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)

The availability of recombinant expression systems for the production of purified human hyaluronidases PH-20 and Hyal-1 facilitated the first detailed analysis of the enzymatic reaction products. The human recombinant enzymes, both expressed by Drosophila Schneider-2 (DS-2) cells, were compared to bovine testicular hyaluronidase (BTH), a commercially available hyaluronidase preparation, which has long been considered a prototype of mammalian hyaluronidases. The conversion of low molecular weight hyaluronic acid (HA) fragments was detected by a capillary zone electrophoresis (CZE) method. Surprisingly, the HA hexasaccharide, which is generally accepted to be the minimum substrate of BTH, was not a substrate of recombinant human PH-20 and Hyal-1. However, HA octasaccharide was converted efficiently by both enzymes, thus representing the minimum substrate for human PH-20 and Hyal-1. Additionally, BTH was shown to catabolize the HA hexasaccharide at pH 4.0 mainly by hydrolysis, while at pH 6.0 transglycosylation prevailed. Human PH-20 was found to catalyze both hydrolysis and transglycosylation of the HA octasaccharide. On the contrary, human Hyal-1 converted the HA octasaccharide mainly by hydrolysis with transglycosylation products occurring only at high substrate concentrations (> or = 500 microM). The differences between the hyaluronidase subtypes and isoenzymes were much more prominent than expected. Obviously, the different hyaluronidase subtypes have evolved into very specialized enzymes with respect to their catalytic mechanism of action.
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PMID:Kinetics of Hyal-1 and PH-20 hyaluronidases: comparison of minimal substrates and analysis of the transglycosylation reaction. 1760 39

Bovine testicular hyaluronidase (BTH) has been used as a spreading factor for many years and was primarily characterized by its enzymatic activity. As recombinant human hyaluronidases are now available the bovine preparations can be replaced by the human enzymes. However, data on the pH-dependent activity of hyaluronidases reported in literature are inconsistent in part or even contradictory. Detection of the pH-dependent activity of PH-20 type hyaluronidases, i.e. recombinant human PH-20 (rhPH-20) and BTH, showed a shift of the pH optimum from acidic pH values in a colorimetric activity assay to higher pH values in a turbidimetric activity assay. Contrarily, recombinant human Hyal-1 (rhHyal-1) and bee venom hyaluronidase (BVH) exhibited nearly identical pH profiles in both commonly used types of activity assays. Analysis of the hyaluronic acid (HA) degradation products by capillary zone electrophoresis showed that hyaluronan was catabolized by rhHyal-1 continuously into HA oligosaccharides. BTH and, to a less extent, rhPH-20 exhibited a different mode of action: at acidic pH (pH 4.5) HA was degraded as described for rhHyal-1, while at elevated pH (pH 5.5) small oligosaccharides were produced in addition to HA fragments of medium molecular weight, thus explaining the pH-dependent discrepancies in the activity assays. Our results suggest a sub-classification of mammalian-type hyaluronidases into a PH-20/BTH and a Hyal-1/BVH subtype. As the biological effects of HA fragments are reported to depend on the size of the molecules it can be speculated that different pH values at the site of hyaluronan degradation may result in different biological responses.
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PMID:Isoenzyme-specific differences in the degradation of hyaluronic acid by mammalian-type hyaluronidases. 1762 8

Hyaluronidases are enzymes that mediate the breakdown of hyaluronan (HA), a large polysaccharide abundant in the extracellular matrix of vertebrate tissues. Six genes have been predicted to encode hyaluronidases in humans, but the protein products of only SPAM1, HYAL1, and HYAL2 have been characterized. We have now expressed the mouse Hyal3 gene product, hyaluronidase 3 (Hyal3), in Baby Hamster Kidney (BHK) cells and demonstrated the presence of multiple forms of Hyal3 ranging from approximately 45 to 56 kDa in expression lysates. Complete and partial digestions of the expressed protein with PNGase F showed three N-linked oligosaccharides accounted for all forms of Hyal3 detected in expression lysates. Most of these oligosaccharides were Endo H sensitive, indicating that they were high mannose or hybrid N-linked oligosaccharides. Subcellular fractionation of Hyal3-expressing BHK cells by density gradient centrifugation revealed most Hyal3 in a low-density vesicular population. Low levels of Hyal3 were detected in higher density vesicles, but no colocalization with the late endosomal/lysosomal marker Lamp1 was found by immunofluorescence microscopy. BHK cells stably expressing Hyal3 had increased acid-active hyaluronidase activity, but no such activity was detected when Hyal3 was transfected into Hyaluronidase 1 (Hyal1)-deficient fibroblasts. Overexpression of Hyal3 in BHK cells increased the Hyal1 protein and mRNA levels, suggesting that the increased hyaluronidase activity in these cells was due to Hyal1 rather than Hyal3. The results indicate that Hyal3 overexpressed in cultured cells lacks intrinsic hyaluronidase activity and that Hyal3 may contribute to HA metabolism by augmenting the activity of Hyal1.
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PMID:Mouse Hyal3 encodes a 45- to 56-kDa glycoprotein whose overexpression increases hyaluronidase 1 activity in cultured cells. 1823 32

Sperm uptake of epididymal sperm adhesion molecule 1 (SPAM1) in vitro has recently been shown to be a marker of sperm maturation, since acquisition of this surface hyaluronidase increases cumulus dispersal efficiency. Here, we demonstrate that this glycosyl phosphatidylinositol-linked sperm antigen, previously shown to be expressed during estrous in the female reproductive tract, is secreted in the uterine and oviductal fluids (ULF and OF respectively) in a 67 kDa form, which can bind to sperm. We show that it can be acquired by caudal sperm from Spam1 null, Spam1-deficient mutant, and wild-type (WT) mice in vitro during incubation in ULF or OF at 37 degrees C, as detected by immunocytochemistry and flow cytometry. SPAM1 binding after ULF incubation was localized predominantly to the acrosome and the mid-piece of the flagella of Spam1 null sperm in a pattern identical to that of WT sperm. After ULF incubation, WT sperm demonstrated a significantly (P<0.001) enhanced hyaluronic acid-binding ability, and the involvement of SPAM1 in this activity was shown by a significant (P<0.001) decrease in binding when sperm were exposed to SPAM1 antiserum-inhibited ULF. Importantly, when Spam1 null sperm were exposed to ULF with SPAM1 accessible (in the presence of pre-immune serum) or inaccessible (in the presence of SPAM1 antiserum) for uptake, there was a significant difference in cumulus dispersal efficiency. Taken together, these results suggest that in the sperm surface remodeling that occurs prior to and during capacitation, the fertilizing competence of sperm is increased via acquisition of SPAM1, and likely other hyaluronidases, from the female tract.
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PMID:Murine SPAM1 is secreted by the estrous uterus and oviduct in a form that can bind to sperm during capacitation: acquisition enhances hyaluronic acid-binding ability and cumulus dispersal efficiency. 1829 22

Sperm uptake of glycosyl phosphatidylinositol (GPI)-linked proteins from luminal fluids has been shown to occur in male and estrous female reproductive tracts. In males, this is attributed to membranous vesicles secreted into the epididymis and prostate. While epididymosomes have been characterized, there have been no reports of the presence of vesicles in female luminal fluids. Here we report the presence of vesicles, characterized as "uterosomes," in the murine estrous female reproductive fluid; and use Sperm Adhesion Molecule 1 (SPAM1/PH-20), a well-known hyaluronidase found in male and female fluids, as a model to investigate vesicle-mediated GPI-linked protein transfer to sperm. Epididymosomes and uterosomes isolated after ultracentrifugation of epididymal (ELF) and uterine luminal fluid (ULF) were analyzed by electron microscopy and shown to be approximately 10-70 and approximately 15-50 nm in diameter. The structural integrity of uterosomes was confirmed by their resistance to hypo-osmotic and freeze/thaw stresses; and immunogold labeling localized SPAM1 to their outer membrane surface, as was the case for epididymosomes. SPAM1 was acquired by caudal sperm during incubation in epididymosomes and uterosomes; uptake was abolished when the GPI anchor was enzymatically cleaved. Sperm analyzed by confocal and transmission electron microscopy (TEM) after incubation in fluorescently labeled vesicles revealed the label on the membrane over the acrosome and midpiece of the flagella, where SPAM1 normally resides. High magnification TEM images demonstrated vesicles juxtaposed to the sperm plasma membrane potentially transferring SPAM1. Taken together, these results implicate vesicular docking as the mechanism of vesicle-mediated GPI-linked protein transfer to sperm from murine reproductive fluids.
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PMID:Investigating the role of murine epididymosomes and uterosomes in GPI-linked protein transfer to sperm using SPAM1 as a model. 1838 48

Hyaluronan is a megadalton glycosaminoglycan polymer critical for maintaining the integrity of the extracellular matrix. It can exist in a protein-bound state with aggregating proteoglycans, where it expands the extracellular matrix and modulates cell-cell interactions. It also exists in lower molecular weight forms that participate in a myriad of biological functions. It is unique in that much of it is degraded within hours of its synthesis. High molecular weight hyaluronan, a reflection of intact healthy tissues, is normally produced by hyaluronan synthases at the plasma membrane. It is catabolized by the action of an extracellular plasma membrane-tethered hyaluronidase that is coordinated with intracellular lysosomal hyaluronidases and exoglycosidases. This occurs in local tissues and lymph, with the remainder being cleared by the sinusoidal liver endothelium upon entering the vascular compartment. Elevated extracellular levels of hyaluronan and its partially catabolized oligomers are found in certain malignancies, potentially due to decoupled synthesis and degradation. Furthermore, partially depolymerized hyaluronan in the extracellular environment may have properties not found in the multivalent high molecular weight polymer in malignancies. Functional perturbations of hyaluronan synthesis and degradation have revealed active roles of the synthases and hyaluronidases in epithelial mesenchymal conversion, stroma and vascular formation, interstitial fluid pressure and chemosensitivity. While at least three confirmed hyaluronidases exist in the human genome (HYAL1, HYALl2 and PH20), functional perturbation of these genes in mice have failed to identify a simple linear catabolic circuit. The family of enzymes responsible for the synthesis and degradation of hyaluronan are being characterized. The fragmented forms of hyaluronan, largely a sign of cellular distress, occur in abundance in many malignancies. These small hyaluronan oligomers are assumed to be largely a result of hyaluronidase activity. Precisely how particular-sized fragments are generated and maintained is not known. Presumably, hyaluronan-binding proteins, in addition to the proteoglycans, participate in this process. Hyaluronidase inhibitors are now recognized, as well as growth factors that enhance the synthetic enzymes. A complete understanding of the anabolic and catabolic systems for hyaluronan may provide new dimensions into our understanding of cancer progression, as well as new opportunities for therapeutic intervention.
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PMID:Hyaluronidases in cancer biology. 1848 30

HYAL-1 (hyaluronoglucosaminidase-1) belongs to the hyaluronidase family of enzymes that degrade hyaluronic acid. HYAL-1 is a marker for cancer diagnosis and a molecular determinant of tumor growth, invasion, and angiogenesis. The regulation of HYAL-1 expression is unknown. Real time reverse transcription-PCR using 11 bladder and prostate cancer cells and 69 bladder tissues showed that HYAL-1 mRNA levels are elevated 10-30-fold in cells/tissues that express high hyaluronidase activity. Although multiple transcription start sites (TSS) for HYAL-1 mRNA were detected in various tissues, the major TSS in many tissues, including bladder and prostate, was at nucleotide 27274 in the cosmid clone LUCA13 (AC002455). By analyzing the 1532 base sequence 5' to this TSS, using cloning and luciferase reporter assays, we identified a TACAAA sequence at position -31 and the minimal promoter region between nucleotides -93 and -38. Mutational analysis identified that nucleotides -73 to -50 (which include overlapping binding consensus sites for SP1, Egr-1, and AP-2), bases C(-71) and C(-59), and an NFkappaB-binding site (at position -15) are necessary for promoter activity. The chromatin immunoprecipitation assay identified that Egr-1, AP-2, and NFkappaB bind to the promoter in HYAL-1-expressing cells, whereas SP1 binds to the promoter in non-HYAL-1-expressing cells. 5-Aza-2'-deoxycytidine treatment, bisulfite DNA sequencing, and methylation-specific PCR revealed that HYAL-1 expression is regulated by methylation at C(-71) and C(-59); both Cs are part of the SP1/Egr-1-binding sites. Thus, HYAL-1 expression is epigenetically regulated by the binding of different transcription factors to the methylated and unmethylated HYAL-1 promoter.
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PMID:Epigenetic regulation of HYAL-1 hyaluronidase expression. identification of HYAL-1 promoter. 1871 11

Hyaluronidases are endoglycosidases that initiate the breakdown of hyaluronan (HA), an abundant component of the vertebrate extracellular matrix. In humans, six paralogous genes encoding hyaluronidase-like sequences have been identified on human chromosomes 3p21.3 (HYAL2-HYAL1-HYAL3) and 7q31.3 (SPAM1-HYAL4-HYALP1). Mutations in one of these genes, HYAL1, were reported in a patient with mucopolysaccharidosis (MPS) IX. Despite the broad distribution of HA, the HYAL1-deficient patient exhibited a mild phenotype, suggesting other hyaluronidase family members contribute to constitutive HA degradation. Hyal3 knockout (Hyal3-/-) mice were generated to determine if HYAL3 had a role in constitutive HA degradation. Hyal3-/- mice were viable, fertile, and exhibited no gross phenotypic changes. X-ray analysis, histological studies of joints, whole-body weights, organ weights and the serum HA levels of Hyal3-/- mice were normal. No evidence of glycosaminoglycan accumulation, including vacuolization, was identified in the Hyal3-/- tissues analyzed. Remarkably, the only difference identified in Hyal3-/- mice was a subtle change in the alveolar structure and extracellular matrix thickness in lung-tissue sections at 12-14 months-of-age. We conclude that HYAL3 does not play a major role in constitutive HA degradation.
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PMID:Hyaluronidase 3 (HYAL3) knockout mice do not display evidence of hyaluronan accumulation. 1876 56

Although sperm entry into the oocyte-cumulus complex and subsequent sperm penetration through the cumulus matrix to reach the oocyte zona pellucida are essential for mammalian fertilization, the molecular mechanism remains controversial. Previously, we have shown that mouse sperm lacking SPAM1 are capable of penetrating the cumulus matrix despite a delayed dispersal of cumulus cells. We also have identified another sperm hyaluronidase, HYAL5, as a candidate enzyme involved in sperm penetration through the cumulus. In the present study, we produced HYAL5-deficient mice to uncover the functional roles of HYAL5 and SPAM1 in fertilization. The HYAL5-deficient mice were fully fertile and yielded normal litter sizes. In vitro fertilization assays demonstrated that HYAL5-deficient epididymal sperm is functionally normal. We thus conclude that HYAL5 may be dispensable for fertilization. Comparative analysis among wild-type, HYAL5-deficient, and SPAM1-deficient epididymal sperm revealed that only SPAM1 is probably involved in sperm penetration through the cumulus matrix. Notably, the loss of SPAM1 resulted in a remarkably increased accumulation of sperm on the surface or outer edge of the cumulus. These data suggest that SPAM1 may function in sperm entry into the cumulus and sperm penetration through the cumulus matrix.
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PMID:Functional roles of mouse sperm hyaluronidases, HYAL5 and SPAM1, in fertilization. 1960 84

The deduced amino acid sequence of bull sperm, SPAM1, suggests that it possesses a transmembrane domain between the hyaluronidase and the putative zona pellucida (ZP) binding domains. The objective of this study was to determine the orientation and localization of SPAM1 in order to understand how it could fulfill these two roles. We report that two isoforms of SPAM1 are present on ejaculated bull spermatozoa: one localized on the anterior portion of the sperm head, and the other on the postacrosomal portion of the head. The first isoform is expressed intracellularly, while the second one is detected at the sperm surface with its hyaluronidase domain facing the extracellular environment. Two-dimensional electrophoresis revealed that the two isoforms have different masses (80 and 70 kDa, respectively), and LC/MS/MS analyses confirmed our previously published deduced amino acid sequence of bovine SPAM1. In addition, this approach showed that the 70-kDa isoform differs from the 80-kDa isoform in its C terminus. Our results suggest that the shorter SPAM1 form originates from the epididymis, while the longer one is produced during spermatogenesis. These results clearly demonstrate that ejaculated bull sperm possess two forms of SPAM1: one (epididymal) expressed at the surface, and one (testicular) that interacts with the ZP after the acrosome exocytosis.
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PMID:SPAM1 isoforms from two tissue origins are differentially localized within ejaculated bull sperm membranes and have different roles during fertilization. 1981 1


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