Hialuronidasa

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Carbohydrate Research 318 (1999) 26–37

Control of enzymatic degradation of hyaluronan by divalent cations
Koen P. Vercruysse, Michael R. Ziebell, Glenn D. Prestwich *
Uni6ersity of Utah, Department of Medicinal Chemistry, 30 South 2000 East, Room 201, Salt Lake City, UT 84112 -5820, USA
Received 11 February 1999; accepted 8 April 1999

Abstract Enzymaticdegradation of hyaluronan (HA) by testicular hyaluronidase (HAase, hyaluronate 4-glucanohydrolase) requires inclusion of mono- or divalent cations in the reaction mixture. Most divalent cations activated HAase with equal potency; however, Cu2 + suppressed degradation, and Ca2 + showed a concentration-dependent regulation of size of the oligosaccharide products. Careful selection of HAase assayparameters is critical for discovery of novel HAase inhibitors and for preparation of controlled-size oligosaccharide fragments. © 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Hyaluronidase; Hyaluronan; Gel-permeation chromatography; Perfusion anion-exchange chromatography

1. Introduction Hyaluronan (HA), a naturally occurring linear polysaccharide comprising b-(1“4)-linkedD-glucuronic acid b-(1“3)-N-acetyl-D-glucosamine disaccharide units (Fig. 1), is found in the extracellular matrix (ECM) of all higher animals, especially in soft connective tissues. This polyanionic polymer has a range of naturally occurring molecular sizes from several hundred to over 10 million daltons (MDa), with an average size of 1–2 MDa [1]. By binding to the HA-binding proteins (HABP) present in theECM, HA participates in defining the interaction and the organization of the complex proteoglycan-based aggregates [2]. HA and its chemically modified derivatives have recently begun to occupy center stage in
* Corresponding author. Fax: +1-801-585-9053. E-mail address: gprestwich@deans.pharm.utah.edu (G.D. Prestwich)

areas ranging from drug delivery to tissue engineering [3]. Cell surfacespossess HA receptors, e.g., CD44 and RHAMM [4], that play important roles in normal physiology and disease states [5,6]. Relatively small HA fragments (oligosaccharides) exhibit different and specific cell-biological properties when compared with the megadalton-sized biopolymer. Oligosaccharide fragments of HA (starting with six disaccharide units, i.e., HA12) have angiogenic potential by stimulatingthe proliferation and migration of endothelial cells [7– 9]. The interaction of HA fragments with CD44 receptors on macrophages has important effects in the early stages of the inflammatory response [10]. The activation of CD44 by HA fragments has been shown to stimulate the gene expression of IL-1b, TNF-a, and IGF-1 [11] of several members of the chemokine gene family [12] and of nitric oxidesynthase [13]. This gene induction seems to

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K.P. Vercruysse et al. / Carbohydrate Research 318 (1999) 26–37

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occur in a nuclear factor (NF) kB-dependent fashion [13,14]. Thus, CD44 is an important component of the signal transduction pathway leading toinflammatory leukocyte recruitment and activation [15]. Small fragments of HA, with their angiogenic and pro-inflammatory potential, are generated through an enzymatic digestion of the high-molecular-mass parent HA molecule. The enzyme responsible for this degradation of HA is called hyaluronidase (HAase; hyaluronate 4-glucanohydrolase, EC 3.2.1.35). HAases comprise a class of enzymes that are ubiquitouslydistributed in prokaryotes and eukaryotes, performing a broad range of biochemical and biological functions. The properties and functions of these enzymes have been extensively reviewed in recent years [16– 18]. HAase is found in the testicular tissues of most mammals and is located on the acrosomal region of spermatozoa. The enzyme plays a major role in the passage of the spermatozoa towards...