Biochemical and molecular characterization of the hyaluronidase from Bothrops atrox Peruvian snake venom.

Snake venoms are a rich source of enzymes such as metalloproteinases, serine proteinases phospholipases A2 and myotoxins, that have been well characterized structurally and functionally. However, hyaluronidases (E.C.3.2.1.35) have not been studied extensively. In this study, we describe the biochemical and molecular features of a hyaluronidase (Hyal-Ba) isolated from the venom of the Peruvian snake Bothrops atrox. Hyal-Ba was purified by a combination of ion-exchange and gel filtration chromatography. Purified Hyal-Ba is a 69-kDa (SDS-PAGE) monomeric glycoprotein with an N-terminal amino acid sequence sharing high identity with homologous snake venom hyaluronidases. Detected associated carbohydrates were hexoses (16.38%), hexosamines (2.7%) and sialic acid (0.69%). Hyal-Ba selectively hydrolyzed only hyaluronic acid (HA; specific activity = 437.5 U/mg) but it did not hydrolyze chondroitin sulfate or heparin. The optimal pH and temperature for maximum activity were 6.0 and 40 °C, respectively, and its Km was 0.31 µM. Its activity was inhibited by EDTA, iodoacetate, 2-mercaptoethanol, TLCK and dexamethasone. Na+ and K+ (0.2 M) positively affect hyaluronidase activity; while Mg2+, Br2+, Ba2+, Cu2+, Zn2+, and Cd2+ reduced catalytic activity. Hyal-Ba potentiates the hemorrhagic and hemolytic activity of whole venom, but decreased subplantar edema caused by an l-amino acid oxidase (LAAO). The Hyal-Ba cDNA sequence (2020 bp) encodes 449 amino acid residues, including the catalytic site residues (Glu135, Asp133, Tyr206, Tyr253 and Trp328) and three functional motifs for N-linked glycosylation, which are conserved with other snake hyaluronidases. Spatial modeling of Hyal-Ba displayed a TIM-Barrel (α/ß) fold and an EGF-like domain in the C-terminal portion. The phylogenetic analysis of Hyal-Ba with other homologous Hyals showed the monophyly of viperids. Further, Hyal-Ba studies may extend our knowledge of B. atrox toxinology and provides insight to improve the neutralizing strategies of therapeutic antivenoms.

Differentiation of Intracellular Hyaluronidase Isoform by Degradable Nanoassembly Coupled with RNA-Binding Fluorescence Amplification.

Hyaluronidase has two cruical isoforms, hyaluronidase-1 (Hyal-1) and hyaluronidase-2 (Hyal-2), which are essential for cellular hyaluronic acid (HA) catabolism to generate different-sized oligosaccharide fragments for performing different physiological functions. In particular, Hyal-1 is the major tumor-derived hyaluronidase. Thus, specific detection of one hyaluronidase isoform, especially Hyal-1, in live cells is of scientific significance but remains challenging. Herein, by use of differentiated tolerance capability of an amphiphilic HA-based nanoassembly to Hyal-1 and Hyal-2, we rationally design a Hyal-1 specific nanosensor, consisting of cholesterylamine-modified HA nanoassembly (CHA) and RNA-binding fluorophores (RBF). The RBF molecules were entrapped in CHA to switch off their fluorescence via aggregation caused quenching. However, CHA can be disassembled by Hyal-1 to release RBF, resulting in fluorescence activation. Moreover, the fluorescence of the released RBF is further enhanced by cytoplasm RNA. Owing to this cascade signal amplification, this nanosensor RBF@CHA displays a significant change of signal-to-background-noise ratio (120-fold) toward 16 µg/mL Hyal-1 in cellular lysates. In contrast, it is resistant to Hyal-2. By virtue of its selective and sensitive characteristics under a complicated matrix, RBF@CHA had been successfully applied for specifically visualizing Hyal-1 over Hyal-2 inside live cells for the first time, detecting a low level of intracellular Hyal-1 and distinguishing normal and cancer cells with different expressions of Hyal-1. This approach would be useful to better understand biological functions and related diseases of intracellular Hyal-1.

Hyaluronidase and Chondroitinase.

Glycosaminoglycans (GAGs) are important constituents of the extracellular matrix that make significant contributions to biological processes and have been implicated in a wide variety of diseases. GAG-degrading enzymes with different activities have been found in various animals and microorganisms, and they play an irreplaceable role in the structure and function studies of GAGs. As two kind of important GAG-degrading enzymes, hyaluronidase (HAase) and chondroitinase (CSase) have been widely studied and increasing evidence has shown that, in most cases, their substrate specificities overlap and thus the "HAase" or "CSase" terms may be improper or even misnomers. Different from previous reviews, this article combines HAase and CSase together to discuss the traditional classification, substrate specificity, degradation pattern, new resources and naming of these enzymes.

Cloning, structural modelling and characterization of VesT2s, a wasp venom hyaluronidase (HAase) from Vespa tropica

Wasp venom is a complex mixture containing proteins, enzymes and small molecules, including some of the most dangerous allergens. The greater banded wasp (Vespa tropica) is well-known for its lethal venom, whose one of the major components is a hyaluronidase (HAase). It is believed that the high protein proportion and activity of this enzyme is responsible for the venom potency. Methods: In the present study, cDNA cloning, sequencing and 3D-structure of Vespa tropica venom HAase were described. Anti-native HAase antibody was used for neutralization assay. Results: Two isoforms, VesT2a and VesT2b, were classified as members of the glycosidase hydrolase 56 family with high similarity (4297 %) to the allergen venom HAase. VesT2a gene contained 1486 nucleotide residues encoding 357 amino acids whereas the VesT2b isoform consisted of 1411 residues encoding 356 amino acids. The mature VesT2a and VesT2b are similar in mass and pI after prediction. They are 39119.73 Da/pI 8.91 and 39571.5 Da/pI 9.38, respectively. Two catalytic residues in VesT2a, Asp107 and Glu109 were substituted in VesT2b by Asn, thus impeding enzymatic activity. The 3D-structure of the VesT2s isoform consisted of a central core (/)7 barrel and two disulfide bridges. The five putative glycosylation sites (Asn79, Asn99, Asn127, Asn187 and Asn325) of VesT2a and the three glycosylation sites (Asn1, Asn66 and Asn81) in VesT2b were predicted. An allergenic property significantly depends on the number of putative N-glycosylation sites. The anti-native HAase serum specifically recognized to venom HAase was able to neutralize toxicity of V. tropica venom. The ratio of venom antiserum was 1:12. Conclusions: The wasp venom allergy is known to cause life-threatening and fatal IgE-mediated anaphylactic reactions in allergic individuals. Structural analysis was a helpful tool for prediction of allergenic properties including their cross reactivity among the vespid HAase.

Active hyaluronidase 2 expression in the granulation tissue formed in the healing process of equine superficial digital flexor tendonitis.

Upregulation of hyaluronidase 2 (HYAL2), one of somatic hyaluronidase (HAase), was demonstrated in granulation tissue during the healing of equine superficial digital flexor tendon injuries. The activity of HAase was assessed by hyaluronan (HA)-containing gel zymography and in situ zymography using frozen sections obtained from normal and injured tendon tissues. Elevated HAase activity was identified in the extract from the tendinopathic tissues, with lower levels of the activity in normal tendons. In situ zymography using fluorescently-labeled HA demonstrated HAase activity in the granulation tissue formed in the injured region. In addition, in situ hybridization analysis indicated that fibroblastic cells in the granulation tissue of the injured tendon actively expressed HYAL2 but not HYAL1. Quantitative RT-PCR further confirmed a higher level of amplicons corresponding to HYAL2 in tendonitis-derived samples. These results suggest that elevated HYAL2 activity in the granulation tissue could participate in controlling the healing process in equine tendonitis.

Isoenzyme-specific differences in the degradation of hyaluronic acid by mammalian-type hyaluronidases.

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.

Devising a pathway for hyaluronan catabolism: are we there yet?

Hyaluronan is a negatively charged, high molecular weight glycosaminoglycan found predominantly in the extracellular matrix. Intracellular locations for hyaluronan have also been documented in cytoplasm, nucleus, and nucleolus. The polymer has an extraordinarily high rate of turnover in vertebrate tissues. The focus here is to formulate a metabolic pathway for hyaluronan degradation using all available data, including the recently acquired information on the hyaluronidase gene family. Such a catabolic scheme has defied explication up to now. In somatic tissues, stepwise processing occurs, from the extracellular high molecular weight space filling, antiangiogenic approximately 107-kDa polymer, to intermediate sized highly angiogenic, inflammatory, and immune-stimulating fragments, and ultimately to tetrasaccharides that are antiapoptotic and potent inducers of heat-shock proteins. It is proposed that the high molecular weight extracellular polymer is tethered to the cell surface by the combined efforts of hyaluronan receptors and hyaluronidase-2 (Hyal-2). The hyaluronan is cleaved to a 20-kDa intermediate-sized fragment, the limit product of Hyal-2 digestion. These fragments are delivered to endosomal- and ultimately lysosomal-like structures. Further catabolism occurs there by Hyal-1, coordinated with the activity of two lysosomal beta-exoglycosidases, beta-glucuronidase and beta-N-acetyl-glucosaminidase. A membrane-associated mini-organelle is postulated, the hyaluronasome, in which coordinated synthetic and catabolic enzyme reactions occur. The hyaluronasome can respond to the physiological states of the cell by a series of membrane-bound and soluble hyaluronan-associated receptors, binding proteins, and cofactors that trigger enzymatic events and signal transduction pathways. These in turn can be modulated by the amounts and sizes of the hyaluronan polysaccharides generated in the catabolic cascade. Most of these highly dynamic interactions remain to be determined. It is also proposed that malignant cells can commandeer some of these interactions for facilitating tumor growth and spread.

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.

Hyaluronidases in tissue invasion.

Hyaluronidases are broadly distributed enzymes with varying substrate specificities, a wide range of pH optima, and different catalytic mechanisms. They may be used by organisms to invade one another. Hyaluronidases have also been invoked as mechanisms for tumor invasion and metastatic spread. In this review, we will concentrate more on the different kinds of hyaluronidases involved in tissue invasion other than cancer metastasis, present some of the rapidly accumulating new data, and address the paradox that both hyaluronidase as well as its hyaluronan substrate are associated with invasion.