Journal of Pharmaceutical and Biomedical Analysis
Capillary electrophoresis: a tool for studying interactions of glycans/proteoglycans with growth factors
Introduction
Heparin and heparan sulfate (HS) are linear heteropolysaccharides composed of repeating disaccharide units which contain glucosamine and glucuronate (GlcA) or its C-5 epimer, the iduronate (IdoA). The disaccharide units may be sulfated at any available hydroxyl group and twelve differently sulfated units have been identified so far (Fig. 1); [1], [2]. Heparin contains higher number of sulfates and IdoA residues than HS and is characterized by the presence of sulfated glucosamine at C-3. Both glycosaminoglycans (GAGs) are biosynthesized as proteoglycans (PGs), i.e. covalently bound to a protein core. HSPGs are integral components of plasma membrane (e.g. syndecan family) and the extracellular matrix (e.g. perlecan). HSPGs via their HS chains participate in several cellular events and biologic processes, such as cell–cell and cell–protein interactions. Furthermore, HS acts as an endogenous receptor for growth factors [3].
Biologic activities of heparin and HS generally depend on interactions of the GAG chain with proteins. These interactions are mediated by distinct oligosaccharide sequences. Binding of heparin/HS oligosaccharide domains to proteins is generally (although not exclusively) ionic, and thus involves positively charged, usually clustered, amino acid residues of the protein components. Conversely, the anionic protein binding domains of GAG chains may differ with regard to structure, degree of binding specificity and organization at the macromolecular level.
Basic fibroblast growth factor (bFGF or FGF-2) is a member of a large family of structurally related proteins that affect the growth, differentiation, migration and survival of a wide variety of cell types. It was initially identified as a 15 kDa protein (pI 9.6) [4] that was later found to represent a proteolytic product of a primary 18 kDa form [5]. Although the binding of heparin and HS has little effect on bFGF structure, it may facilitate the self-association of bFGF molecules into dimer and higher-order oligomers [6]. It has been proposed that bFGF has two separate receptor binding sites, which might allow a single bFGF to bind to two receptors or to interact with a single receptor in two separate positions [7]. HSPGs can increase the affinity of bFGF for its receptors and potentially act as a bridge to facilitate dimerization of receptors [8], [11].
Interaction of bFGF with heparin/HS protects the growth factor against heat and acid denaturation, and protease cleavage [9]. Heparin and HS oligosaccharides that bind bFGF have been found to be rich in IdoA(2-OSO3−) containing disaccharides, with affinity increasing with chain length. The minimal bFGF binding sequence in HS has been identified as a pentasaccharide, which contains the disaccharide units IdoA (2-OSO3−)–GlcNSO3− or IdoA(2-OSO3−)–GlcNSO3−–(6-OSO3−) [10]. Accordingly, binding studies involving chemically modified heparins or HS preparations have shown that 2-OSO3− and N-sulfonyl groups are important for their interaction with bFGF. It should be pointed out that carboxyl groups of heparin hexuronic acid contribute to bFGF interaction and that small non-sulfated di- and trisaccharides compete with heparin for binding to bFGF. It is, therefore, obvious that distinct structural domains are necessary for the interaction of heparin/HS with bFGF.
In order to study the interactions of HS with bFGF, we developed a method combining the high-resolution efficiency of capillary electrophoresis (CE) with the specificity of hepatitinase against HS for monitoring the ability of the variously sulfated HS oligosaccharides to bind bFGF.
Section snippets
Biologic material and chemicals
HS (Na+ salt) from bovine intestinal mucosa, the various sulfated heparin- and HS-derived Δ-disaccharides and tris[hydroxymethyl]aminomethane were from Sigma (St. Louis, MO, USA). Heparitinase (heparin lyase III, EC 4.2.2.8) from Flavobacterium heparenium was purchased from Seikagaku Kogyo (Tokyo, Japan). bFGF was obtained from Serotec (Oxford, UK). All other chemicals used were of analytical reagent grade.
Enzymic degradation of GAGs and interaction with bFGF
Enzymic treatment of HS was performed as previously described [12], [13]. In brief, HS
Results and discussion
Capillary zone electrophoresis (CZE) analysis of standard Δ-disaccharides under the conditions described showed the complete separation of all 12 differently sulfated Δ-disaccharides within 17 min (Fig. 2), as previously described by our research group [1]. It has been well documented that treatment of HS with heparin/HS lyases I, II and III in combination results in almost complete degradation of HS (>92%) to Δ-disaccharides [1], [2]. Therefore, determination of the disaccharide composition of
Concluding remarks
In the present report, we describe a simple CZE method that can be used to resolve the variously sulfated HS groups of oligosaccharides. Combining all three heparin/HS lyases the disaccharide composition of HS can be easily determined. Partial degradation of HS with heparitinase resulted in five Δ-oligosaccharide groups of different electrophoretic mobility. Incubation of degraded HS with bFGF followed by CZE analysis revealed that this growth factor interacts with specific groups of HS-derived
Acknowledgements
Dr Fotini Lamari thanks the Hellenic State Scholarships Foundation (IKY) for the financial support.
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