Venom Hyaluronidase Research Articles

Snake venom hyaluronidase: an evidence for isoforms and extracellular matrix degradation.

The present study attempts to establish the isoforms of hyaluronidase enzyme and their possible role in the spreading of toxins during envenomation. Screening of venoms of 15 snakes belonging to three different families revealed varied hyaluronidase activity in ELISA-like assay, but with relatively similar pH and temperature optima. The zymograms of individual venoms showed varied activity banding patterns and indicated the presence of at least two molecular forms of the enzyme. During envenomation, activity of hyaluronidase is considered crucial for the spreading of toxins and is presumed to distort the integrity of extracellular matrix through the degradation of hyaluronic acid in it. This property has been addressed through localization of hyaluronic acid in human skin and muscle tissue sections using the probe, biotinylated hyaluronic acid binding protein. Faint and discontinuous staining pattern of hyaluronidase treated tissue sections over intense staining of untreated tissue sections confirm the selective degradation of hyaluronic acid in extracellular matrix and thus provide an evidence for the spreading property of the enzyme.

Antibody binding to venom carbohydrates is a frequent cause for double positivity to honeybee and yellow jacket venom in patients with stinging-insect allergy

Background: Up to 50% of patients with stinging-insect allergy have double-positive RAST results to honeybee and yellow jacket (YJ) venom. True double sensitization and crossreactivity through venom hyaluronidases are considered main reasons for this multiple reactivity. Objective: We investigated the role of antibodies against cross-reactive carbohydrate determinants in venom double positivity. Methods: CAP inhibition experiments were performed with crude oilseed rape (OSR) and timothy grass pollen extracts and a neoglycoprotein construct displaying a MUXF glycan, as present in pineapple-stem bromelain (MUXF-BSA). CAP to OSR was used as a rough measure for carbohydrate-specific IgE in individual sera. Results: CAP results to OSR pollen were positive in 2 of 14 single-positive honeybee venom sera, 2 of 16 single-positive YJ venom sera, and 33 (80.5%) of 41 double-positive sera (P < .00001, χ2 test). CAP inhibition was performed in 16 selected patients with a CAP class of 3 or higher to both venoms. In 9 of 11 patients with a highly positive CAP result to OSR (CAP score to OSR > CAP score to second venom), pollen extracts, MUXF-BSA, or both were able to completely inhibit IgE binding to one of the venoms, whereas this was not the case in 5 patients with a negative or weakly positive CAP result to OSR (CAP score to OSR < CAP score to second venom). Conclusions: The data suggest that carbohydrate-specific IgE is a major cause for the double positivity to honeybee and YJ venom seen in patients with Hymenoptera allergy. Because these antibodies may have low clinical relevance, they may severely impede the correct diagnosis of Hymenoptera venom allergy. (J Allergy Clin Immunol 2001;108:1045-52.)

The dual functions of GPI-anchored PH-20: hyaluronidase and intracellular signaling.

The ovulated mammalian oocyte is surrounded by the "cumulus ECM", composed of cells embedded in an extracellular matrix that is rich in hyaluronic acid (HA). The cumulus ECM is a viscoelastic gel that sperm must traverse prior to fertilization. Mammalian sperm have a GPI-anchored hyaluronidase which is known as PH-20 and also as SPAM 1. PH-20 is located on the sperm surface, and in the lysosome-derived acrosome, where it is bound to the inner acrosomal membrane. PH-20 appears to be a multifunctional protein; it is a hyaluronidase, a receptor for HA-induced cell signaling, and a receptor for the zona pellucida surrounding the oocyte. The zona pellucida recognition function of PH-20 was discovered first. This function is ascribed to the inner acrosomal membrane PH-20, which appears to differ biochemically from the PH-20 on the sperm surface. Later, when bee venom hyaluronidase was cloned, a marked cDNA sequence homology with PH-20 was recognized, and it is now apparent that PH-20 is the hyaluronidase of mammalian sperm. PH-20 is unique among the hyaluronidases in that it has enzyme activity at both acid and neutral pH, and these activities appear to involve two different domains in the protein. The neutral enzyme activity of plasma membrane PH-20 is responsible for local degradation of the cumulus ECM during sperm penetration. Plasma membrane PH-20 mediates HA-induced sperm signaling via a HA binding domain that is separate from the hyaluronidase domains. This signaling is associated with an increase in intracellular calcium and as a consequence, the responsiveness of sperm to induction of the acrosome reaction by the zona pellucida is increased. There is extensive evidence that GPI-anchored proteins are involved in signal transduction initiated by a diverse group of cell surface receptors. GPI-anchored proteins involved in signaling are often associated with signaling proteins bound to the cytoplasmic leaflet of the plasma membrane, typically Src family, non-receptor protein tyrosine kinases. PH-20 appears to initiate intracellular signaling by aggregating in the plasma membrane, and a 92-kDa protein may be the cell signaling molecule linked to PH-20.

Characterization of Hyaluronidase Isolated from Agkistrodon contortrix contortrix (Southern Copperhead) Venom

Snake venoms are a rich source of enzymes including many hydrolytic enzymes. Some enzymes such as phospholipase A2, proteolytic enzymes, and phosphodiesterases are well characterized. However many enzymes, such as the glycosidase, hyaluronidase, have not been studied extensively. Here we describe the characterization of snake venom hyaluronidase. In order to determine which venom was the best source for isolation of the enzyme, the hyaluronidase activity of 19 venoms from Elapidae, Viperidae, and Crotalidae snakes was determined. Since Agkistrodon contortrix contortrix venom showed the highest activity, this venom was used for purification of hyaluronidase. Molecular weight was determined by matrix-assisted laser desorption ionization mass spectroscopy and was found to be 59,290 Da. The molecular weight value as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 61,000 Da. Substrate specificity studies indicated that the snake venom enzyme was specific only for hyaluronan and did not hydrolyze similar polysaccharides of chondroitin, chondroitin sulfate A (chondroitin 4-sulfate), chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C (chondroitin 6-sulfate), chondroitin sulfate D, chondroitin sulfate E, or heparin. The enzyme is an endo-glycosidase without exo-glycosidase activity, as it did not hydrolyze p-nitrophenyl-β-d-glucuronide or p-nitrophenyl-N-acetyl-β-d-glucosaminide. The main hydrolysis products from hyaluronan were hexa- and tetrasaccharides with N-acetylglucosamine at the reducing terminal. The cleavage point is at the β1,4-glycosidic linkage and not at the β1,3-glycosidic linkage. Thus, snake venom hyaluronidase is an endo-β-N-acetylhexosaminidase specific for hyaluronan.

Crystal Structure of Hyaluronidase, a Major Allergen of Bee Venom

Background: Hyaluronic acid (HA) is the most abundant glycosaminoglycan of vertebrate extracellular spaces and is specifically degraded by a β-1,4 glycosidase. Bee venom hyaluronidase (Hya) shares 30% sequence identity with human hyaluronidases, which are involved in fertilization and the turnover of HA. On the basis of sequence similarity, mammalian enzymes and Hya are assigned to glycosidase family 56 for which no structure has been reported yet. Results: The crystal structure of recombinant (Baculovirus) Hya was determined at 1.6 Å resolution. The overall topology resembles a classical (β/α) 8 TIM barrel except that the barrel is composed of only seven strands. A long substrate binding groove extends across the C-terminal end of the barrel. Cocrystallization with a substrate analog revealed the presence of a HA tetramer bound to subsites −4 to −1 and distortion of the −1 sugar. Conclusions: The structure of the complex strongly suggest an acid–base catalytic mechanism, in which Glu113 acts as the proton donor and the N-acetyl group of the substrate is the nucleophile. The location of the catalytic residues shows striking similarity to bacterial chitinase which also operates via a substrate-assisted mechanism.

N-Glycan Analysis by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of Electrophoretically Separated Nonmammalian Proteins: Application to Peanut Allergen Ara h 1 and Olive Pollen Allergen Ole e 1

A method has been developed which allows the analysis of glycoproteins separated by SDS-PAGE. The procedure, though applicable to N-glycosylated glycoproteins of any origin, is particularly devised for glycoproteins potentially containing fucose in α1,3-linkage to the reducing GlcNAc as may be found in plants and invertebrates, e.g., insects and parasitic helminths. Starting with an established procedure for mass spectrometric peptide mapping, the analysis of N-glycans by matrix-assisted laser desorption/ionization mass spectrometry involved the use of peptide:N-glycosidase A, a triphasic microcolumn for sample cleanup, and a new matrix mixture consisting of 2,5-dihyhydroxybenzoic acid, 1-hydroxyisoquinoline, and arabinosazone. The method was tested on proteins with N-glycans of known structure, i.e., as horseradish peroxidase, zucchini ascorbate oxidase, soybean agglutinin, honeybee venom hyaluronidase, bovine ribonuclease B, and bovine fetuin. An electrophoretic band corresponding to 4 μg of glycoprotein was generally sufficient to allow detection of the major N-glycan species. As an additional benefit, a peptide mass map is generated which serves to identify the analyzed protein. The method was applied to glycoprotein allergens whose glycan structures were unknown. Ara h 1 and Ole e 1, major allergens from peanut and olive pollen, respectively, contained mainly xylosylated N-glycans with the composition Man3(−4)XylGlcNAc2 in the case of Ara h 1 and GlcNAc1–2Man3XylGlcNAc2 in the case of Ole e 1 where also some GlcNAc0–2Man3XylFucGlcNAc2 was found.

Production d'hyaluronidase par les cellules cancéreuses humaines en culture

The presence of hyaluronidase was detected at pH 3.8 in eight out of twelve human cancer cell line culture media. Eight cell lines derived from primary tumours and four from metastases. In three culture media the enzymatic activity was lower than 0.035 pU/cell/h. In five others (in a hepatoma cell line and in four metastasis-derived cell lines) the activity was higher than 0.057 pU/cell/h. A tumour-derived fibroblast culture was negative. The optimal activity was observed at a pH comprised between 3.6 and 4. Salt inhibition of hyaluronidase was reversible. The enzyme was denaturated by a 10-min heating at 70 °C. The enzyme was not strictly specific for hyaluronan hydrolysis but also digested chondroitin sulfates. PH20, a spermatozoid protein that has homologies with the bee venom hyaluronidase, was not expressed by cell lines tested.

Molecular cloning and characterization of rat sperm surface antigen 2B1, a glycoprotein implicated in sperm-zona binding.

The rat sperm surface antigen, 2B1, that has been proposed to play a key role in sperm adhesion to the zona pellucida, has been cloned and its entire cDNA sequenced. Northern blot analysis indicates that 2B1 is encoded by a 2.2-kb RNA transcript that is abundantly expressed in the testis. The deduced protein sequence contains 512 amino-acid residues with a strong candidate signal sequence and C-terminal transmembrane domain. Data base searches reveal a high degree of sequence similarity to guinea pig, rabbit, monkey, and human PH20 sperm surface antigens, and a lower degree of similarity to honey bee and whiteface hornet venom hyaluronidases. Rat 2B1 antigen also possesses hyaluronidase activity, suggesting that it is a bifunctional protein with putative roles in the dispersion of cumulus oophorus cells as well as zona adhesion. However, while it would appear that 2B1 is the rat homologue of the guinea pig PH20 antigen, they differ in a number of important biochemical respects (including their mode of attachment to the sperm membrane and distribution between soluble and membrane-bound fractions), as well as in their localization on the sperm membrane. Expression of regions of the 2B1 protein in recombinant bacterial cells has allowed a preliminary mapping of the 2B1 epitope, and has provided more definitive information on the endoproteolytic processing of 2B1 during epididymal transit.

Yellow jacket venom allergens, hyaluronidase and phospholipase: Sequence similarity and antigenic cross-reactivity with their hornet and wasp homologs and possible implications for clinical allergy

Three known allergens of yellow jacket (Vespula vulgaris) venom are antigen 5, hyaluronidase, and phospholipase. Yellow jacket antigen 5 has been previously cloned and expressed in bacteria; it contains 204 amino acid residues, and it has 69% and 60% sequence identities with the homologous proteins of white-faced hornet (Dolichovespula maculata) and wasp (Polistes annularis), respectively. These studies are now extended to yellow jacket hyaluronidase and phospholipase; they contain 331 and 300 amino acid residues, respectively, and they show 92% and 67% sequence identity with their homologs of white-faced hornet. Tests with the natural and the recombinant vespid allergens in mice indicate partial antigenic cross-reactivity of their homologous proteins at both B- and T-cell levels. There is greater cross-reactivity among hornet and yellow jacket allergens than that among hornet or yellow jacket and wasp allergens. The order of cross-reaction of the three vespid allergens is hyaluronidase > antigen 5 > phospholipase. The continuous (linear) B-cell epitopes of vespid allergens show greater cross-reactivity than their discontinuous epitopes do. The discontinuous B-cell epitopes are immunodominant for all vespid allergens. The low degree of cross-reactivity of the immunodominant discontinuous B-cell epitopes of vespid allergens should be taken into consideration in selection of venoms for immunotherapy of patients with sensitivity to multiple vespids. (J A LLERGY C LIN I MMUNOL 1996;98:588-600.)

Biochemical Characterization of a Glycosylphosphatidylinositol-Linked Hyaluronidase on Mouse Sperm

On the basis of DNA homology to bee venom hyaluronidase, it was recently suggested that the GPI-linked mammalian sperm antigen, PH-20, may function as a cell surface hyaluronidase [Gmachl, M., & Kreil, G. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 3569-3573]. We have quantified the activity of the soluble acrosomal hyaluronidase of mouse sperm and further demonstrate the existence of a membrane-bound hyaluronidase, detected on both acrosome-intact and acrosome-reacted mouse sperm, distinct from the soluble form of the enzyme. The membrane-bound hyaluronidase was specifically released by PI-PLC, indicating that it is GPI linked. Acrosome-intact and acrosome-reacted sperm released several polypeptides (68, 44, 39, 34, 17, and 15 kDa) when treated with PI-PLC. In addition, GPI-linked polypeptides unique to acrosome-intact or to acrosome-reacted sperm were identified. Fractionation of the PI-PLC-released components from acrosome-reacted sperm using size exclusion chromatography revealed a single peak of hyaluronidase activity which comigrates with a 68 kDa GPI-linked protein present in these fractions. Taken together, these data demonstrate the existence of at least two isoforms of hyaluronidase: a soluble form within the acrosomal vesicle which is released during acrosomal exocytosis and a GPI-linked form which is present on the surface of both acrosome-intact and acrosome-reacted sperm. Both forms may be necessary for successful penetration of the extracellular vestments that surround the egg prior to fertilization.

Isolation and characterization of stonefish venom stonustoxin and hyaluronidase: R. Yuen, H. E. Khoo, B. Cai, F. J. Ghadessy and C. H. Poh (Department of Biochemistry, National University of Singapore, Kent Ridge, Singapore 0511)

Isolation and characterization of stonefish venom stonustoxin and hyaluronidase: R. Yuen, H. E. Khoo, B. Cai, F. J. Ghadessy and C. H. Poh (Department of Biochemistry, National University of Singapore, Kent Ridge, Singapore 0511)

Dispersion of cumulus cells surrounding mice oocytes by stonefish (Synanceja horrida) venom hyaluronidase: S. Vasuthevan, T. A. Bongso, H. E. Khoo, R. Yuen and C. H. Poh (Departments of Obstetrics and Gynaecology, and Biochemistry, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511).

Dispersion of cumulus cells surrounding mice oocytes by stonefish (Synanceja horrida) venom hyaluronidase: S. Vasuthevan, T. A. Bongso, H. E. Khoo, R. Yuen and C. H. Poh (Departments of Obstetrics and Gynaecology, and Biochemistry, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511).

Identification of the end-products of stonefish (Synanceja horrida) venom hyaluronidase action on hyaluronic acid: K. Sugahara, K. Takeda, X. Yamada, A. Masashi, M. Sugiura, R. Yuen, H. E. Khoo and C. H. Poh (Department of Physiological Chemistry, Kobe Women's College of Pharmacy, Kobe, Japan; Department of Biochemistry, National University of Singapore).

Identification of the end-products of stonefish (Synanceja horrida) venom hyaluronidase action on hyaluronic acid: K. Sugahara, K. Takeda, X. Yamada, A. Masashi, M. Sugiura, R. Yuen, H. E. Khoo and C. H. Poh (Department of Physiological Chemistry, Kobe Women's College of Pharmacy, Kobe, Japan; Department of Biochemistry, National University of Singapore).

Inhibitory effects of flavonoids on several venom hyaluronidases

In vitro studies showed that the flavonoid aglycones apigenin, luteolin and kaempferol inhibited the hyaluronidase activity of five different venoms dose-dependently. They were also able to delay the venom action when injected into mice. Naringenin, catechin and flavonoid glycosides had no effect. The flavonoids with unsubstituted hydroxyl groups at C-positions 5, 7 and 4', a double bond between carbons 2 and 3, as well as a ketone group at position 4, exhibited potent inhibitory actions on the venom hyaluronidases.

The hyaluronidase activities of some Southeast Asian snake venoms

The hyaluronidase activities of venoms of snakes indigenous to Southeast Asia were investigated. With the exception of the venom of the Malayan krait Bungarus candidus, the elapid venoms had either little or no hyaluronidase activities, whereas the viperid venoms possessed considerable activity. A component of Russell's viper venom with hyaluronidase activities had a mol. wt of approximately 14,000. Neither MP4, a monoclonal antibody raised against the purified Russell's viper venom hyaluronidase toxin, nor a monospecific polyclonal antivenom neutralized the hyaluronidase activities of this purified hyaluronidase component of crude Russell's viper venom. The Russell's viper venom hyaluronidase activities was labile on heating and storage. The significance of these observations to envenomation and antivenom production is discussed.

Antibodies to purified bee venom proteins and peptides: I. Development of a highly specific RAST for bee venom antigens and its application to bee sting allergy

IgE antibodies to purified proteins and peptides from honeybee venom have been measured by the RAST. Trace amounts (<0.1%) of the major venom protein phospholipase A 2 (PLA 2) grossly distorted the measurement of IgE antibody to the other venom proteins, acid phosphatase (Acid P) and hyaluronidase (HYAL), and overemphasized their importance. Reduction of antigen coupled to the cellulose paper discs, which were used in the assay, diluted out the contaminating PLA 2 without apparent loss in sensitivity. The reduction of disc-bound antigen increased the competition between IgE and IgG antibodies but did not affect measurement of IgE antibodies in sera taken from 35 untreated patients who had a history of general allergic reactions to bee stings. In 54% of sera from bee venom-allergic patients, the greatest IgE antibody response was to PLA 2. In all, IgE antibodies to PLA 2 were present in 91% of these sera. IgE antibodies to Acid P, HYAL, or melittin were present in 60%, 51%, and 31% of sera, respectively, and accounted for the highest level of binding in 17%, 17%, and 6% of these. Only 6% of sera were positive for whole venom but negative for the isolated antigens. A low level of IgE antibody was found to peptide 401 in 6% of sera. No IgE antibodies were found to apamin. While confirming the central role played by PLA 2 in bee sting allergy, these results show that other venom components are also important in some patients.

The venom system and venom hyaluronidase of the African honeybee ( Apis mellifera adansonii)

African honeybees ( Apis mellifera adansonii) have smaller venom glands and less venom hyaluronidase than Italian ( A. m. ligustica) bees. There are changes, paralleling those in Italian bees, in venom reservoir volume and in the amounts of hyaluronidase in the venom system of African honeybees of different ages.

Characterization of lizard venom hyaluronidase and evidence for its action as a spreading factor

1. 1. Hyaluronidase was isolated from the lizard ( Heloderma horridum horridum) crude venom. The chemical properties were characterized and compared to the same enzyme from other sources. 2. 2. The enzyme was found to be a single polypeptide chain with a molecular weight of 63,000 daltons. It possesses an isoelectric point and pH optimum of 5.0, and was observed to be extremely temperature sensitive. 3. 3. The role of hyaluronidase as a spreading factor which serves to aid in the diffusion of toxins has been suspected for a long time; yet no experimental proof has been offered until now. 4. 4. It was shown that hyaluronidase promotes the spread of the hemorrhagic area in mice when injected with hemorrhagic toxin. 5. 5. Thus experimental evidence is supplied for the first time that the enzyme plays a role as a “spreading factor” in the toxic action of venom.

Purification and partial characterization of hyaluronidase from five pace snake ( Agkistrodon acutus) venom

—A hyaluronidase (EC 3.2.1. 35) was isolated and purified from Agkistrodon acutus venom. The purification procedure included CM-Sephadex C-50 chromatography, gel-filtration on Sephadex G-75 and CM-Sephadex C-25 chromatography. The purified preparation of the enzyme was homogeneous on polyacrylamide gel electrophoresis at pH 4.3, a 45-fold purification being obtained. The hyaluronidase was a glycoprotein (positive PAS staining) with a molecular weight of 33,000 and a pI of 10.3. No hemorrhagic activity was found. The hyaluronidase had an optimum pH of 3.5–5.0 and an optimum temperature of 37°C. It was heat sensitive, was more stable in the acidic than in the neutral region, and lost its activity in the alkaline region. Fe 2+, Cu 2+ and heparin inhibited the venom hyaluronidase. The K m value for hyaluronic acid was 6.2 × 10 −3 mg/ml.

A convenient plate assay for the quantitation of hyaluronidase in hymenoptera venoms

A simple plate assay for hyaluronidase activity in biological samples is described. Hyaluronic acid is incorporated into agarose gels and the enzyme is allowed to diffuse from punched wells. The undigested hyaluronic acid is then precipitated with cetylpyridinium chloride and the diameters of the clear circles are proportional to the logarithm of the enzyme concentration applied to the well. The assay was utilized to examine commercially available hymenoptera venoms, manufactured for use in allergy diagnosis and treatment, for their content of hyaluronidase as a measure of lot-to-lot consistency. The assay permits the analyses of a large number of samples with good reproducibility, without the need for any special instrumentation. Based on the quantity of purified hyaluronidase reported in honey bee venom (T. P. King, A. K. Sobotka, L. Kochoumian, and L. M. Lichtenstein, 1976, Arch. Biochem. Biophys. 172, 661–671) we estimate that the assay should detect 70 ng/ml of purified honey bee venom hyaluronidase.