Chemical modification of hyaluronidase as a means to regulate its inhibition by heparin

The modification of hyaluronidase by aldehydodextran regulates inhibition of the enzyme by heparin. A 70–90% modification of the surface amino groups of hyaluronidase results in sharp conformational changes and a substantial decrease of its inhibition by heparin, whereas hyaluronidase derivatives with a modification degree of 96–100% are practically uninhibited.     

Resistance of Dextran-Modified Hyaluronidase to Inhibition by Heparin

Properties of native and aldehyde dextran-modified hyaluronidase (with surface amino group modification about 98%) were investigated. Optimal endoglycosidase activity of the native enzyme was observed at 0.15 M NaCl and pH 5.5 and electrostatic interactions influenced the enzyme activity. The inhibitory effect of heparin on hyaluronidase activity slightly differed at pH 5.5 (1.5-fold inhibition) and 7.5 (1.2-fold inhibition). Ionic strength of the reaction medium only slightly influenced the effect of heparin. Modification of hyaluronidase with dextran increased hydrophobic interactions and steric hindrance. Conjugation with dextran increased the resistance of hyaluronidase activity to denaturing agents (urea, guanidinium hydrobromide) and extended the optimal conditions for maximal endoglycosidase activity (pH 4.5-6.5, the range of NaCl concentration from 0.1 to 0.3 M). The conjugation also reduced electrostatic effects on the active site of hyaluronidase and efficacy of heparin inhibition. At pH 7.5 the enzyme was almost insensitive to heparin. The resistance of dextran-modified hyaluronidase to heparin points to approaches for subsequent studies of the heparin binding site of this enzyme and biomedical trial of the stabilized enzyme for the treatment of acute cardiovascular lesions.     

Inhibition of hyaluronidase by fully O-sulfonated glycosaminoglycans.

We report a new flow injection assay (FIA) method for determining hyaluronidase activity and the inhibitory effects of chemical fully O-sulfonated glycosaminoglycans on this enzyme. The products of enzymatic action on hyaluronidase can be detected by FIA using fluorometric detection with the fluorogenic reagent 2-cyanoacetamide. The major products derived from hyaluronan by the action of mammalian testicular hyaluronidase (a hydrolyase) were confirmed by (1)H NMR spectroscopy and capillary electrophoresis. The FIA method was next applied to the assay of hyman urinary hyaluronidase activity and the screening of hyaluronidase inhibitors. The human urinary hyaluronidase activity measured ranged from 46 to 59 turbidity reducing units/mg protein. Among the glycosaminoglycans only heparin showed hyaluronidase inhibition. Chemically O-sulfonated glycosaminoglycans showed IC(50) values of hyaluronidase inhibition that correlated with the degree of O-sulfonation. Heparin was found to inhibit hyaluronidase activity noncompetitively, while chemically O-sulfonated HA strongly inhibited hyaluronidase through both competitive and noncompetitive effects.     

Role of the glycosaminoglycan microenvironment of hyaluronidase in regulation of its endoglycosidase activity

The glycosaminoglycan microenvironment of testicular hyaluronidase was simulated by multipoint covalent attachment of the enzyme to glycans as a result of benzoquinone activation. The efficiency of their binding was assessed using gel chromatography, ultrafiltration, titration of surface amino groups of the enzyme, electrophoresis, as well as judging by the value of residual endoglycosidase activity and its inhibition with heparin. Copolymer glycosaminoglycans, such as dermatan sulfate and heparin, inactivated the endoglycosidase activity as a result the C-5 epimerization of hexuronic acid. It was shown that glucuronic acid and, to a lesser extent, N-acetylglucosamine determine the specificity of hyaluronidase. The chondroitin-sulfate microenvironment made the enzyme resistant to heparin inhibition because the equatorial orientation of the OH groups is similar to that in hyaluronic acid. Model experiments with dextran and dextran sulfate showed that sulfation of the glycan chain increased its rigidity, thus hampering the stabilizing effect on hyaluronidase. The effect of chondroitin sulfate on the endoglycosidase activity of hyaluronidase had additive character and did not directly affect the small fragment of the active site of the enzyme located at the bottom of a groove. The glycosaminoglycan microenvironment of hyaluronidase, containing an iduronic acid residue, the 1-3 and 1-4 glycosidic bond, inactivated the hyaluronidase activity of the enzyme, whereas simple polymers (such as gluco- and galactoaminoglycans) potentiated it due to a similar way of linking—(1e-4e) and (1e-3e). To understand the nature of these interactions in detail, the effect of oligomeric glycosaminoglycan fragments and their derivatives on hyaluronidase should be studied.     

Inhibition of <Emphasis Type="Italic">Naja naja</Emphasis> Venom Hyaluronidase by Plant-Derived Bioactive Components and Polysaccharides

The inhibitory effect of several bioactive compounds on the activity of hyaluronidase enzyme purified from Naja naja venom was investigated in vitro. Compounds were found to inhibit the hyaluronidase activity dose dependently. Among glycosaminoglycans, heparin, heparan sulfate, and dermatan sulfate showed maximum inhibition compared to chondroitin sulfates. Different molecular forms of chitosan inhibit the enzyme, and inhibition appears to depend on the chain length. In addition, plant-derived bioactive compounds also inhibited the activity of hyaluronidase dose dependently. Among those tested, aristolochic acid, indomethacin, quercetin, curcumin, tannic acid, and flavone exhibited inhibition, with aristolochic acid and quercetin completely inhibiting the enzyme activity. It is concluded that the inhibitors of hyaluronidase could be used as potent first aid agents in snakebite therapy. Furthermore, these inhibitors not only reduce the local tissue damage but also retard the easy diffusion of systemic toxins and hence increase survival time.     

Histochemical demonstration of acidic glycosaminoglycans in the cell nuclei of the iris and other tissues.

Using histochemical methods, the presence of acidic glycosaminoglycans in the cell nuclei of 51 human irides and a series of monkey organs was demonstrated. In general, these substances are sensitive to testicular hyaluronidase and chondroitinase ABC and also to Streptomyces hyaluronidase, when using special staining methods. The specificity of testicular hyaluronidase was tested by inhibition with heparin. By simultaneously staining with alcian blue and Feulgen, acidic glycosaminoglycans can be distinguished from the nucleic acids. Sporadically, hyaluronidase-resistant substances with a specific acidic glycosaminoglycan stainability occur. We assume the existence of various acidic glycosaminoglycans in the cell nuclei. Aging changes were not traceable with constancy.     

Evidence for essential lysines in heparin cofactor II

Covalent modification with pyridoxal 5'-phosphate was used to study the function of lysyl residues in heparin cofactor II, a heparin-dependent plasma protease inhibitor. Reduction of the Schiff base with sodium borohydride resulted in modification of 3-4 lysyl residues of heparin cofactor II at high concentrations of pyridoxal 5'-phosphate, one of which was protected in the presence of heparin. The antithrombin activity of modified heparin cofactor II was enhanced compared to the native protein. However, the heparin cofactor activity for thrombin inhibition was reduced significantly or completely eliminated in the modified protease inhibitor depending on the extent of phosphopyridoxylation. In contrast to native heparin cofactor II, the modified protease inhibitor did not bind to a heparin-agarose column. The results suggest that lysyl residues are essential for heparin cofactor activity during thrombin inhibition.     

Purification and partial characterization of hyaluronidase from stonefish (Synanceja horrida) venom.

1. A marine hyaluronidase was purified 261-fold from the stonefish (Synanceja horrida) crude venom using Sephacryl S-200 HR and heparin affinity-gel chromatography. 2. Stonefish hyaluronidase has a pI of 9.2, a mol. wt of 62,000 and it was purified to a very high spec. act. of 1.6 x 10(6) NFU/mg protein. 3. It was heat sensitive and was inhibited by Cu2+, Hg2+ and heparin. 4. Stonefish hyaluronidase did not contain any haemorrhagic or lethal activity. 5. The N-terminal sequence of stonefish hyaluronidase has been determined to be A-P-S-X-D-E-G-N-K-K-A-D-N-L-L-V-K-K-I-N.     

Binding of heparin to human antithrombin III activates selective chemical modification at lysine 236. Lys-107, Lys-125, and Lys-136 are situated within the heparin-binding site of antithrombin III

A new water-soluble color reagent, 4-N,N-dimethylaminoazobenzene-4'-isothiocyano-2'-sulfonic acid (S-DABITC), was used to identify lysine residues of antithrombin III which participate in the binding of heparin. Antithrombin, modified with S-DABITC in the presence and absence of low molecular weight heparin (Mr 5000) was reduced, carboxymethylated, and digested with trypsin. The digest was analyzed by high-performance liquid chromatography and monitored at 465 nm. In the absence of heparin, four major colored peptides (T1, T2, T3, and T4) were identified. When antithrombin was preincubated with heparin (2-fold by weight), followed by S-DABITC modification, the recovery of peptide T4 remained unchanged, but the recoveries of T1, T2, and T3 were reduced by 93, 86, and 98%, respectively. In addition, a new colored peptide, TA, appeared. Amino acid sequencing of peptides T1, T2, T3, and TA localized S-DABITC modification sites as Lys-136, Lys-125, Lys-107, and Lys-236, respectively. Thus, binding of heparin to human antithrombin diminished S-DABITC modification at Lys-107, Lys-125, and Lys-136, but at the same time enhanced S-DABITC modification at Lys-236. This phenomenon was further characterized by varying the molar ratio of heparin/antithrombin (from 0.04 to 20). The shielding of Lys-125 and Lys-136 was inversely proportional to the activation of Lys-236. At a heparin/antithrombin molar ratio of 1, the extent of shielding of Lys-125 and Lys-136 and the unmasking of Lys-236 were 25-33%. This shielding-unmasking effect correlated with enhanced antithrombin inhibition of thrombin. We conclude that Lys-107, Lys-125, and Lys-136 are situated within the heparin-binding site of human antithrombin and that binding of heparin to antithrombin causes a conformational change of antithrombin that leads to the exposure of Lys-236 for S-DABITC modification.     

东亚钳蝎毒透明质酸酶的纯化和部分性质的研究

用CM-SephadexC50,CM-SephadexC25和SephadexG-75凝胶过滤,从东亚钳蝎毒中提纯蝎毒透明质酸酶,应用低pH系统不连续聚丙烯酰胺凝胶圆盘电泳,SDS-不连续聚丙烯酰胺凝胶垂直板电泳鉴定均为单一条带,活力提高34倍,产率为12％,纯品无出血活性,无神经毒性。用凝胶过滤法和SDS电泳法测得分子量为54000,PAS染色证实为糖蛋白。 纯化的透明质酸酶的最适pH为4.5～6.5,最适温度为37℃,该酶对热的稳定性比蛇毒透明质酸酶高一些,但在碱性环境中也易失活。0.15MNaCl对酶活性有明显稳定作用,Fe~(2+)、Fe~(3+)及肝素对酶活性有明显的抑制作用,Cu~(2+)对酶活力也有一定影响。     

Evidence that arginine-129 and arginine-145 are located within the heparin binding site of human antithrombin III

Arginyl residues of human antithrombin III have been implicated to involve in the heparin binding site [Jorgensen, A. M., Borders, C. L., & Fish, W. W. (1985) Biochem, J. 231, 59-63]. We have performed chemical modification of antithrombin with (p-hydroxyphenyl)glyoxal (HPG) in order to determine the locations of these arginine residues. Antithrombin was modified with 12 mM HPG in the absence and presence of heparin (2-fold by weight to antithrombin). In the absence of heparin, about 3-4 mol of arginines/mol of antithrombin were modified within 60 min, and the modification led to the loss of 95% of the inhibitor's heparin cofactor activity as well as heparin-induced fluorescence enhancement and 50% of its progressive inhibitory activity. In the presence of heparin, the extent of modification was diminished by 30% and modified antithrombin retained approximately 70% of its heparin cofactor activity. Peptide mapping and subsequent sequence analysis revealed that selective HPG modification occurred at Arg129 and Arg145 and that their modifications were protected upon binding of heparin to antithrombin. We conclude that Arg129 and Arg145 are situated within the heparin binding site of human antithrombin III.     

Do electrostatic interactions determine glycation of hyaluronidase derivatives with N-acetylhexosamines?

Using N-acetylglucosamine and N-acetylgalactosamine as model agents for glycation of native hyaluronidase and its chondroitin sulfate modified form it has been shown that the modified enzyme exhibited higher inactivation than the native enzyme, while heparin caused similar inhibition of both forms. Such effect could be attributed to the development of electrostatic interactions as the modified hyaluronidase had altered surface electrostatic potential after chondroitin sulfate binding. However, variations in ionic strength of the medium containing enzyme derivatives have shown that their endoglycosidase activity changed in a similar manner and the effect on glycation represents a multifactor process. N-acetylhexosamines are natural labels of endothelial glycocalyx degradation products. Interaction of the hyaluronidase forms with charged hyaluronan fragments revealed significantly higher inactivation of the modified enzyme compared with the native enzyme. The glycation pattern observed in this study was opposite to that observed with mono- and disaccharides. Thus, it appears that the investigated hyaluronidase derivatives represent an informative enzymatic test in vivo for determination of the dominant type of glycation agents in blood circulation and their origin.     

Heparin-sepharose affinity chromatography for purification of bull seminal-plasma hyaluronidase.

Bull seminal-plasma hyaluronidase was purified 180-fold by chromatography on concanvalin A-Sepharose, heparin Sepharose, Sephadex G-200 and Sephacryl S-200. With hyaluronic acid as the substrate, the specific activity and turnover number of purified hyaluronidase were 3.63 mumol/min per mg (104000 National Formulary units/mg of protein) and 214 min-1 (mol of product formed/mol of enzyme per min) respectively. Polyacrylamide-gel electrophoresis indicated that the purified enzyme migrated as a single band on 7.5 and 10% (w/v) gels at pH 4.3 and 5.3. Bull seminal-plasma hyaluronidase was markedly inhibited by hydroxylamine, phenylhydrazine and semicarbazide. Purified hyaluronidase (1.25 munits; 1 unit = 1 mumol of N-acetylglucosamine liberated/min at 37 degrees C) dispersed the cumulus clot of rabbit ova in 1 h at 22 degrees C.     

Importance of lysine 125 for heparin binding and activation of antithrombin

The anticoagulant sulfated polysaccharide, heparin, binds to the plasma coagulation proteinase inhibitor, antithrombin, and activates it by a conformational change that results in a greatly increased rate of inhibition of target proteinases. Lys125 of antithrombin has previously been implicated in this binding by chemical modification and site-directed mutagenesis and by the crystal structure of a complex between antithrombin and a pentasaccharide constituting the antithrombin-binding region of heparin. Replacement of Lys125 with Met or Gln in this work reduced the affinity of antithrombin for full-length heparin or the pentasaccharide by 150-600-fold at I = 0.15, corresponding to a loss of 25-33% of the total binding energy. The affinity decrease was due both to disruption of approximately three ionic interactions, indicating that Lys125 and two other basic residues of antithrombin act cooperatively in binding to heparin, and to weakened nonionic interactions. The mutations caused a 10-17-fold decrease in the affinity of the initial, weak binding step of the two-step mechanism of heparin binding to antithrombin. They also increased the reverse rate constant of the second, conformational change step by 10-50-fold. Lys125 is thus a major heparin-binding residue of antithrombin, contributing an amount of binding energy comparable to that of Arg129, but less energy than Lys114. It is the first residue identified so far that has a critical role in the initial recognition of heparin by antithrombin, but also appreciably stabilizes the heparin-induced activated state of the inhibitor. These effects are exerted by interactions of Lys125 with the nonreducing end of the heparin pentasaccharide.     

The role of hyaluronic acid in intercellular adhesion of cultured mouse cells

Aggregation of cultured mouse cells was measured by the rate of disappearance of particles from a suspension of single cells. Treatment with several enzymes which degrade hyaluronic acid (testicular hyaluronidase, streptomyces hyaluronidase, streptococcal hyaluronidase and chondroitinase ABC) inhibited the aggregation of SV-3T3 and several other cell types. Since streptomyces and streptococcal hyaluronidases are specific for hyaluronic acid, it is suggested that hyaluronic acid is involved in the observed aggregation. Hyaluronidase-induced inhibition of aggregation was complete in the absence of divalent cations, but only partial in their presence. This finding is consistent with the hypothesis that two separate mechanisms are responsible for aggregation; one dependent upon and the other independent of calcium and magnesium. Aggregation was also inhibited by high levels of hyaluronic acid. A similar effect was obtained with fragments of hyaluronic acid consisting of six sugar residues or more. Chondroitin (desulfated chondroitin 6-sulfate) and to a lesser extent desulfated dermatan sulfate also inhibited aggregation. Other glycosaminoglycans (chondroitin 4-sulfate, chondroitin 6-sulfate, heparin and heparan sulfate) had little or no effect on aggregation. It is suggested that the hyaluronic acid inhibits aggregation by competing with endogenous hyaluronic acid for cell surface binding sites.     

Hyaluronidase treatment of coronary glycocalyx increases reactive hyperemia but not adenosine hyperemia in dog hearts

Because adenosine is commonly used for inducing maximal coronary hyperemia in the clinic, it is imperative that adenosine-induced hyperemia (AH) resembles coronary hyperemia that can be attained by endogenous stimuli. In the present study we hypothesized that coronary reactive hyperemia (RH) is limited compared with AH due to the presence of the glycocalyx and that the AH response is therefore unable to detect glycocalyx modifications. In anesthetized open-chest dogs, blood flow and pressure were measured in the left circumflex artery. RH after 15-s occlusion was compared with an intracoronary infusion of adenosine (650 microg; AH) during control conditions and after intracoronary treatment of the glycocalyx with hyaluronidase (20.000 U, 2 x 20 min; n = 6) or heat-inactivated hyaluronidase (n = 5). During control, coronary conductance during RH was 1.49 +/- 0.15 ml.mmHg(-1).min(-1) and 76 +/- 7% of coronary conductance during AH (P < 0.05). After hyaluronidase, RH conductance increased (P < 0.01) by 43 +/- 13% and became 93 +/- 4% of AH conductance (P = NS). Heat-inactivated hyaluronidase had no effect on RH and AH conductance. Our results demonstrate that adenosine-induced coronary hyperemia profoundly exceeds RH and that the difference is virtually abolished on selective removal of the glycocalyx. It is concluded that, compared with RH, adenosine-induced coronary hyperemia is not affected by modification of the glycocalyx. This glycocalyx insensitivity should be taken into account when using adenosine-induced coronary hyperemia as a marker for vasodilating capacity to an ischemic stimulus.     

The heparin binding site of human antithrombin III. Selective chemical modification at Lys114, Lys125, and Lys287 impairs its heparin cofactor activity

Heparin binds to human antithrombin III and accelerates its inhibitory activity in the blood coagulation system. Previous reports (Rosenberg, R. D., and Damus, P. S. (1973) J. Biol. Chem. 248, 6490-6505; Pecon, J. M., and Blackburn, M. N. (1984) J. Biol. Chem. 259, 935-938) have shown that selective chemical modification of a limited number of lysine residues in antithrombin III causes drastic loss of its heparin cofactor activity. We have performed chemical modification of antithrombin III with trinitrobenzene sulfonic acid in order to determine the location of these lysine residues. When antithrombin III was treated with 100 M excess of trinitrobenzene sulfonic acid for 10 min, about 3.2 mol of amino group per mol of antithrombin III were modified. The heparin cofactor activity dropped to about 25%, whereas the progressive inhibitory activity (in the absence of heparin) remained essentially intact (about 95%). The modified amino groups were identified to be Lys114 (75%), Lys125 (94%), and Lys287 (96%). These results were obtained by comparing and analyzing the cyanogen bromide fragments derived from native antithrombin III and the 10-min modified antithrombin III. When antithrombin III was pretreated with heparin, followed by trinitrobenzene sulfonic acid modification, the extent of modification at Lys114 and Lys125 decreased from 75% and 94% to 20% and 40%, respectively, whereas the modification at Lys287 remained nearly quantitative (greater than 95%). Based on these results, we conclude that Lys114 and Lys125 are essential for the heparin cofactor activity of human antithrombin III.     

Inhibition of boar sperm hyaluronidase activity by tannic acid reduces polyspermy during in vitro fertilization of porcine oocytes

The present study was conducted to examine the effects of three polyphenols (tannic acid, apigenin and quercetin) on hyaluronidase activity and in vitro fertilization (IVF) parameters. Among them, tannic acid showed by far the strongest potency for blocking hyaluronidase activity extracted from preincubated boar sperm, causing a dose-dependent inhibition over the range of 2-10 microg/ml. When cumulus-intact and cumulus-free oocytes were inseminated in IVF medium containing tannic acid, the penetration and the polyspermy rates were significantly decreased in the presence of 10 microg/ml tannic acid compared with those in the absence of tannic acid, and the addition of 5 microg/ml tannic acid significantly reduced the polyspermy rate (p < 0.05) compared with that of the control while maintaining the high penetration rate. However, apigenin and quercetin had no effect on the rate of polyspermy. Interestingly, the incidence of polyspermy was significantly reduced in oocytes inseminated with sperm pretreated with 5 microg/ml tannic acid (p < 0.05), although the pretreatment of oocytes had no effect against the polyspermy after insemination with untreated sperm. Treatment with tannic acid caused neither a protective proteolytic modification of the zona pellucida matrix before fertilization, nor a reduction of the proteolytic activity of acrosomal contents or the number of zona-bound spermatozoa. These data suggest that an appropriate concentration of tannic acid prevents polyspermy through the inhibition of sperm hyaluronidase activity during IVF of porcine oocytes.     

Indole carboxamides inhibit bovine testes hyaluronidase at pH 7.0 and indole acetamides activate the enzyme at pH 3.5 by different mechanisms

Hyaluronidases are enzymes controlling many crucial physiological processes. Imbalanced enzymatic activity is connected with severe diseases. Because there is limited availability of drugs modulating hyaluronidase activity, the search for hyaluronidase interacting compounds is getting more and more important. A series of fifteen indole carboxamides and acetamides were synthesized and tested on inhibition of bovine testes hyaluronidase. In vitro assays were performed using stains-all at pH 7 and the Morgan-Elson reaction at pH 3.5. At neutral pH, the most active inhibitory compound was N-(Pyridin-4yl)-[5-bromo-1-(4-fluorobenzyl)indole-3-yl]carboxamide (20) with an IC₅₀ value of 46 μM. Surprisingly, inhibition of all compounds was completely abolished by a decrease in pH. At pH 3.5 the activity of the enzyme was increased up to 134% by compound N-(4,6-Dimethylpyridin-2yl)-(1-ethylindole-3-yl)acetamide (24) at a concentration of 100 μM. The known activating effect of bovine serum albumine (BSA) on hyaluronidase activity was verified in the assay and compared to the effect of compound 24. Structure-activity relationships are discussed and a model is proposed, which explains the increase in activity at pH 3.5 by bonding of the protonated form of N-(4,6-Dimethylpyridin-2yl)-(1-ethylindole-3-yl)acetamide (24) to hyaluronic acid. The bonding results in an elongated form of the substrate with easier enzymatic access.     

Catalytic properties of testicular hyaluronidase after gamma-irradiation

The effect of gamma-irradiation on ovine testicular hyaluronidase was studied in aqueous solution. Following irradiation, hyaluronidase is inhibited, and the kinetics of inhibition follow a pattern in which Km and Vmax decline as radiation dose is increased. It was indicated that the binding affinity of the residual activity of hyaluronidase with substrate is enhanced and depends upon radiation damage. Effects of various agents such as pH, salts, PCMB and glutathione on irradiated hyaluronidase have been compared with non-irradiated enzyme. The irradiated hyaluronidase was more sensitive to inhibition by CuSO4 than the non-irradiated enzyme. The residual activity after irradiation is less refractory to FeCl3 inhibition and less sensitive to NaCl stimulation compared to non-irradiated hyaluronidase. pH response curves of ovine testicular hyaluronidase show two maxima which become more evident after irradiation.