Elsevier

Carbohydrate Polymers

Volume 85, Issue 2, 6 May 2011, Pages 303-311
Carbohydrate Polymers

pH effects on the hyaluronan hydrolysis catalysed by hyaluronidase in the presence of proteins: Part II. The electrostatic hyaluronan – Protein complexes

https://doi.org/10.1016/j.carbpol.2011.02.007Get rights and content

Abstract

Hyaluronan (HA) hydrolysis catalysed by hyaluronidase (HAase) is enhanced when bovine serum albumin (BSA) is present and competes with HAase to form electrostatic complexes with HA. At 1 g L−1 HA and BSA concentrations, BSA is able to form three types of complexes with HA depending on pH ranging from 2.5 to 6: insoluble neutral complexes at low pH values, sedimentable slightly charged complexes at pH near 4 and soluble highly charged complexes at pH near 5. The BSA content, charge and solubility of the HA–BSA complexes increase when pH is increased up to the pI of BSA. The normalised charge excess does not exceed 20% for the sedimentable complexes and 40% for the soluble complexes. It has been shown that the sedimentable slightly charged HA–BSA complexes are the most efficient to compete with HAase and release it. All the HA–BSA complexes are hydrolysable by HAase. The HA–BSA binding site shows that one BSA molecule is associated with 85–170 HA carboxyl groups, depending on pH. Similar results have been obtained for lysozyme over an extended pH domain, including the neutrality.

Introduction

Hyaluronan (HA) is a linear high-molar-mass polysaccharide composed of repeating d-glucuronic acid-β(1,3)-N-acetyl-d-glucosamine disaccharide units linked together through β(1,4) glycosidic bonds. Since its discovery in the vitreous humor by Meyer in 1936, it has been well established that HA is widely distributed in the extra-cellular matrix (ECM) of vertebrate tissues where it is involved in cellular adhesion, mobility, proliferation and differentiation (Catterall, 1995, Delpech et al., 1997, Girish and Kemparaju, 2007, Kennedy et al., 2002, Laurent, 1987, Rooney et al., 1995). HA is the substrate of hyaluronidase (HAase). HAases are present in mammals, insects, parasites and bacteria (Csóka, Frost, & Stern, 1997) and catalyse the cleavage of HA into oligosaccharides. Recently, it has been demonstrated that properties and functions of HA strongly depend on the chain size (David-Raoudi et al., 2008, Deschrevel et al., 2008b, Stern et al., 2006) and that HA fragments and native HA may have opposite roles, especially in regards to angiogenesis (Deed et al., 1997, West and Kumar, 1989, West et al., 1985). It is now established that the action of HAase plays a role in cancer development (Lokeshwar et al., 1996, Mio and Stern, 2002, Rooney et al., 1995, Stern, 2008). More generally, the action of HAases is very important because their presence in the ECM often results in the degradation of the polysaccharide, altering its structure and consequently its function. Understanding the interactions between polysaccharides and proteins, or polysaccharides and enzymes, are thus of fundamental importance. Crystal structure and molecular modeling of such specific assemblies (with short oligosaccharides) have recently been reviewed (Imberty, Lortat-Jacob, & Perez, 2007), offering very important progresses in that field.

At pH 4, HA and HAase can form non-specific electrostatic HA–protein complexes (Deschrevel et al., 2008a, Lenormand et al., 2008) because HA is negative and HAase is positive. Moreover, it has been shown that HAase is no longer catalytically active when electrostatically complexed with HA (Astériou et al., 2006, Deschrevel et al., 2008a, Lenormand et al., 2007). In fact, various proteins, like bovine serum albumin (BSA) or lysozyme (LYS), can also form electrostatic complexes with HA (Gacesa et al., 1981, Moss et al., 1997, Xu et al., 2000). Moreover, Gold (1982) 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 HAase activity. However, the enhancement depends on the protein concentration. It is thus very important to understand how proteins, simultaneously present in the tissue, may modulate the HAase action.

We have shown that BSA is able to form complexes with HA at pH 4 and is able to compete with HAase when the two proteins are simultaneously present (Deschrevel et al., 2008a, Lenormand et al., 2008). At pH 4, we have shown that HAase activity depends on the BSA concentration in a very atypical manner. BSA can enhance or suppress HAase activity. At low concentrations, BSA forms complexes with HA and prevents the complexation of HAase. At high concentrations, BSA forms dense complexes with HA hindering a great number of hydrolysable sites on the HA molecule. At pH 5.25, the action of BSA is similar but less efficient than at pH 4 (Lenormand, Tranchepain, Deschrevel, & Vincent, 2009) because, pH being close to the isoelectric pH (pI) of BSA (pI = 5.2 (Xu et al., 2000)), the number of positive charges borne by the BSA molecule and required to form electrostatic complexes with the polyanionic HA molecule is low.

The question of the BSA ability to form electrostatic complexes with HA under physiological pH conditions is of importance for the HAase modulation in the ECM, and we are now engaged in a detailed study of the HAase activity as a function of pH (Lenormand, Deschrevel, & Vincent, 2010a). In fact, HA forms two types of complex with HAase: the first one is the classical catalytic enzyme–substrate complex which involves hydrogen bonds, electrostatic and Van der Waals interactions in a precise 3D arrangement and the second one is the non-specific complex that HA is able to form with a great variety of proteins, such as BSA, LYS, and HAase (Deschrevel et al., 2008a, Lenormand et al., 2007, Lenormand et al., 2008) through electrostatic interactions. The actual pH-dependence is thus the result of the two following pH-dependences: the intrinsic pH-dependence of the enzyme action and the pH-dependence of the formation of the non-specific electrostatic HA–protein complexes (Lenormand et al., 2010a). The enhancement of the HAase activity in the presence of non-catalytic proteins thus requires two conditions: (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.

We have shown (Lenormand et al., 2010a) that BSA is able to control the enhancement/suppression of the HAase activity at pH values ranging from 3.5 to 5.25. For an HA concentration of 1 g L−1 and an HAase concentration of 0.5 g L−1, the optimal BSA concentration varies from 0.6 to 2 g L−1 according to pH. At pH higher than 4.5, BSA can still form complexes with HA, but the release of HAase requires more BSA molecules, corresponding to BSA concentrations higher than 4 g L−1. The fact that BSA can enhance the HAase activity on a pH domain ranging from 3.5 to 5.25 means that: (i) BSA is able to compete with HAase to form complexes with HA, (ii) HAase is able to form complexes with HA over this pH domain, and (iii) HAase is catalytically active over this pH domain when BSA limits its complexation with HA. All of this have been considered in a theoretical model (Vincent & Lenormand, 2009) which clearly explains the functioning of the HA–HAase–BSA system. Nevertheless, BSA is not able to enhance HAase activity at physiological pH. We have shown (Lenormand et al., 2010a) that LYS, which has a pI value higher than that of BSA, is able to enhance HAase activity at physiological pH. That means that HA–HAase complexes can be formed at physiological pH. It should thus be important to study the HA–BSA, HA–LYS and HA–HAase complexes as a function of pH, in order to see which type of complex is concerned.

Most of the work on protein–polyelectrolyte complexes is reviewed by Schmitt, Sanchez, Desobry-Banon, and Hardy (1998), Cooper, Dubin, Kayimazer, and Turksen (2005) and Imberty et al. (2007). 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 solvatation and can produce turbidity. Numerous studies have been devoted to the complexes formed between HA and proteins (for details, see Lenormand et al., 2010a). Xu et al. (2000) studied HA/BSA mixtures and showed the electrostatic nature of the interactions between the two molecules and the strong influence of pH on solubility of the HA–BSA complex. They determined the stoichiometry, viscosity and size of the HA–BSA complexes at pH near 5 and proposed an interesting model for HA–BSA complex formation showing the change from a worm-like structure to spherical particules by intra-polymer then interpolymer interactions when BSA concentration is increased. Grymonpré, Staggemeier, Dubin, and Mattison (2001) studied the effects of both pH and ionic strength on the existence and solubility of the HA–BSA complexes. They showed that the electrostatic HA–BSA complexes can be formed even at a pH higher than the pI of the protein thanks to the presence of charge patches.

Other papers reported pH effects on complexes formed between another anionic polysaccharide (heparin) and BSA, between another protein (silk fibroin) and HA (Malay, Bayraktar, & Batigün, 2007), or between a cationic polyelectrolyte and BSA (Kaibara et al., 2000, Mattison et al., 1998). In our group, we have studied the HA–BSA complexes formed at pH 4 and low ionic strength (Lenormand et al., 2008). We have determined the conditions for existence, the nature, the stoichiometry and the size of the complexes, with respect to the BSA over HA ratio. We have shown that HA and BSA can form three types of electrostatic complexes: highly charged soluble complexes, slightly charged sedimentable complexes and neutral insoluble complexes at the phase separation.

Existence and nature of HA–BSA complexes being related to the charge borne by the two macromolecules, they are pH dependent. In complement to the important work of Grymonpré et al. (2001) and Xu et al. (2000), and to our own work (Deschrevel et al., 2008a, Lenormand et al., 2008, Lenormand et al., 2009, Lenormand et al., 2010a), the present paper deals with the detailed study of the HA–protein (BSA or LYS) complexes as a function of pH and presents their nature, solubility, stoichiometry, size and composition, in order to establish a relationship between the HAase activity (Lenormand et al., 2010a) and the existence of the electrostatic nonspecific HA–protein complexes. In addition, one of the objectives of the paper is to determine among these complexes, which type is the most efficient to compete with the HA–HAase complex and to release HAase?

Section snippets

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 69,000 g mol−1. HA, BSA, LYS and HAase were used without any further

Results

In all the experiments, no salt was added in the solution. The ionic strength was thus very low and only due to the presence of the counterions of the biopolymers and pH adjustments. It was of the order of magnitude of 10−3 mol L−1 and thus very different from the physiological level. For sake of simplicity, the term “low ionic strength” was used throughout the text.

Discussion and conclusion

At 1 g L−1 HA and BSA concentrations and low ionic strength, BSA is able, depending on pH, to form three types of electrostatic complexes with HA: insoluble neutral complexes at pH values lower than 3.2, sedimentable slightly charged complexes at pH near 4 and decreasing from pH 3.2 to 5.3 and soluble highly charged complexes at pH near 4.5 and increasing from pH 3.2 to 5.3. The HA–BSA complexes are more charged and soluble when pH is increased up to 5.2, i.e. the pI of BSA, because the BSA

Acknowledgements

We thank Dr. Annie Steinchen-Sanfeld and Vishwas Purohit for critical readings of the manuscript.

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