Polyanion Binding Site Research Articles

Identification of indole-based activators of insulin degrading enzyme

Insulin degrading enzyme (IDE) is a zinc metalloprotease that cleaves numerous substrates among which amyloid-β and insulin. It has been linked through genetic studies to the risk of type-2 diabetes (T2D) or Alzheimer's disease (AD). Pharmacological activation of IDE is an attractive therapeutic strategy in AD. While IDE inhibition gave paradoxal activity in glucose homeostasis, recent studies, in particular in the liver suggest that IDE activators could be also of interest in diabetes. Here we describe the discovery of an original series of IDE activators by screening and structure-activity relationships. Early cellular studies show that hit 1 decreases glucose-stimulating insulin secretion. Docking studies revealed it has an unprecedented extended binding to the polyanion-binding site of IDE. These indole-based pharmacological tools are activators of both Aβ and insulin hydrolysis by IDE and could be helpful to explore the multiple roles of IDE.

Preformulation Characterization and the Effect of Ionic Excipients on the Stability of a Novel DB Fusion Protein

Shigella ssp cause bacillary dysentery (shigellosis) which has high global morbidity in young children and the elderly. The virulence of Shigella relies upon a type III secretion system (T3SS) which injects host altering effector proteins into targeted intestinal cells. The Shigella T3SS contains two components, invasion plasmid antigen D (IpaD) and invasion plasmid antigen B (IpaB), that were previously identified as broadly protective antigens. When IpaD and IpaB were co-expressed to give the DB fusion (DBF) protein, vaccine efficacy was further improved. Biophysical characterization under various pH conditions showed that DBF is most stable at pH 7 and 8 and loses its conformational integrity at 48 and 50 °C respectively. Forced degradation studies revealed significant effects on the secondary structure, tertiary structure and conformational stability of DBF. In the presence of phosphate buffers as well as other anionic excipients, DBF demonstrated a concentration dependent conformational stabilization. Molecular docking revealed potential polyanion binding sites in DBF that may interact with phytic acid. These sites can be exploited to stabilize the DBF protein. This work highlights potential destabilizing and stabilizing factors, which not only improves our understanding of the DBF protein but helps in future development of a stable Shigella vaccine.

Size and Flexibility Define the Inhibition of the H3N2 Influenza Endonuclease Enzyme by Calix[n]arenes.

Inhibition of H3N2 influenza PA endonuclease activity by a panel of anionic calix[n]arenes and β-cyclodextrin sulfate has been studied. The joint experimental and theoretical results reveal that the larger, more flexible and highly water-soluble sulfonato-calix[n]arenes have high inhibitory activity, with para-sulfonato-calix[8]arene, SC8, having an IC50 value of 6.4 μM. Molecular docking calculations show the SC8 can interact at both the polyanion binding site and also the catalytic site of H3N2 influenza PA endonuclease.

Investigating the dynamics and polyanion binding sites of fibroblast growth factor-1 using hydrogen-deuterium exchange mass spectrometry.

In this study, we examined the local dynamics of acidic fibroblast growth factor (FGF-1) as well as the binding sites of various polyanions including poly-sulfates (heparin and low MW heparin) and poly-phosphates (phytic acid and ATP) using hydrogen-deuterium exchange mass spectrometry (HX-MS). For local dynamics, results are analyzed at the peptide level as well as in terms of buried amides employing crystallographic B-factors and compared with a residue level heat map generated from HX-MS results. Results show that strand 4 and 5 and the turn between them to be the most flexible regions as was previously seen by NMR. On the other hand, the C-terminal strands 8, 9, and 10 appear to be more rigid which is also consistent with crystallographic B-factors as well as local dynamics studies conducted by NMR. Crystal structures of FGF-1 in complex with heparin have shown that heparin binds to N-terminal Asn18 and to C-terminal Lys105, Tryp107, Lys112, Lys113, Arg119, Pro121, Arg122, Gln127, and Lys128 indicating electrostatic forces as dominant interactions. Heparin binding as determined by HX-MS is consistent with crystallography data. Previous studies have also shown that other polyanions including low MW heparin, phytic acid and ATP dramatically increase the thermal stability of FGF-1. Using HX-MS, we find other poly anions tested bind in a similar manner to heparin, primarily targeting the turns in the lysine rich C-terminal region of FGF-1 along with two distinct N-terminal regions that contains lysines and arginines/histidines. This confirms the interactions between FGF-1 and polyanions are primary directed by electrostatics.

Inositol phosphates and phosphoinositides activate insulin-degrading enzyme, while phosphoinositides also mediate binding to endosomes

Insulin-degrading enzyme (IDE) hydrolyzes bioactive peptides, including insulin, amylin, and the amyloid β peptides. Polyanions activate IDE toward some substrates, yet an endogenous polyanion activator has not yet been identified. Here we report that inositol phosphates (InsPs) and phosphatdidylinositol phosphates (PtdInsPs) serve as activators of IDE. InsPs and PtdInsPs interact with the polyanion-binding site located on an inner chamber wall of the enzyme. InsPs activate IDE by up to ∼95-fold, affecting primarily Vmax The extent of activation and binding affinity correlate with the number of phosphate groups on the inositol ring, with phosphate positional effects observed. IDE binds PtdInsPs from solution, immobilized on membranes, or presented in liposomes. Interaction with PtdInsPs, likely PtdIns(3)P, plays a role in localizing IDE to endosomes, where the enzyme reportedly encounters physiological substrates. Thus, InsPs and PtdInsPs can serve as endogenous modulators of IDE activity, as well as regulators of its intracellular spatial distribution.

Recognition of Malondialdehyde-modified Proteins by the C Terminus of Complement Factor H Is Mediated via the Polyanion Binding Site and Impaired by Mutations Found in Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is a severe thrombotic microangiopathy characterized by uncontrolled complement activation against endothelial and blood cells. Mutations in the C-terminal target recognition domains 19-20 of complement regulator factor H (FH) are strongly associated with aHUS, but the mechanisms triggering disease onset have remained unresolved. Here we report that several aHUS-related mutations alter the binding of FH19-20 to proteins where lysines have reacted with malondialdehyde (MDA). Although FH19-20 did not interact with MDA-modified hexylamine, lysine-containing peptides, or a proteolytically degraded protein, it bound to MDA-modified polylysine. This suggests that FH19-20 recognizes only clustered MDA adducts. Binding of MDA-modified BSA to FH19-20 was ionic by nature, depended on positive residues of FH19-20, and competed with the polyanions heparin and DNA. This could not be explained with the mainly neutral adducts known to form in MDA modification. When positive charges of lysines were eliminated by acetic anhydride instead of MDA, the acetylated BSA started to bind FH19-20. Together, these results indicate that negative charges on the modified proteins dominate the interaction with FH19-20. This is beneficial for the physiological function of FH because by binding to the negative charges of the modified target, FH could prevent excess complement activation initiated by naturally occurring antibodies recognizing MDA epitopes with multiple different structures. We propose that oxidative stress leading to formation of MDA adducts is a common feature for triggers of aHUS and that failure of FH in protecting MDA-modified surfaces from complement activation is involved in the pathogenesis of the disease.

The Interaction of Coagulation Factor XI with Polyphosphate

AbstractAbstract 498The plasma zymogen factor XI (FXI) is a homodimer of 80 kDa subunits. During blood coagulation, each subunit is activated by cleavage of the Arg369-Ile370 bond by factor XIIa (FXIIa) or thrombin. Initially, one subunit of the FXI dimer is activated to create the species 1/2FXIa, followed by activation of the second subunit, generating FXIa. Initial rates of activation of the first subunit are relatively low (∼8000 M−1.sec−1 for FXIIa and 120 M−1.sec−1 for thrombin). Rates for activation of the second subunit are even lower (1200 M−1.sec−1 for FXIIa and 35 M−1.sec−1 for thrombin), suggesting that conformational changes accompanying activation of a subunit reduce the efficiency of activation of its partner. The slow rate of FXI activation in solution strongly suggests that a cofactor is required for protease activation in vivo. FXI activation by FXIIa or thrombin is enhanced by polyanions such as dextran sulfate (DS). In addition, polyanions induce FXI activation by FXIa (autoactivation). Polymers of inorganic phosphate (polyP) released from platelet dense granules accelerate FXI activation by thrombin or FXIa (Blood 2011;118:6963), and likely represent the physiologic counterpart to DS. PolyP (4 μM) increased the initial rate of FXI activation by FXIIa ∼30-fold (300,000 M−1.sec−1), and by a-thrombin ∼3600-fold (440,000 M−1.sec−1). Furthermore, polyP induced FXI autoactivation in a manner similar to DS. Each FXI subunit contains two polyanion binding sites (residues Arg250, Lys252, Lys253, Lys255 on the A3 domain, and Lys529, Arg530, Arg532 on the catalytic domain). Both sites bind heparin, and are required for normal heparin-mediated enhancement of FXIa inhibition by antithrombin. FXI lacking the A3 domain site (FXIΔA3), but not FXI lacking the protease domain site (FXIΔCD), is activated slowly in the presence of DS compared to wild type FXI (FXIWT). Interestingly, both FXIΔA3 and FXIΔCD are activated slowly compared to FXIWT in the presence of polyP, and a species lacking both sites (FXIΔA3/CD) has an even greater defect, indicating FXI’s interaction with polyP is different from its interaction with DS.The FXI gene arose from a duplication of the gene for the monomeric protein prekallikrein (PK). The observations that the dimeric structure of FXI is highly conserved across species, and that the ancestral molecule is a monomer, strongly indicate that the dimer is important for a specific aspect of FXI function. It was recently reported that monomeric forms of FXI are activated slowly compared to dimeric FXI in solution or in the presence of DS (J Biol Chem 2008;283:18655). FXI dimer formation is mediated through a hydrophobic interface involving Leu284, Ile290, and Tyr329, and an interchain disulfide bond involving Cys321. A Ser substitution for Cys321 in combination with an Ala substitution for Leu284 or Ile290 results in the monomeric species FXIC321S, L284A and FXIC321S, I290A. Rates of FXIC321S, L284A or FXIC321S, I290A activation by FXIIa were significantly lower than for FXIWT in the presence of polyP. However, this defect was not observed during activation by thrombin or FXIa, demonstrating that the dimeric structure is not a prerequisite for zymogen activation on polyP. FXI-deficient (FXI−/−) mice are more resistant to arterial thrombus formation induced by vessel injury with ferric chloride than are wild type mice. FXIWT, FXIC321S, L284A and FXIC321S, I290A were transiently expressed in FXI−/− mice by hydrodynamic tail vein injection. While the three proteins were expressed at comparable levels, only FXIWT completely reconstituted the wild type phenotype in the ferric chloride thrombosis model. In summary, polyP is a strong candidate for a cofactor to support FXI activation in vivo. The interaction of FXI with this polyanion differs from its interaction with DS. The dimeric structure of FXI appears to be required for normal protease function in vivo, and for FXIIa-mediated FXI activation, but not for thrombin- or FXIa-mediated activation in the presence of polyP. Considering that the FXI homolog PK is a monomer that is activated efficiently by FXIIa, and that FXII deficiency is not associated with a significant phenotype, our results suggest that the FXI dimeric structure is required for a function distinct from zymogen activation. Disclosures:No relevant conflicts of interest to declare.

Complement Regulator Factor H Mediates a Two-step Uptake of Streptococcus pneumoniae by Human Cells

Streptococcus pneumoniae, a human pathogen, recruits complement regulator factor H to its bacterial cell surface. The bacterial PspC protein binds Factor H via short consensus repeats (SCR) 8-11 and SCR19-20. In this study, we define how bacterially bound Factor H promotes pneumococcal adherence to and uptake by epithelial cells or human polymorphonuclear leukocytes (PMNs) via a two-step process. First, pneumococcal adherence to epithelial cells was significantly reduced by heparin and dermatan sulfate. However, none of the glycosaminoglycans affected binding of Factor H to pneumococci. Adherence of pneumococci to human epithelial cells was inhibited by monoclonal antibodies recognizing SCR19-20 of Factor H suggesting that the C-terminal glycosaminoglycan-binding region of Factor H mediates the contact between pneumococci and human cells. Blocking of the integrin CR3 receptor, i.e. CD11b and CD18, of PMNs or CR3-expressing epithelial cells reduced significantly the interaction of pneumococci with both cell types. Similarly, an additional CR3 ligand, Pra1, derived from Candida albicans, blocked the interaction of pneumococci with PMNs. Strikingly, Pra1 inhibited also pneumococcal uptake by lung epithelial cells but not adherence. In addition, invasion of Factor H-coated pneumococci required the dynamics of host-cell actin microfilaments and was affected by inhibitors of protein-tyrosine kinases and phosphatidylinositol 3-kinase. In conclusion, pneumococcal entry into host cells via Factor H is based on a two-step mechanism. The first and initial contact of Factor H-coated pneumococci is mediated by glycosaminoglycans expressed on the surface of human cells, and the second step, pneumococcal uptake, is integrin-mediated and depends on host signaling molecules such as phosphatidylinositol 3-kinase.

Utilising ion mobility-mass spectrometry to interrogate macromolecules: Factor H complement control protein modules 10–15 and 19–20 and the DNA-binding core domain of tumour suppressor p53

Ion mobility-mass spectrometry (IM-MS) has been used to study the gas-phase structures of three proteins: the DNA-binding core domain of p53 (amino acid residues 94–312) and two fragments of complement control protein factor H (regions 10–15 and 19–20) which are involved in important cellular processes. We report collision cross-sections as a function of charge state for these systems, following electrospray ionisation from ‘native’ conditions. The DNA-binding core domain of p53 contains a zinc ion that is postulated to have an important role in retaining functionality. We have chelated the zinc from the protein using phenanthroline and observed the conformational change in collision cross-section. Factor H region 10–15 has been interrogated and cross-sections have been elucidated for this recombinant protein that contains a number of covalently bound N-acetylglucosamine sugar molecules. Factor H region 19–20 is proposed to be an important polyanion-binding site for the intact factor H molecule (regions 1–20, 1213 amino acid residues in total) and characterising its structure by IM-MS has provided further information to the overall structure of the Factor H protein. This work employs two different IM-MS instruments: the commercially available Waters Synapt HDMS instrument, and our Mobility Q-TOF (“MoQTOF”) developed in house. Collision cross-sections obtained from identical molecular species differ to some extent between the two instruments. We observe some good agreement and some variation, with a general trend being that systems examined on the MoQTOF possess and present more compact collision cross-sections than observed with the same species on the Synapt. We attribute these differences principally to slight variations in solution conditions, effecting solution conformations and also to the differences in the source conditions and the ion transmission to the mobility devices affecting gas-phase conformations.

Polyanion-induced self-association of complement factor H.

Factor H is the primary soluble regulator of activation of the alternative pathway of complement. It prevents activation of complement on host cells and tissues upon association with C3b and surface polyanions such as sialic acids, heparin, and other glycosaminoglycans. Here we show that interaction with polyanions causes self-association forming tetramers of the 155,000 Da glycosylated protein. Monomeric human factor H is an extended flexible protein that exhibits an apparent size of 330,000 Da, relative to globular standards, during gel filtration chromatography in the absence of polyanions. In the presence of dextran sulfate (5000 Da) or heparin an intermediate species of apparent m.w. 700,000 and a limit species of m.w. 1,400,000 were observed by gel filtration. Sedimentation equilibrium analysis by analytical ultracentrifugation indicated a monomer Mr of 163,000 in the absence of polyanions and a Mr of 607,000, corresponding to a tetramer, in the presence of less than a 2-fold molar excess of dextran sulfate. Increasing concentrations of dextran sulfate increased binding of factor H to zymosan-C3b 4.5-fold. This result was accompanied by an increase in both the decay accelerating and cofactor activity of factor H on these cells. An expressed fragment encompassing the C-terminal polyanion binding site (complement control protein domains 18-20) also exhibited polyanion-induced self-association, suggesting that the C-terminal ends of factor H mediate self-association. The results suggest that recognition of polyanionic markers on host cells and tissues by factor H, and the resulting regulation of complement activation, may involve formation of dimers and tetramers of factor H.

A new map of the C3b/c/d- and polyanion-binding sites of factor H

A new map of the C3b/c/d- and polyanion-binding sites of factor H

Disease-associated Sequence Variations Congregate in a Polyanion Recognition Patch on Human Factor H Revealed in Three-dimensional Structure

Mutations and polymorphisms in the regulator of complement activation, factor H, have been linked to atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis, and age-related macular degeneration. Many aHUS patients carry mutations in the two C-terminal modules of factor H, which normally confer upon this abundant 155-kDa plasma glycoprotein its ability to selectively bind self-surfaces and prevent them from inappropriately triggering the complement cascade via the alternative pathway. In the current study, the three-dimensional solution structure of the C-terminal module pair of factor H has been determined. A binding site for a fully sulfated heparin-derived tetrasaccharide has been delineated using chemical shift mapping and the C3d/C3b-binding site inferred from sequence comparisons and computational docking. The resultant information allows assessment of the likely consequences of aHUS-associated amino acid substitutions in this critical region of factor H. It is striking that, excepting those likely to perturb the three-dimensional structure, aHUS-associated missense mutations congregate in the polyanion-binding site delineated in this study, thus potentially disrupting a vital mechanism for control of complement on self-surfaces in the microvasculature of the kidney. It is intriguing that a single nucleotide polymorphism predisposing to age-related macular degeneration occupies another region of factor H that harbors a polyanion-binding site.

A positively charged cluster in the epidermal growth factor-like domain of Factor VII-activating protease (FSAP) is essential for polyanion binding

FSAP (Factor VII-activating protease) is a novel plasma-derived serine protease that regulates haemostasis as well as vascular cell proliferation. FSAP undergoes autoactivation in the presence of polyanionic macromolecules such as heparin and RNA. Competition experiments suggest that RNA and heparin bind to the same or overlapping interaction sites. A proteolysis approach, where FSAP was hydrolysed into smaller fragments, was used to identify the polyanion-binding site. The EGF (epidermal growth factor)-like domains EGF2 and EGF3 of FSAP are the major interaction domains for RNA. The amino acids Arg170, Arg171, Ser172 and Lys173 within the EGF3 domain were essential for this binding. This is also the region with the highest positive net charge in the protein and is most probably located in an exposed loop. It is also highly conserved across five species. Disruption of disulphide bridges led to the loss of RNA and heparin binding, indicating that the three-dimensional structure of the EGF3 domain is essential for binding to negatively charged heparin or RNA. The identification of polyanion-binding sites will help to define the role of FSAP in the vasculature.

Hemolytic Uremic Syndrome

Hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy defined by thrombocytopenia, nonimmune microangiopathic hemolytic anemia, and acute renal failure. The most frequent form is associated with infections by Shiga-like toxin-producing bacteria (STEC-HUS). Rarer cases are triggered by neuraminidase-producing Streptococcus pneumoniae (pneumococcal-HUS). The designation of aHUS is used to refer to those cases in which an infection by Shiga-like toxin-producing bacteria or S. pneumoniae can be excluded. Studies performed in the last two decades have documented that hyperactivation of the complement system is the pathogenetic effector mechanism leading to the endothelial damage and the microvascular thrombosis in aHUS. Recent data suggested the involvement of the complement system in the pathogenesis of STEC-HUS and pneumococcal-HUS as well. Clinical signs and symptoms may overlap among the different forms of HUS; however, pneumococcal-HUS and aHUS have a worse prognosis compared with STEC-HUS. Early diagnosis and identification of underlying pathogenetic mechanism allows instating specific support measures and therapies. In clinical trials in patients with aHUS, complement inhibition by eculizumab administration leads to a rapid and sustained normalization of hematological parameters with improvement in long-term renal function. This review summarizes current concepts about the epidemiological findings, the pathological and clinical aspects of STEC-HUS, pneumococcal-HUS, and aHUS, and their diagnosis and management.

Human milk lactoferrin binds two DNA molecules with different affinities

Evidence is presented that lactoferrin (LF), an Fe 3+-binding glycoprotein, possesses two DNA-binding sites with different affinities for specific oligonucleotides (ODNs) ( K d1=8 nM; K d2∽0.1 mM). The high affinity site became labeled after incubation with affinity probes for DNA-binding sites; like the antibacterial and polyanion-binding sites, this site was shown to be located in the N-terminal domain of LF. Interaction of heparin with the polyanion-binding site inhibits the binding of ODNs to both sites. These data suggest that the DNA-binding sites of LF coincide or overlap with the known polyanion and antimicrobial domains of the protein.

Human milk lactoferrin binds ATP and dissociates into monomers.

The physiological role of lactoferrin (LF) is still unclear, but it has been suggested to be responsible for primary defence against microbial infections. Many different unique functions have been attributed to LF, including DNA and RNA binding, and transport into the nucleus, where LF binds to specific DNA sequences and activates transcription. Here we present evidence that in addition to the above (and below) mentioned functions LF binds ATP with a stoichiometry of 1 mole of nucleotide per mole of the protein and a Kd = 0.3 mM. The ATP-binding site is localized in the C-terminal domain of LF, in contrast to the antibacterial and polyanion-binding sites, which are located in the N-terminal domain. Binding of ATP by LF leads to dissociation of its oligomeric forms and to a change of the protein's interaction with polysaccharides, DNA and proteins.

Interaction of nucleotides with acidic fibroblast growth factor (FGF-1).

A wide variety of nucleotides are shown to bind to acidic fibroblast growth factor (aFGF) as demonstrated by their ability to (1) inhibit the heat-induced aggregation of the protein, (2) enhance the thermal stability of aFGF as monitored by both intrinsic fluorescence and CD, (3) interact with fluorescent nucleotides and displace a bound polysulfated naphthylurea compound, suramin, (4) reduce the size of heparin-aFGF complexes, and (5) protect a reactive aFGF thiol group. The binding of mononucleotides, diadenosine compounds (ApnA), and inorganic polyphosphates to aFGF is enhanced as the degree of phosphorylation of these anions is increased with the presence of the base reducing the apparent binding affinity. The nature of the base appears to have much less effect. Photoactivatable nucleotides (8N3-ATP, 2N3-ATP, 8N3-GTP, and 8N3-Ap4A) were employed to covalently label the aFGF nucleotide binding site. In general, Kd's in the low micromolar range are observed. Protection against 90% displacement is observed at several hundred micromolar nucleotide concentration. Using 8N3-ATP as a prototypic reagent, photolabeled aFGF was proteolyzed with trypsin and chymotrypsin and labeled peptides were isolated and sequenced resulting in the identification of 10 possible labeled amino acids (Y8, G20, H21, T61, K112, K113, S116, R119, R122, H124). On the basis of the crystal structure of bovine aFGF, eight of the prospective labeled sites appear to be dispersed around the perimeter of the growth factor's presumptive polyanion binding site. On residue (T61) is more distally located but still proximate to several positively charged residues, and another (Y8) is not locatable in crystal structures. Using heparin affinity chromatography, at least three distinct photolabeled aFGF species were resolved. These labeled complexes display diminished affinity for heparin and a reduced ability to stimulate mitogenesis even in the presence of polyanions such as heparin. In conclusion, nucleotides bind apparently nonspecifically to the polyanion binding site of aFGF but nevertheless are capable of modulating the protein's activity. Evidence for the presence of a second or more extended polyanion binding site and the potential biological significance of these results in terms of potential natural ligands of aFGF are also discussed but not resolved.

Regulation of Alternative Pathway Complement Activation by Glycosaminoglycans: Specificity of the Polyanion Binding Site on Factor H

The discrimination between activators and nonactivators of the alternative pathway of complement depends on the affinity of the control factor H for C3b molecules covalently associated with the target. The affinity of factor H for the C3b-target complex is regulated by a positively charged site at or near the 13th short consensus repeat (SCR) domain of factor H. In this study we have analyzed the ability of different glycosaminoglycans and other negatively charged macromolecules to interact with the factor H polyanion recognition site and to enhance binding of H to the C3b-target complex. Strongest enhancement of factor H binding to zymosan-C3b was observed by the highly sulphated glycoconjugates dextran sulphate (m.w. = 5,000), heparin, chondroitin sulphate A and carrageenan (types III and IV). DNA also enhanced H binding. Removal of N-linked sulphates or reduction of the size of heparin decreased its enhancing effect on H binding. Little or no effect was seen with chondroitin sulphate C, keratan sulphate, hyaluronic acid, colominic acid (bacterial polysialic acid) or negatively charged polypeptides. The results show that the interaction of the polyanion binding site on factor H with glycosaminoglycans depends upon the number, orientation and polymeric arrangement of sulphate groups and suggest that most, but not all, sulphated glycosaminoglycans participate in the protection of host tissues from complement damage by promoting inactivation of tissue-bound C3b.

Sucralfate and soluble sucrose octasulfate bind and stabilize acidic fibroblast growth factor

The actions of the anti-ulcer drug sucralfate have been proposed to be mediated through interaction with fibroblast growth factors (Folkman, J., Szabo, S., Strovroff, M., McNeil, P., Li, W. and Shing, Y. (1991) Ann. Surg. 214, 414–427). We show here that acidic fibroblast growth factor (aFGF; FGF-1) binds in vitro to both the soluble potassium salt and the insoluble aluminum salt of sucrose octasulfate, as demonstrated by a variety of biophysical techniques. Similar to the well-described interaction and stabilization of aFGF by heparin, soluble sucrose octasulfate (SOS) stabilizes aFGF against thermal, urea and acidic pH-induced unfolding as determined by a combination of circular dichroism, fluorescence spectroscopy and differential scanning calorimetry. In addition, SOS also enhances the mitogenic activity of aFGF and partially protects the protein's three cysteine residues from copper-catalyzed oxidation. SOS competes with heparin and suramin for the aFGF polyanion binding site as measured by both fluorescence and light scattering based competitive binding assays. Front-face fluorescence measurements show that the native, folded form of aFGF binds to the insoluble aluminum salt of sucrose octasulfate (sucralfate). Moreover, sucralfate stabilizes aFGF against thermal and acidic pH-induced unfolding to the same extent as observed with SOS. Thus, due to their high charge density, SOS and sucralfate bin and stabilize aFGF via interaction with the aFGF polyanion binding site.

Physical Stabilization of Acidic Fibroblast Growth Factor by Polyanions

Acidic fibroblast growth factor (aFGF) is markedly stabilized by heparin. Partially due to the heterogeneity of heparin preparations, the nature of the aFGF polyanion binding site is still ill-defined. We have, therefore, investigated a wide variety of well-defined polyanions in terms of their ability to stabilize human recombinant aFGF (15-154) against thermal denaturation. The specificity of the interaction between aFGF and polyanions is shown to be remarkably weak with a surprising number of polyanions (including small phosphorylated and sulfated compounds as well as highly charged biopolymers) able to induce physical stability. Temperature-dependent fluorescence and circular dichroism measurements show that many of these polyanionic compounds stabilize aFGF to the same extent as heparin. The ability of these agents to protect the three free thiol groups of aFGF from copper-catalyzed oxidation was also explored and significant protection was observed. The extent and electrostatic requirements of the protein′s polyanion binding site were probed by the use of a series of well-defined heparin fragments and differentially phosphorylated inositol compounds. A tetrasaccharide fragment of heparin is the smallest unit of heparin capable of stabilizing aFGF against thermal denaturation. Increasing phosphorylation of inositol compounds (up to six phosphate groups per molecule) enhances the thermal stability of aFGF. These results are discussed in the context of a model of human aFGF based on the X-ray crystal structure of the bovine protein and previous studies hy others of the heparin binding site of both acidic and basic FGF.