Role of the Glycosaminoglycan Microenvironment of Hyaluronidase in Regulation of Its Endoglycosidase Activity

A. V. Maksimenko^*, Y. V. Schechilina, and E. G. Tischenko

Institute of Experimental Cardiology, Russian Cardiology Research Center, 3-ya Cherepkovskaya ul. 15a, Moscow 121552, Russia; fax: (095) 415-2962; E-mail: cclibr@comcor.ru

^* To whom correspondence should be addressed.

Received June 1, 2002; Revision received July 25, 2002

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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 alpha1-3 and alpha1-4 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--beta(1e-4e) and beta(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.

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KEY WORDS:hyaluronidase, glycosaminoglycans, dermatan sulfate, heparin, chondroitin sulfate, hyaluronic acid, microenvironment, regulation of endoglycosidase activity

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