Testicular Hyaluronidase Research Articles

Method of hyaluronidase immobilization to produce an oral dosage form

Hyaluronidase is an enzyme that helps maintain an optimal balance of hyaluronic acid in connective tissue and ensures full tissue regeneration. Currently, hyaluronidase drugs are used in various fields of medicine. Meanwhile, there are no oral preparations based on hyaluronidase on the pharmaceutical market due to its low bioavailability in this method of administration. One of the options to increase enzyme bioavailability is electron-beam pegylation, which involves obtaining drugs with a modified pharmacokinetic profile in the direction of increasing enteral bioavailability, as well as having additional protection from the action of proteolytic enzymes. Aim of the study was using the electron beam pegylation method to obtain a laboratory batch of a preparation based on highly purified hyaluronidase, which can be further used in the development of an oral drug product. Material and methods. Hyaluronidase, which was isolated from bovine testicular hyaluronidase and polyethylene glycols of different molecular weight, was used in the experimental study. Experiments were carried out using a pulsed linear accelerator ILU-10 for enzyme electron beam immobilization on a water-soluble polymer matrix of polyethylene glycol molecules. The formation of hyaluronidase and polyethylene glycols conjugates was confirmed by exclusion chromatography. Results. As a result of accelerated electron irradiation of hyaluronidase and polyethylene glycol of molecular mass 1500 Da conjugates of enzyme and polymer matrix were formed. After electron-beam pegylation, the specific activity of the enzyme was retained. Conclusions. A laboratory batch of prototype pharmaceutical substance based on immobilised hyaluronidase on polyethylene glycol was obtained.

Garcinol: A novel and potent inhibitor of hyaluronidase enzyme

Extracellular matrix (ECM) contains hyaluronic acid (HA) as its integral part that is involved in numerous functional activities within the body. Degradation of HA by hyaluronidase enzyme involved in many pathophysiological conditions such as asthma, arthritis, COPD and in venom spreading during envenomation. Inhibitor of hyaluronidase enzyme has a wide range of application along with the hyaluronan-hyaluronidase system. In this present study, we have evaluated the inhibitory effect of garcinol against hyaluronidase from Hippasa partita spider venom (HPHyal), bovine testicular hyaluronidase (BTH) and human serum hyaluronidase. Garcinia indica fruit rind has been used to isolate the active component garcinol. Garcinol has been used in treatment of diverse ailments. Garcinol has exhibited anti-oxidant, anti-inflammatory, HAT inhibition and miRNA deregulator in development and progression of cancers. Experimental data have shown that garcinol completely inhibited all the three tested hyaluronidase enzymes. The inhibition was found to be non-competitive pattern with reversible type. In the docking study, garcinol with hyaluronidase enzyme has been stabilized by hydrogen bonding and hydrophobic interactions. Thus, garcinol could be a potent novel inhibitor of hyaluronidase enzyme which can be further used for pharmacotherapeutic applications.

Enhancing hyaluronidase enzyme activity: Insights from advancement in bovine and ovine testicular hyaluronidase purification

This essay investigates the use of an affinity resin named Capto lentil lectin for the purification of bovine and ovine testicular hyaluronidase. Hyaluronidase, an enzyme that degrades hyaluronic acid, is used widely in medical fields like dermatology, orthopedics, and ophthalmology. The research highlights the importance of optimizing the purification process to increase enzyme activity and purity. A new purification method is proposed, which begins with ammonium sulfate precipitation, followed by Blue Sepharose and Capto Lentil Lectin chromatography. This novel approach significantly increases the yield, purity, and activity of the enzyme. This study paves the way for further research into improving the purification process. The study further discusses challenges in identifying hyaluronidase bands using SDS-PAGE and highlights the necessity of using Western blotting for precise results.

Structural identification and quantification of unreported sialylated N-glycans in bovine testicular hyaluronidase by LC-ESI-HCD-MS/MS

Bovine testicular hyaluronidase (BTH), which accelerates the absorption and dispersion of drugs by decomposing hyaluronan in subcutaneous tissues, has been used in medical applications, including local anesthesia, ophthalmology, and dermatosurgery. The requirement of N-glycans for the activity of human hyaluronidase has been reported, and BTH has greater activity than human hyaluronidase. However, the N-glycan characteristics of BTH are unclear. From a commercial BTH source containing additional proteins, purified BTH (pBTH) was obtained using size exclusion chromatography, and the structures and quantities of its N-glycans were analyzed using liquid chromatography (LC)-electrospray ionization-higher energy collisional dissociation (HCD)-tandem mass spectrometry (MS/MS). In pBTH, 32 N-glycans were identified, with 12 sialylations (39.0% of total N-glycan content), nine core-fucosylations (31.5%), six terminal galactosylations (14.6%), five high-mannosylations (13.7%), and four bisecting N-acetylglucosamine structures (7.8%). The presence of sialylated glycopeptides in pBTH was confirmed by nano-LC-HCD-MS/MS analysis. The absolute quantity of all N-glycans was calculated as 1.4 pmol (0.6 pmol for sialylation) in pBTH (1.0 pmol). The sialylation level (related to half-life, thermal stability, resistance to proteolysis, and solubility) was 24.4 times higher than that of human hyaluronidase. The hyaluronan degradation activity of de-sialylated pBTH decreased to 41.2 ± 4.2%, showing that sialylated N-glycans were required for pBTH activity as well. This is the first study to identify and quantify 32 N-glycans of pBTH and investigate their structural roles in its activity. The presence of larger amounts of sialylated N-glycans in pBTH than in human hyaluronidase suggests a greater utilization of pBTH.

Biochemical and histological study of spermatogenic activity in male albino rats treated with Cantharis Q

Cantharidin is a bioactive toxin produced by the blister beetles commonly called Spanish fly. This natural compound is a bicyclic monoterpene (C10H12O4 ) that has clinical application as an anti-inflammatory, antiviral as well as antitumor agent and has been widely used to treat a variety of skin-related diseases. There are several reports of consumption of dried blister beetles and their traditional formulations due to a long history of the supposed aphrodisiac property of cantharidin. The present study, therefore, aimed at investigating the biochemical and histological changes in the testes of male albino rats of Wistar strain due to oral intake of Cantharis Q, a crude alcoholic extract of blister beetle Lytta vesicatoria used as mother tincture and as diluted potentized form in homeopathy. The experimental rats were given Cantharis Q dosage of 500 µg/Kg body weight for 60 days and the subsequent autorecovery was also studied after 30 days without treatment. The treated rats showed a significant decrease in the weight of testis, seminiferous tubular diameter and sperm count. Activity of testicular acid phosphatase (ACP), alkaline phosphatase (ALP), hyaluronidase (HL) enzyme and content of cholesterol, total protein, DNA and RNA decreased significantly whereas activity of beta-glucuronidase (BG) enzyme and glycogen was significantly increased. Spermatogenesis stages VIII, IX and X showed sloughing of round spermatids into the tubular lumen. The animals kept for autorecovery showed only partial improvement. Thus, it could be concluded that the treatment had a negative impact on the physiological function of testis of albino rat causing detrimental effects on the spermatogenic activity

Identification, quantification, and structural role of N-glycans in two highly purified isoforms of sheep testicular hyaluronidase

Animal-derived hyaluronidase, which hydrolyzes the polysaccharide hyaluronic acid, has been used in medical applications despite its limited purity. Additionally, the N-glycan characterization of sheep testicular hyaluronidase (STH) and its structural role remain poorly understood. In this study, STH was purified from the commercially available STH preparation (containing at least 14 impurity proteins) using heparin-affinity chromatography followed by size exclusion chromatography. The structure and quantity of N-glycans of STH were investigated using liquid chromatography-electrospray ionization-high energy collision dissociation-tandem mass spectrometry. Two isoforms, H3S1 and H3S2, of STH were obtained (purity >98 %) with a yield of 3.4 % and 5.1 %, respectively. Fourteen N-glycans, including nine core-fucosylated N-glycans (important for the stability and function of glycoproteins), were identified in both H3S1 and H3S2, with similar quantities of each N-glycan. The amino acid sequences of the proteolytic peptides of H3S1 and H3S2 were compared with those reported in STH. The hyaluronic acid-degrading activity of deglycosylated H3S1 and H3S2 was reduced to 70.8 % and 71.1 % compared to that (100 %) of H3S1 and H3S2, respectively. This is the first report of N-glycan characterization of two highly purified isoforms of STH. These H3S1 and H3S2 will be useful for medical use without unwanted effects of partially purified STH.

Modified hyaluronic acid with enhanced resistance to degradation

A mild and efficient reduction of negatively charged glucuronate units of hyaluronic acid (HA) into less polar glucose units has not been reported yet. However, this modification could significantly affect physical and chemical properties. Here we show a one-pot procedure where HA is converted into its derivate with carboxyl groups reduced to primary alcohols (HA-Red) without severe polymer degradation. Optimized synthesis aimed at aqueous solutions allowed the preparation of polysaccharides with molecular weights up to 1000 kDa. The chemical structure of HA-Red was proved by 2-dimensional NMR methodologies, FT-IR, LC-MS and SECMALLS. The final materials were exposed to a higher temperature or digested with bovine testicular hyaluronidase (BTH). Obtained data proved higher stability of HA-Red compared to HA, and significant dependence of stability on the degree of modification was observed in most cases. Preliminary in vitro studies showed no negative effects of HA-Red on the growth of 3T3 fibroblasts, which may be promising for applications requiring biodegradable and biocompatible HA derivatives with increased resistance to degradation.

РЕЗУЛЬТАТЫ ЭКСПЕРИМЕНТОВ ПО РАЗРАБОТКЕ ВЫСОКООЧИЩЕННОЙ ГИАЛУРОНАТ-ЭНДО-β-N-АЦЕТИЛГЕКСОЗАМИНИДАЗЫ В КАЧЕСТВЕ ФАРМАЦЕВТИЧЕСКОЙ СУБСТАНЦИИ ДЛЯ ЛЕКАРСТВЕННОГО ПРЕПАРАТА

Enzyme preparations, including hyaluronidase, are widely used in medical practice. Most drugs with this active substance on the pharmaceutical market are produced mainly from testicular hyaluronidase, the raw material for which are bovine testes. The technologies of enzyme preparation production are quite well known and diverse. However, the creation of a different method will allow the highly purified hyaluronate-endo-β-N-acetylhexosaminidase with high specific enzymatic activity. Objective: develop a technology and obtain a laboratory batch of the highly purified pharmaceutical substance hyaluronate-endo-β-N-acetylhexosaminidase. Materials and Methods. In the experimental study, preparations of bovine testes extracts were taken to obtain highly purified hyaluronate-endo-β-N-acetylhexosaminidase. In the first stage the protein spectrum of the testes extracts was studied, proteins were fractionally precipitated, and substances of different chemical nature were fractionated by chromatography methods. At the second stage, the development of a method to obtain the pharmaceutical substance hyaluronate-endo-β-N-acetylhexosaminidase was carried out using the methods described further in the work. Results. Based on the results obtained during the experimental study, P-II, a preparation of bovine testes extract produced by Samson-Med LLC (St. Petersburg), was chosen as the initial substance of crude hyaluronidase for further studies. The most optimal is the purification scheme, which includes as the main method affinity chromatography on heparin-sepharose using affinity elution with heparin solution in buffer with a concentration of 3 mg/ml. Conclusions As a result of the experimental study, the optimal scheme was presented to obtain the pharmaceutical substance HEBNA with high enzymatic total activity of 289 (unit of action) was presented, and a laboratory batch of highly purified hyaluronate-endo-β-N-acetylhexosaminidase was obtained.

Hyaluronan Hydrogels: Rheology and Stability in Relation to the Type/Level of Biopolymer Chemical Modification.

BDDE (1,4-butanediol-diglycidylether)-crosslinked hyaluronan (HA) hydrogels are widely used for dermo-aesthetic purposes. The rheology and stability of the gels under physiological conditions greatly affect their clinical indications and outcomes. To date, no studies investigating how these features are related to the chemistry of the polymeric network have been reported. Here, four available HA-BDDE hydrogels were studied to determine how and to what extent their rheology and stability with respect to enzymatic hydrolysis relate to the type and degree of HA structural modification. 1H-/13C-NMR analyses were associated for the quantification of the “true” HA chemical derivatization level, discriminating between HA that was effectively crosslinked by BDDE, and branched HA with BDDE that was anchored on one side. The rheology was measured conventionally and during hydration in a physiological medium. Sensitivity to bovine testicular hyaluronidase was quantified. The correlation between NMR data and gel rheology/stability was evaluated. The study indicated that (1) the gels greatly differed in the amounts of branched, crosslinked, and overall modified HA, with most of the HA being branched; (2) unexpectedly, the conventionally measured rheological properties did not correlate with the chemical data; (3) the gels’ ranking in terms of rheology was greatly affected by hydration; (4) the rheology of the hydrated gels was quantitatively correlated with the amount of crosslinked HA, whereas the correlations with the total HA modification level and with the degree of branched HA were less significant; (5) increasing HA derivatization/crosslinking over 9/3 mol% did not enhance the stability with respect to hyaluronidases. These results broaden our knowledge of these gels and provide valuable information for improving their design and characterization.

Kinetic theory of hyaluronan cleavage by bovine testicular hyaluronidase in standard and crowded environments

In this paper, we introduce a comprehensive kinetic model describing the enzymatic cleavage of hyaluronan (HA) by bovine testicular hyaluronidase (BTH). Our theory focuses specifically on the late stage of the hydrolysis, where the concentrations of a limited number of oligomers may be determined experimentally with accuracy as functions of time.The present model was applied to fit different experimental sets of kinetic data collected by capillary electrophoresis at two HA concentrations and three concentrations of PEG crowder (0, 10, 17% w/w). Our theory seems to apply universally, irrespective of HA concentration and crowding conditions, reproducing to an excellent extent the time evolution of the individual molar fractions of oligomers. Remarkably, we found that the reaction mechanism in the late degradation stage essentially reduces to the cleavage or transfer of active dimers. While the recombination of dimers is the fastest reaction, the rate-limiting step turns out to be invariably the hydrolysis of hexamers. Crowding, HA itself or other inert, volume-excluding agents, clearly boosts recombination events and concomitantly slows down all fragmentation pathways.Overall, our results bring a novel and comprehensive quantitative insight into the complex reaction mechanism underlying enzymatic HA degradation. Importantly, rationalizing the effect of crowding not only brings the intricate conditions of in-vivo settings a little closer, but also emerges as a powerful tool to help pinpointing relevant kinetic pathways in complex systems.

Dimers and Trimers of Chondroitin in Molecular Docking of Bovine Testicular Hyaluronidase

Molecular docking of a 3D model of bovine testicular hyaluronidase was performed with dimers and trimers of chondroitin. On the molecular surface of a hyaluronidase globule, eight sites for ligand binding were established. Sites 6, 3, and 1 of the enzyme structure were found to be the most important sites since chondroitin binding therewith distorts the protein 3D structure. The interactions of enzyme structure with chondroitin ligands are mainly determined by electrostatic forces. For free hyaluronidase molecule (without ligands) at temperatures above 300 K conformational transfers leading to the enzyme inactivation were noted. The inactivation was mainly manifested through irreversible attraction of the protein fragment surrounding the Glu-105 residue and the one between Arg-59 and Arg-96, where a temperature decrease did not induce the restoration of the initial view of 3D structure of the enzyme molecule. Binding of chondroitin ligands to the enzyme structure at positions 6, 3, and 1 increased its denaturation temperature by approximately 10 degrees. The most significant effect of enzyme structure stabilization was provided by chondroitin ligand binding with hyaluronidase at site 6. This effect of protein structure stabilization exceeded the one of chondroitin sulfate trimers against the enzyme inhibition by heparin tetramers, which required binding of four to five chondroitin sulfate ligands on the molecular surface of the enzyme.

Construction of saturated odd- and even-numbered hyaluronan oligosaccharide building block library

Hyaluronic acid (HA) and its oligosaccharides (o-HAs) have been receiving much attention as medical treatments. Even-numbered o-HAs with two different reducing ends (HA2nAN and HA2nNA, n = 1, 2, 3…) can be prepared using bovine testicular hyaluronidase (BTH) and leech hyaluronidase (LHase) respectively, while preparation of odd-numbered o-HAs (HA2n+1AA and HA2n+1NN, n = 1, 2, 3…) is relatively difficult. A new enzymatic method was developed to prepare odd-numbered o-HAs in this study, taking advantage of differences in the degradation of glycosidic linkages by BTH and LHase. Through quantitative analysis of the yield of even-numbered o-HAs, a dynamic model for LHase hydrolysis was established, providing optimal preparation conditions for HA2nNA (n = 1–5). Then HA2nNA (n = 2–5) was further degraded by BTH to produce odd-numbered o-HAs. The above o-HAs were separated and purified by combining high-performance Q-Sepharose ion-exchange (Q HP) and size exclusion column chromatography steps.

Dermatan sulfate oligosaccharides having reducing end 2, 5-anhydro-d-talose inhibit bovine testicular hyaluronidase activity

Dermatan sulfate oligosaccharides having reducing end 2,5-anhydro-d-talose were prepared by partial N-deacetylation of dermatan sulfate polysaccharide with hydrazine followed by deamination with nitrous acid, and the effect of these oligosaccharides on the activity of bovine testicular hyaluronidase was investigated. Hydrolysis activity and transglycosylation activity of this enzyme were assessed in an independent reaction system by analyzing the products by HPLC. Dermatan sulfate oligosaccharides inhibited hyaluronan hydrolysis by bovine testicular hyaluronidase. Kinetic analysis of the hydrolysis reaction revealed that the inhibition mode by dermatan sulfate oligosaccharides was as competitive as that previously shown by chondroitin sulfate oligosaccharides. Transglycosylation of hyaluronan by bovine testicular hyaluronidase, as a reverse reaction of hydrolysis of hyaluronan, was also inhibited. These inhibitory effects were dependent on the dose and sulfation degree of dermatan sulfate.

Hyaluronan-based hydrogels as dermal fillers: The biophysical properties that translate into a "volumetric" effect.

Biophysical and biochemical data on hyaluronan (HA)-based dermal fillers strongly support their optimal use and design to meet specific requisites. Here, four commercially available (in Europe) HA “volumetric” fillers, among the most used in the clinical practice, have been characterized in vitro. Analyses revealed the highest amounts of water-soluble HA reported so far and provided hydrodynamic data for these soluble polymeric fractions. Volumetric gels exhibit a wide range of rigidity with most of them showing G’ values around 200-300Pa. They greatly differ in cohesivity. 1mL of gel hydrates up to 2.4–3.2mL. The products completely solubilize due to Bovine Testicular Hyaluronidase (BTH)’s action, thus predicting in vivo complete resorption. For the first time, filler degradation due to reactive oxygen species (ROS) was studied by rheological measurements and a rank in stability was established. Studies using Human Dermal Fibroblasts (HDF) indicated a positive biological response to the HA networks. Further, gel capacity to prompt collagen I, elastin and aquaporin3 synthesis was demonstrated, thus suggesting a positive effect on skin elasticity and hydration, besides the physical volumetric action. The findings are the first wide assessment of features for the volumetric class of HA-fillers and include first data on their resistance to degradation by ROS and biological effects on HDF. The study represents a valuable contribution to the understanding of HA-fillers, useful to optimize their use and manufacture.

Clinical Applications of Hyaluronidase.

Hyaluronidases are enzymes that degrade hyaluronic acid, which constitutes an essential part of the extracellular matrix. Initially discovered in bacteria, hyaluronidases are known to be widely distributed in nature and have been found in many classes including insects, snakes, fish and mammals. In the human, six different hyaluronidases, HYAL1-4, HYAL-P1 and PH-20, have been identified. PH-20 exerts the strongest biologic activity, is found in high concentrations in the testicles and can be localized on the head and the acrosome of human spermatozoa. Today, animal-derived bovine or ovine testicular hyaluronidases as well as synthetic hyaluronidases are clinically applied as adjuncts to increase the bioavailability of drugs, for the therapy of extravasations, or for the management of complications associated with the aesthetic injection of hyaluronic acid-based fillers. Further applications in the fields of surgery, aesthetic medicine, immunology, oncology, and many others can be expected for years to come. Here, we give an overview over the molecular and cellular mode of action of hyaluronidase and the hyaluronic acid metabolism, as well as over current and potential future clinical applications of hyaluronidase.

Chondroitin: a natural biomarker with immense biomedical applications.

Naturally extracted glycosaminoglycan chondroitin sulphate is the reactive product of N-acetylgalactosamine and d-glucuronic acid. Chondroitin sulfate (CS) extracted from Scophthalmus maximus, H. scabra, E. fraudatrix, M. magnum, and H. mexicana has shown remarkable anticoagulant, articular cartilage repair, corneal lesion healing, antidiabetic, and antiproliferative effects. Also, platinum and strontium nanoparticles of chondroitin sulfate are effective in osteoarthritis and exert anti-HSV2 and anti-angiogenic properties. A combination of chondroitin sulfate and RNA lipolexes demonstrates gene silencing effects in liver fibrosis. Chondroitin sulfate has also been used as a carrier for loxoprofen hydrogel preparation. Oligosaccharides of chondroitin sulfate showed effective inhibition of bovine testicular hyaluronidase enzyme as an antibacterial agent during pregnancy. Monoclonal antibody-recognized chondroitin sulfate A was effectively used to treat ameloblastoma. Selenium-chondroitin sulfate nanoparticles demonstrated positive effects in therapy of Kashin–Beck disease (KBD) and osteoarthritis.

Thermostabilization of Hyaluronidase by Chondroitin Ligands in Molecular Docking

Speedup of present high-molecular drug derivatives developments is based on the computational methods harmonic application. Such glycosidase as hyaluronidase has been functionated among multifarious glycosaminoglycan microenvironment in blood circulation of organism. It is important for elucidation of action mechanism of biosystem components (on vascular wall) and productive obtaining of hyaluronidase derivatives of cardiological destination the using of computer aided calculations for investigation of protein-glycosaminoglycan interactions. The aim of our study became the molecular docking fulfillment for 3D model of bovine testicular hyaluronidase with short-chain dimer and trimer chondroitin ligands. We used the molecular docking of hyaluronidase with chondroitin ligands for theoretical determination of biocatalyst conformational stability. At temperatures higher than 300 K free/native hyaluronidase (without ligands) displayed conformational transitions leading to its inactivation. Predominant manifestation of these transitions was an irreversible attraction developing between the protein area proximate to Glu-105, on the one hand, and to Arg-59 and Arg-96, on the other. Lowering of temperature did not restore the initial 3D structure of hyaluronidase. Binding of chondroitin ligands at ch 6, ch 3 and ch 1 sites stabilized the enzyme, increasing its denaturation temperature by 10°C. The highest degree of stabilization was achieved after chondroitin binding to ch 6. This effect was higher than that for chondroitin sulfate trimers concerning the enzyme inhibition by heparin tetramer, which requires binding of 4-5 chondroitin sulfate ligands to the enzyme surface. Molecular docking of 3D model of bovine testicular hyaluronidase with chondroitin dimers and trimers has revealed eight sites for their binding to the enzyme surface. At biological concentrations of these ligands the most important binding sites for them are ch 6, ch 3 and ch 1. Binding at these sites induces a reversible deformation of the protein 3D structure. Interactions between 3D model of bovine hyaluronidase with chondroitin ligands are based predominantly on electrostatic forces. Chondroitin ligands stabilized 3D structure of hyaluronidase after binding predominantly at chondroitin 6 as well as chondroitin 3 and chondroitin 1 positions and their effect was higher than chondroitin sulfate upon enzyme inhibition by heparin tetramer. Stabilizing effects of chondroitin and chondroitin sulfate ligands are fundamental for further direct theoretical comparative investigation of impact produced by these ligands.

Electrostatic Interactions in Docking a 3D Model of Bovine Testicular Hyaluronidase with a Chondroitin Sulfate Trimer and a Heparin Tetramer

The molecular docking of a 3D-model of bovine testicular hyaluronidase with glycosaminoglycan ligands is performed. A chondroitin sulfate trimer and a heparin tetramer were used as ligands. Methods of computational chemistry are applied to elucidate the regulation of hyaluronidase functioning in an organism when the heparin ligand inactivates the biocatalyst, and the chondroitin sulfate ligand protects the enzyme structure. Eight ligand binding sites are identified on the molecular surface of the enzyme, each of which is equally capable of interacting with chondroitin sulfate trimers and heparin tetramers via electrostatic interactions. It is found that reversible and irreversible conformational changes in the enzyme 3D structure can occur depending on the positioning of negatively charged ligands on its globule (under different conditions, they can either stabilize or inactivate the biocatalyst). Binding sites whose occupancy is sufficient for preventing irreversible deformations of the enzyme conformation upon introducing the heparin ligand into the active site are identified on the molecular surface of hyaluronidase. The interaction of glycosaminoglycan ligands with hyaluronidase is mainly determined by electrostatic forces.

Molecular Docking of Short-Chain Glycosaminoglycan Ligands in 3D Model of Bovine Testicular Hyaluronidase

1. <span style=font-size:11.0pt;mso-ansi-language: EN-US>Abstract Computational analysis of molecular docking of 3D model of bovine testicular hyaluronidase in silico with a trimer (hexasaccharide) chondroitin sulfate and tetramer (octasaccharide) heparin has demonstrated eight significant binding sites (cs1-cs8) for these ligands. Interactions with heparin that inactivates the enzyme and protective effects of chondroitin sulfate were examined. Binding sites have been identified which are critical for stabilization of the protein structure after chondroitin sulfate binding: at positions cs2, cs4, cs7 and cs8 or cs1, cs2, cs4, cs7 and cs8. Occupation of these sites is theoretically sufficient for prevention of irreversible molecular deformations after heparin inclusion into active site. The presence of sensitivity sites on hyaluronidase globule indicates the possibility of regulating the enzyme function via coupling glycosaminoglycan ligands thus initiating delicate/fine formation of effective electrostatic potential. Interactions between glycosaminoglycan ligands and hyaluronidase are based primarily on electrostatic forces. 2. <span style=font-size:11.0pt;mso-ansi-language: EN-US>Keywords: <span style=font-size:10.0pt;mso-ansi-language: EN-US>Bovine Testicular Hyaluronidase; Chondroitin Sulfate; Docking; Glycosaminoglycan Ligands; Heparin

Effect of the hyaluronidase microe nvironment on the enzyme structure–function relationship and computational study of the in silico molecular docking of chondroitin sulfate and heparin short fragments to hyaluronidase

The review addresses the biochemical interactions of hyaluronidases with components of the natural microenvironment. The effect of subtle structural differences between ligands on the enzyme structure–function relationship regulation is noted. Docking of chondroitin sulfate (CS) trimers (hexasaccharides) and heparin tetramers (octasaccharides) to the 3D model of the bovine testicular hyaluronidase (BTH) was performed by computational chemistry methods in order to elucidate the mechanism of regulation of the enzyme functioning in the body (using virtual screening, molecular dynamics, and calculation of surface electrostatic potential of protein complexes). Several binding sites for glycosaminoglycan (GAG) ligands were found to occur on the hyaluronidase surface. They are identical for CS trimers and heparin tetramers. The calculations showed the possibility of both reversible and irreversible conformational changes of the 3D structure of BTH, depending on the arrangement of negatively charged ligands on its globule. When the changes are irreversible, Glu-149 and Asp-147, which are key amino acid residues for the catalytic activity of BTH, can migrate from the vicinity of the native enzyme active site to the periphery of the protein molecule, thus inducing enzyme inactivation. The interaction of the GAG ligands with the BTH active site is mainly caused by electrostatic forces. Four or five binding sites of the chondroitin sulfate trimer proved to be critical for stabilization of the enzyme structure. Their occupation was sufficient for preventing irreversible deformation of the BTH molecule upon the insertion of the heparin ligand into the active site cavity. Protein stabilization is accompanied by the formation of a particular form of the surface electrostatic potential.