pH effects on the hyaluronan hydrolysis catalysed by hyaluronidase in the presence of proteins: Part I. Dual aspect of the pH-dependence
Introduction
Hyaluronan (HA) is a polysaccharide present in the extra cellular matrix (ECM) of connective, growing and tumour tissues where it is involved in cellular adhesion, mobility and differentiation (Catterall, 1995, Delpech et al., 1997, Kennedy et al., 2002, Laurent, 1987, Rooney et al., 1995, Girish and Kemparaju, 2007). It is a high-molar-mass polyelectrolyte composed of d-glucuronic acid-β(1,3)-N-acetyl-d-glucosamine disaccharide units linked together through β(1,4) glycosidic bonds. HA is the substrate of hyaluronidase (HAase). HAases are present in mammals, insects, parasites and bacteria (Csóka et al., 1997). In the human body, HAase is found in various organs (testis, skin, liver, kidney, uterus, etc.) and body fluids (plasma, sperm, urine, etc.). The purification of the first human HAase (Frost et al., 1997) along with much recent work has greatly increased our knowledge of human HAases (Csóka et al., 2001). More generally, three types of HAases exist, classified according to their cleavage mechanism (Meyer, 1971); one of them, of the testicular type, which includes human HAases, hydrolyses the glycosidic bonds in the β(1,4) position to produce, usually, oligosaccharides. As HA oligosaccharides (4 to 25 disaccharides) have an angiogenic action (Rooney et al., 1995, West et al., 1985, West and Kumar, 1989) contrary to native HA (Deed et al., 1997), the ratio between high molecular weight HA and low molecular weight HA plays a role in cancer development (Stern, 2008, Mio and Stern, 2002). This ratio is at least partly controlled by the action of HAase (Delpech et al., 1997, Mio and Stern, 2002, Cameron, 1966).
Interactions between proteins and polyelectrolytes have long been of interest in several domains: controlling textures in the food industry (Tolstoguzov, 1986), enzyme immobilization (Kokufuta, 1992) and protein purification (Dubin et al., 1994). Interactions between DNA and proteins have also been extensively studied (Tsodikov et al., 2001). Most of the work on protein–polysaccharide complexes has been reviewed by Schmitt et al., 1998, Cooper et al., 2005. Two main classes can be distinguished for the electrostatic complexes: i) neutral insoluble complexes at the phase separation when the net positive charge of one of the biomacromolecules exactly compensates the net negative charge of the other, and ii) charged complexes, when an excess of charge exists, which are maintained in suspension by solvation and can produce turbidity. Although the binding of synthetic polyelectrolytes to one another generally involves non-specific interactions, the binding of DNA to proteins often involves specific interactions leading to recognition; in the latter case, the non-repetitive sequence of DNA induces the affinity and specificity of the binding. HA, like DNA, is a polyanion and several HA-binding proteins exist such as CD44 (Kennedy et al., 2002), Rhamm (Kennedy et al., 2002), TSG-6 (Parkar et al., 1998), and hyaluronectin (Delpech et al., 1993). However, the repetitive primary structure of HA may also allow non-specific binding with proteins, as is the case for other polysaccharides (Seyrek et al., 2003, Laos et al., 2007).
The ability of HA and proteins to form non-specific complexes has long been known. The Mucin Clot Prevention (Robertson et al., 1940) and the turbidimetric methods (Dorfman and Ott, 1948) have been developed to assay HAase by measuring the disappearance of the turbidity resulting from complexes formed between long HA chains and albumin under acidic conditions. Since then, numerous studies have characterised the complexes formed between HA and bovine serum albumin (BSA). Xu et al. (2000) studied mixtures of HA and BSA and showed both the electrostatic nature of the interactions between the two molecules and the strong influence of pH on the solubility of the HA–BSA complex. HA–BSA complexes were also studied by Grymonpré et al. (2001) who focused particularly on the effects of both pH and ionic strength on the existence and solubility of the complexes. There have also been reports that BSA can activate HAase. Gacesa et al. (1981) found that serum proteins enhance the catalytic activity of HAase and that albumin has the greatest effect. Gold (1980) observed that BSA is able to activate both human liver HAase and bovine testicular HAase at pH 4. Maingonnat et al. (1999) showed that BSA and other proteins such as hyaluronectin, hemoglobin and immunoglobulins are able to enhance tumour HAase activity at pH 3.8 and hence to increase the sensitivity of the HAase detection. Such an enhancement of HAase activity is also dependent on the concentration of these proteins (Maingonnat et al., 1999).
Polysaccharides and proteins are thus able to form complexes in well delimited pH domains (Schmitt et al., 1998) by establishing electrostatic bonds between the negatively charged polysaccharide and the positively charged protein. Bovine testicular HAase, which is commercially available and belongs to the same class as human HAases, has been chosen as a model for our studies (Vincent et al., 2003, Lenormand et al., 2008, Deschrevel et al., 2008). We have shown that at pH 4, HA and HAase, in addition to forming enzyme–substrate type complexes for the catalytic reaction, can form non-specific complexes because HA is negative and HAase is positive (Lenormand et al., 2008, Deschrevel et al., 2008). Although enzyme–substrate type complexes are stabilized by hydrogen bonds, electrostatic and Van der Waals interactions, non-specific complexes are mainly stabilized by electrostatic interactions. It has also been shown that HAase is no longer catalytically active when complexed with HA only via electrostatic interactions (Deschrevel et al., 2008, Lenormand et al., 2007, Astériou et al., 2006). When HA is in excess, the HAase preferentially forms electrostatic non-specific complexes with HA and the HAase activity is nil. When HAase is in excess, all the HA molecules are electrostatically complexed with HAase, and the HAase enzymes are in two classes: the first corresponds to electrostatically complexed HAase which is inactive and the second corresponds to free HAase which can catalyse the hydrolysis of the HA molecules electrostatically complexed by HAase. HAase activity thus depends on the concentration of its substrate in a very unusual manner (Astériou et al., 2006). The mechanism responsible for this has been clearly shown by modelling (Lenormand et al., 2007, Vincent and Lenormand, 2009). In the following text, unless otherwise mentioned, by “complexes” we mean “non-specific electrostatic complexes” and by “HAase” we mean “bovine testicular HAase”.
BSA is also able to form complexes with HA and to compete with HAase when the two proteins are present at the same time: the addition of BSA in a medium containing HAase and an excess of HA induces the release of HAase from the complex and the recovery of its catalytic activity (Lenormand et al., 2008, Deschrevel et al., 2008). We have shown at pH 4 that HAase activity depends on the BSA concentration in a very particular manner: at low BSA concentrations, the HA hydrolysis rate increases up to a maximum as the BSA concentration is increased, and at high BSA concentrations, the HA hydrolysis rate decreases as the BSA concentration is increased (Lenormand et al., 2008, Deschrevel et al., 2008, Lenormand et al., 2009). BSA is thus able to complex HA to either enhance or suppress HAase activity. At low concentrations, BSA forms complexes with HA and prevents the complexation of HAase. The higher the BSA concentration, the higher the HAase activity. At much higher concentrations, BSA forms denser complexes with HA thereby masking increasing numbers of hydrolysable sites on the HA molecule and thus lowering HAase activity. At pH 5.25, the action of BSA is similar but less effective than at pH 4 (Lenormand et al., 2009) because this pH is close to the isoelectric point (pI) of BSA (pI = 5.2 (Xu et al., 2000)) and hence the number of positive charges on the BSA molecule that allow the formation of electrostatic complexes with the polyanionic HA molecule is low. According to some authors, however, this complexation can occur at higher pH values because of positive patches on the protein surface (Mattison et al., 1998, Park et al., 1992). This raises the question: is BSA able to form electrostatic complexes with HA under physiological pH and does this involve positive patches?
The increase in HAase activity in the presence of non-catalytic proteins has two requirements: i) the protein has to be able to form a complex with HA and ii) this complex has to be more stable than the electrostatic complex formed between HA and HAase. It would be interesting to learn whether the control of HAase activity by proteins can occur in near physiological conditions, at least regarding pH. Lysozyme (LYS) can form complexes with HA at pH 7.5 (Van Damme et al., 1994, Moss et al., 1997) and this protein is present in the ECM of cartilage which also contains HA. Its precise role in this tissue has not yet been established. LYS has a pI close to 10.5 (Hoon Han and Lee, 1997) which is much higher than that of BSA. According to Van Damme et al. (1994), the formation of HA–LYS complexes depends on the ionic strength and is optimal at 5–10 mM salt although complexes still form at 60 mM. The low dissociation constant of this complex (Kd = 1–2 10− 8 M) allows LYS to prevent or reverse the formation of link-protein–HA complexes (Blanco and Pita, 1985). This raises the question of whether LYS is able to prevent or reverse the formation of HA–HAase complexes and thus enhance the HAase activity at physiological pH.
Here we investigate systematically the relationship between HAase activity and pH as modulated by two proteins, BSA and LYS.
Section snippets
Results
The results concern the double influence of pH and protein concentration on the HAase activity. Experiments reported concern BSA, then LYS.
Discussion and conclusion
The enhancement/suppression of HAase activity by BSA – the BSA-dependence – was here found to occur at pH values ranging from 3.5 to 4.5. In this pH range, the BSA-dependences are similar whatever the pH value. There are essentially two sorts of behaviour: i) at low BSA concentrations, HAase activity increases as BSA concentration increases, and ii) at high BSA concentrations, HAase activity decreases as BSA concentration increases. Such enhancement/suppression of HAase was also found for LYS.
Materials
Bovine testicular HAase (H 3884, lot 38H7026), BSA (A 3675, lot 78H1399), LYS (L 6876, lot 051K7028) and sodium hyaluronate from human umbilical cord (H 1876, lot 127H0482) were obtained from Sigma. The molar mass of HA was close to 1 Mda. More precisely, the number-average molar mass (Mn) of HA was 0.967 106 g mol− 1 and its polydispersity index, which represents its degree of homogeneity, was 1.45. The molar mass of BSA was 69 103 g mol− 1 and that of LYS was 14.3 103 g mol− 1. HA, BSA, LYS and HAase
Acknowledgements
We thank Dr. Annie Steinchen-Sanfeld and Dr. Wiswas Purohit for critical readings of the manuscript. We are grateful to the French Research and Technology Ministry for the fellowship granted to Dr. Hélène Lenormand.
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