and that prevention of its binding to CD44 inhibits PDGF-BB-induced cell growth.
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PMID:Growth factor regulation of hyaluronan synthesis and degradation in human dermal fibroblasts: importance of hyaluronan for the mitogenic response of PDGF-BB. 1732 21

Prostate cancer progression can be predicted in human tumor biopsies by abundant hyaluronan (HA) and its processing enzyme, the hyaluronidase HYAL1. Accumulation of HA is dictated by the balance between expression levels of HA synthases, the enzymes that produce HA polymers, and hyaluronidases, which process polymers to oligosaccharides. Aggressive prostate tumor cells express 20-fold higher levels of the hyaluronan synthase HAS3, but the mechanistic relevance of this correlation has not been determined. We stably overexpressed HAS3 in prostate tumor cells. Adhesion to extracellular matrix and cellular growth kinetics in vitro were significantly reduced. Slow growth in culture was restored either by exogenous addition of hyaluronidase or by stable HYAL1 coexpression. Coexpression did not improve comparably slow growth in mice, however, suggesting that excess hyaluronan production by HAS3 may alter the balance required for induced tumor growth. To address this, we used a tetracycline-inducible HAS3 expression system in which hyaluronan production could be experimentally controlled. Adjusting temporal parameters of hyaluronan production directly affected growth rate of the cells. Relief from growth suppression in vitro but not in vivo by enzymatic removal of HA effectively uncoupled the respective roles of hyaluronan in growth and angiogenesis, suggesting that growth mediation is less critical to establishment of the tumor than early vascular development. Collectively results also imply that HA processing by elevated HYAL1 expression in invasive prostate cancer is a requirement for progression.
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PMID:Inducible hyaluronan production reveals differential effects on prostate tumor cell growth and tumor angiogenesis. 1750 71


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