Application of the four-component Ugi condensation for the preparation of sulfated glycoconjugate libraries

A focused library of novel, sulfated glycoconjugates was synthesized by utilizing carbohydrate-derived blocks in the four-component Ugi condensation. Library members comprise a sulfated monosaccharide linked by various spacers to either an aromatic or monosulfated moiety, or a second sulfated monosaccharide. The affinities of these heparan sulfate (HS) mimetics for the HS-binding fibroblast growth factors FGF-1 and FGF-2 were measured via a surface plasmon resonance solution affinity assay.     

A surface plasmon resonance-based solution affinity assay for heparan sulfate-binding proteins

A surface plasmon resonance-based solution affinity assay is described for measuring the K _d of binding of heparin/heparan sulfate-binding proteins with a variety of ligands. The assay involves the passage of a pre-equilibrated solution of protein and ligand over a sensor chip onto which heparin has been immobilised. Heparin sensor chips prepared by four different methods, including biotin–streptavidin affinity capture and direct covalent attachment to the chip surface, were successfully used in the assay and gave similar K _d values. The assay is applicable to a wide variety of heparin/HS-binding proteins of diverse structure and function (e.g., FGF-1, FGF-2, VEGF, IL-8, MCP-2, ATIII, PF4) and to ligands of varying molecular weight and degree of sulfation (e.g., heparin, PI-88, sucrose octasulfate, naphthalene trisulfonate) and is thus well suited for the rapid screening of ligands in drug discovery applications. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

A focused antibody library for improved hapten recognition

The topography of the antigen-binding site as well as the number and the positioning of the antigen contact residues are strongly correlated with the size of the antigen with which the antibody interacts. On the basis of these considerations, we have designed a focused scFv repertoire biased for haptens, designated the cavity library. The hapten-specific scFv, FITC8, was used as a scaffold for library construction. FITC8, like other hapten binders, displays a characteristic cavity in its paratope into which the hapten binds. In five of the six complementarity-determining regions, diversity-carrying residues were selected rationally on the basis of a model structure of FITC8 and on known antibody structure-function relationships, resulting in variation of 11 centrally located, cavity-lining residues. L3 was allowed to carry a more complex type of diversity. In addition, length variation was introduced into H2, as longer versions of this loop have been shown to correlate with increased hapten binding. The library was screened, using phage display, against a panel of five different haptens, yielding diverse and highly specific binders to four of the antigens. Parallel selections were performed with a library having diversity spread onto a greater area, including more peripherally located residues. This resulted in the isolation of binders, which, in contrast to the clones selected from the cavity library, were not able to bind to the soluble hapten in the absence of the carrier protein. Thus, we have shown that by focusing diversity to the hotspots of interaction a library with improved hapten-binding ability can be created. The study supports the notion that it is possible to create antibody libraries that are biased for the recognition of antigens of pre-defined size.     

Cytokines and growth factors cross-link heparan sulfate

The glycosaminoglycan heparan sulfate (HS), present at the surface of most cells and ubiquitous in extracellular matrix, binds many soluble extracellular signalling molecules such as chemokines and growth factors, and regulates their transport and effector functions. It is, however, unknown whether upon binding HS these proteins can affect the long-range structure of HS. To test this idea, we interrogated a supramolecular model system, in which HS chains grafted to streptavidin-functionalized oligoethylene glycol monolayers or supported lipid bilayers mimic the HS-rich pericellular or extracellular matrix, with the biophysical techniques quartz crystal microbalance (QCM-D) and fluorescence recovery after photobleaching (FRAP). We were able to control and characterize the supramolecular presentation of HS chains—their local density, orientation, conformation and lateral mobility—and their interaction with proteins. The chemokine CXCL12α (or SDF-1α) rigidified the HS film, and this effect was due to protein-mediated cross-linking of HS chains. Complementary measurements with CXCL12α mutants and the CXCL12γ isoform provided insight into the molecular mechanism underlying cross-linking. Fibroblast growth factor 2 (FGF-2), which has three HS binding sites, was also found to cross-link HS, but FGF-9, which has just one binding site, did not. Based on these data, we propose that the ability to cross-link HS is a generic feature of many cytokines and growth factors, which depends on the architecture of their HS binding sites. The ability to change matrix organization and physico-chemical properties (e.g. permeability and rigidification) implies that the functions of cytokines and growth factors may not simply be confined to the activation of cognate cellular receptors.     

The heparin/heparan sulfate-binding site on apo-serum amyloid A. Implications for the therapeutic intervention of amyloidosis

Serum amyloid A isoforms, apoSAA1 and apoSAA2, are apolipoproteins of unknown function that become major components of high density lipoprotein (HDL) during the acute phase of an inflammatory response. ApoSAA is also the precursor of inflammation-associated amyloid, and there is strong evidence that the formation of inflammation-associated and other types of amyloid is promoted by heparan sulfate (HS). Data presented herein demonstrate that both mouse and human apoSAA contain binding sites that are specific for heparin and HS, with no binding for the other major glycosaminoglycans detected. Cyanogen bromide-generated peptides of mouse apoSAA1 and apoSAA2 were screened for heparin binding activity. Two peptides, an apoSAA1-derived 80-mer (residues 24-103) and a smaller carboxyl-terminal 27-mer peptide of apoSAA2 (residues 77-103), were retained by a heparin column. A synthetic peptide corresponding to the CNBr-generated 27-mer also bound heparin, and by substituting or deleting one or more of its six basic residues (Arg-83, His-84, Arg-86, Lys-89, Arg-95, and Lys-102), their relative importance for heparin and HS binding was determined. The Lys-102 residue appeared to be required only for HS binding. The residues Arg-86, Lys-89, Arg-95, and Lys-102 are phylogenetically conserved suggesting that the heparin/HS binding activity may be an important aspect of the function of apoSAA. HS linked by its carboxyl groups to an Affi-Gel column or treated with carbodiimide to block its carboxyl groups lost the ability to bind apoSAA. HDL-apoSAA did not bind to heparin; however, it did bind to HS, an interaction to which apoA-I contributed. Results from binding experiments with Congo Red-Sepharose 4B columns support the conclusions of a recent structural study which found that heparin binding domains have a common spatial distance of about 20 A between their two outer basic residues. Our present work provides direct evidence that apoSAA can associate with HS (and heparin) and that the occupation of its binding site by HS, and HS analogs, likely caused the previously reported increase in amyloidogenic conformation (beta-sheet) of apoSAA2 (McCubbin, W. D., Kay, C. M., Narindrasorasak, S., and Kisilevsky, R. (1988) Biochem. J. 256, 775-783) and their amyloid-suppressing effects in vivo (Kisilevsky, R., Lemieux, L. J., Fraser, P. E., Kong, X., Hultin, P. G., and Szarek, W. A. (1995) Nat. Med. 1, 143-147), respectively.     

The role of angiogenic growth factors in organogenesis

Angiogenic growth factors are a class of molecules which exert a fundamental role in the process of blood vessel formation. Besides vasculogenic and angiogenic properties, these compounds mediate a complex series of patterning activities during organogenesis. Angiogenic factors cooperate in the growth and development of embryo tissues in a cross-talk between endothelial cells and tissue cells. It is well established that many tissue-derived factors are involved in blood vessel formation, but there is now emerging evidence that angiogenic factors and endothelial cells themselves represent a crucial source of instructive signals to non-vascular tissue cells during organ development. Thus, angiogenic factors and endothelial cell signalling are currently believed to provide fundamental cues for cell fate specification, embryo patterning, organ differentiation and postnatal tissue remodelling. This review article will summarize some of the recent advances in our understanding of the role of angiogenic factors and endothelial cells as effectors in organ formation.     

Characterization of annexin A1 glycan binding reveals binding to highly sulfated glycans with preference for highly sulfated heparan sulfate and heparin

Annexin A1 is a multifunctional, calcium-dependent phospholipid binding protein involved in a host of processes including inflammation, regulation of neuroendocrine signaling, apoptosis, and membrane trafficking. Binding of annexin A1 to glycans has been implicated in cell attachment and modulation of annexin A1 function. A detailed characterization of the glycan binding preferences of annexin A1 using carbohydrate microarrays and surface plasmon resonance served as a starting point to understand the role of glycan binding in annexin A1 function. Glycan array analysis identified annexin A1 binding to a series of sulfated oligosaccharides and revealed for the first time that annexin A1 binds to sulfated non-glycosaminoglycan carbohydrates. Using heparin/heparan sulfate microarrays, highly sulfated heparan sulfate/heparin were identified as preferred ligands of annexin A1. Binding of annexin A1 to heparin/heparan sulfate is calcium- but not magnesium-dependent. An in-depth structure-activity relationship of annexin A1-heparan sulfate interactions was established using chemically defined sugars. For the first time, a calcium-dependent heparin binding protein was characterized with such an approach. N-Sulfation and 2-O-sulfation were identified as particularly important for binding.     

Heparin-mimetic sulfated peptides with modulated affinities for heparin-binding peptides and growth factors

Heterogeneity in the composition and in the polydispersity of heparin has motivated the development of homogeneous heparin mimics, and peptides of appropriate sequence and chemical function have therefore recently emerged as potential replacements for heparin in selected applications. Here, we report the assessment of the binding affinities of multiple sulfated peptides (SPs) for a set of heparin-binding peptides (HBPs) and for vascular endothelial growth factor isoform 165 (VEGF165); these binding partners have application in the selective immobilization of proteins and in hydrogel formation through non-covalent interactions. Sulfated peptides were produced via solid-phase methods, and their affinity for the HBPs and VEGF165 was assessed via affinity liquid chromatography (ALC), surface plasmon resonance (SPR), and in selected cases, isothermal titration calorimetry (ITC). The shortest peptide, SP(a), showed the highest affinity binding of HBPs and VEGF165 in both ALC and SPR measurements, with slight exceptions. Of the investigated HBPs, a peptide based on the heparin-binding domain of human platelet factor 4 showed greatest binding affinities toward all of the SPs, consistent with its stronger binding to heparin. The affinity between SP(a) and PF4(ZIP) was indicated via SPR (K(D)=5.27 microM) and confirmed via ITC (K(D)=8.09 microM). The binding by SP(a) of both VEGF and HBPs suggests its use as a binding partner to multiple species, and the use of these interactions in assembly of materials. Given that the peptide sequences can be varied to control binding affinity and selectivity, opportunities are also suggested for the production of a wider array of matrices with selective binding and release properties useful for biomaterials applications.     

Clinical applications of angiogenic growth factors and their inhibitors

Promoting the formation of new collateral vessels in ischemic tissues using angiogenic growth factors (therapeutic angiogenesis) is a an exciting frontier of cardiovascular medicine. Conversely, inhibition of the action of key regulators of angiogenesis, such as VEGF, constitutes a promising approach for the treatment of solid tumors and intraocular neovascular syndromes. These concepts are being tested now in clinical trials.     

EG-VEGF and the concept of tissue-specific angiogenic growth factors

The endothelium of the vascular beds is extremely diverse and exquisitely distinct with respect to the specific tissue compartment served by the vessels. The molecular identity and function of the instructive signals that tailor the tissue-specific endothelial phenotype have been largely undefined. Presumably, a complex, integrated network of signals derived from the tissue parenchyma and/or stromal compartments is responsible. Recently, we identified a novel angiogenic mitogen, endocrine-gland-derived vascular endothelial growth factor, EG-VEGF, with a selective activity and very distinct expression pattern. Human EG-VEGF is expressed by steroid producing cells in the adrenal gland, placenta, testis and ovary, and is a mitogen for endothelial cells derived from these microvascular beds. EG-VEGF may represent the first of a novel class of tissue-specific angiogenic factors that function to regulate and fine-tune endothelial cell growth, structural and functional properties. The identification of other selective angiogenic molecules will allow insight into exciting, basic developmental issues and increase our armamentarium of factors for therapeutic angiogenic and anti-angiogenic strategies.     

A method for glycoconjugate synthesis

7-Formylheptyl glycosides of 2-acetamido-2-deoxy--d-glucopyranose andO--l-rhamnopyranosyl-(1 3)-O--l-rhamnopyranose were synthesized and were coupled by reductive amination to bovine serum albumin and aminopropyl glass, respectively.     

Characterization of growth factor-binding structures in heparin/heparan sulfate using an octasaccharide library

Heparan sulfate (HS) chains interact with various growth and differentiation factors and morphogens, and the most interactions occur on the specific regions of the chains with certain monosaccharide sequences and sulfation patterns. Here we generated a library of octasaccharides by semienzymatic methods by using recombinant HS 2-O-sulfotransferase and HS 6-O-sulfotransferase, and we have made a systematic investigation of the specific binding structures for various heparin-binding growth factors. An octasaccharide (Octa-I, DeltaHexA-GlcNSO(3)-(HexA-GlcNSO(3))(3)) was prepared by partial heparitinase digestion from completely desulfated N-resulfated heparin. 2-O- and 6-O-sulfated Octa-I were prepared by enzymatically transferring one to three 2-O-sulfate groups and one to three 6-O-sulfate groups per molecule, respectively, to Octa-I. Another octasaccharide containing 3 units of HexA(2SO(4))-GlcNSO(3)(6SO(4)) was prepared also from heparin. This octasaccharide library was subjected to affinity chromatography for interactions with fibroblast growth factor (FGF)-2, -4, -7, -8, -10, and -18, hepatocyte growth factor, bone morphogenetic protein 6, and vascular endothelial growth factor, respectively. Based upon differences in the affinity to those octasaccharides, the growth factors could be classified roughly into five groups: group 1 needed 2-O-sulfate but not 6-O-sulfate (FGF-2); group 2 needed 6-O-sulfate but not 2-O-sulfate (FGF-10); group 3 had the affinity to both 2-O-sulfate and 6-O-sulfate but preferred 2-O-sulfate (FGF-18, hepatocyte growth factor); group 4 required both 2-O-sulfate and 6-O-sulfate (FGF-4, FGF-7); and group 5 hardly bound to any octasaccharides (FGF-8, bone morphogenetic protein 6, and vascular endothelial growth factor). The approach using the oligosaccharide library may be useful to define specific structures required for binding to various heparin-binding proteins. Octasaccharides with the high affinity to FGF-2 and FGF-10 had the activity to release them, respectively, from their complexes with HS. Thus, the library may provide new reagents to specifically regulate bindings of the growth factors to HS.     

Mesoderm induction and blood island formation by angiogenic growth factors and embryonic inducing factors

Factors which induce mesoderm, including endothelium lined cavities and primitive blood cells in omnipotent amphibian ectoderm, have been isolated from different sources. Recently it was shown that angiogenic factors, which belong to the protein families of the heparin binding growth factors (acidic and basic fibroblast growth factor) and the transforming growth factors (TGF-beta 1 and -beta 2), also induce mesodermal tissues in amphibian ectoderm. In triturus ectoderm, capillary like endothelial networks are induced preferentially by the transforming growth factors. The relationship between growth factors and inducing factors is discussed.     

Neomycin inhibits the angiogenic activity of fibroblast and epidermal growth factors

Three model peptides have been studied in an effort to understand the molecular basis for the fusogenic potency of foamy virus. These peptides corresponded to a 23 amino acid helical segment close to the amino terminus, a shortened 17 amino acid, more hydrophobic homolog of this peptide, and an 18-amino-acid peptide close to or within the transmembrane domain. The peptides have a conformation containing both alpha-helical and beta-structure in aqueous solution but are predominantly alpha-helical in solutions of trifluoroethanol, as assessed by circular dichroism. In common with other viruses, the most fusogenic peptide was the one closest to the amino terminus. However, unlike several other fusion peptides that have been studied previously, this peptide did not promote increase negative membrane curvature as assessed by effects of the peptide on lipid polymorphism. Nevertheless, the foamy virus fusion peptide promotes membrane fusion, apparently by a mechanism that is independent of changes in membrane curvature. We demonstrate that there is a synergistic action in the promotion of membrane fusion between the peptide from the amino terminal region and the one from the region adjacent to the transmembrane segment.     

A novel method for glycoconjugate synthesis

2-Chloroethyl 1-thio--d-galactopyranoside and the corresponding 1-thio--d-glucopyranoside have been found to be suitable glycosylating agents for the preparation of different types of glycoconjugates. Glycosylation of bovine serum albumin and chymotrypsin were chosen as examples of an application of the described compounds. The glycosylating agents can modify not only amino groups, but also alkyl hydroxyl and aryl hydroxyl groups, as was shown in experiments with model water soluble and water insoluble polyacrylamide copolymers.     

Isolation and characterization of low sulfated heparan sulfate sequences with affinity for lipoprotein lipase

Lipoprotein lipase (LPL), which is an important enzyme in lipid metabolism, binds to heparan sulfate (HS) proteoglycans. This interaction is crucial for several aspects of LPL function, such as intracellular/extracellular transport and high capacity attachment to cell surfaces. Retention of LPL on the capillary walls, and elsewhere, via HS chains is most likely affected by the quality and quantity of HS present. Earlier studies have demonstrated that LPL interacts with highly sulfated HS and heparin oligosaccharides. Since such structures are relatively rare in endothelial HS, we have re-addressed the question of physiological ligand structures for LPL by affinity purification of end-labeled oligosaccharides originating from heparin and HS on immobilized LPL. By a combination of chemical modification and fragmentation of the bound material we identified that the bound fraction contained modestly sulfated oligosaccharides with an average sulfation of one O-sulfate per disaccharide unit and tolerates N-acetylated glucosamine residues. Therefore LPL, containing several clusters of positive charges on each subunit, may constitute an ideal structure for a protein that needs to bind with reasonable affinity to a variety of modestly sulfated sequences of the type that is abundant in HS chains.     

Mitogenic activity of acidic fibroblast growth factor is enhanced by highly sulfated oligosaccharides derived from heparin and heparan sulfate

The mitogenic activity of acidic fibroblast growth factor (aFGF) is potentiated by the highly sulfated hexasaccharide [IdoUA,2S-GlcNS,6S]₂-[GlcUA-GlcNS,6S] the structural repetitive unit of lung heparin chains. On a mass basis, the effect of both heparin and oligosaccharide are equivalent whereas on a molar basis, heparin, which contains about seven hexasaccharide repeats, is more efficient. On the other hand, a pentasulfated tetrasaccharide or di- and trisulfated disaccharides are much less effective in potentiating aFGF activity than the hexasaccharide. If the growth factor is pre-incubated with the hexasaccharide at pH 7.2 and then exposed to pH 3.5 the 306/345 nm fluoresence ratio is similar to that of native aFGF indicating that the oligosaccharide stabilizes a native conformation of the protein. Heparan sulfates extracted from various mammalian tissues were also able to potentiate aFGF mitogenic activity. On a mass basis they were in general less efficient than heparin; however, heparan sulfate prepared from medium conditioned by 3T3 fibroblasts is more efficient than heparin both on a mass and molar basis. A highly sulfated oligosaccharide isolated after digestion of pancreas heparan sulfate with heparitinase I is more active than the intact molecule, reaching a potentiating effect equivalent to that of lung heparin, whereas an N-acetylated oligosaccharide isolated after nitrous acid degradation is inactive. These data suggest that the mitogenic activity of aFGF is primarily potentiated by interacting with highly sulfated regions of heparan sulfates chains.Abbreviations aFGF,bFGF acidic and basic fibroblast growth factor - DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum - U,2S-(14)-GlcNS,6S O--L-ido(ene-pyranosyluronic acid 2-O-sulfate)-(14)-2-sulfoamino-2-deoxy-D-glucose-6-O-sulfate - U-(14)-GlcNS,6S O-(ene-pyranosyluronic acid)-(14)-2-sulfoamino-2-deoxy-D-glucose-6-O-sulfate - IdoUA iduronic acid - GlcUA glucuronic acid - GlyUA uronic acid; GlcNAcN-acetylglycosamine - GlcNS N-sulfated glucosamine - GlcNS,6S N,6-disulfated glucosamine - Gal galactose - Xyl xylose - Ser serine - HS heparan Sulfate     

Sequence analysis of heparan sulfate epitopes with graded affinities for fibroblast growth factors 1 and 2.

Proteins that belong to the fibroblast growth factor (FGF) family regulate proliferation, migration, and differentiation of many cell types. Several FGFs, including the prototype factors FGF-1 and FGF-2, depend on interactions with heparan sulfate (HS) proteoglycans for activity. We have assessed tissue-derived HS fragments for binding to FGF-1 and FGF-2 to identify the authentic saccharide motifs required for interactions. Sequence information on a range of N-sulfated HS octasaccharides spanning from low to high affinity for FGF-1 was obtained. All octasaccharides with high affinity for FGF-1 (> or =0.5 m NaCl required for elution) contained an internal IdoUA(2-OSO(3))-GlcNSO(3)(6-OSO(3))-IdoUA(2-OSO(3))-trisaccharide motif. Octasaccharides with a higher overall degree of sulfation but lacking the specific trisaccharide motif showed lower affinity for FGF-1. FGF-2 was shown to bind to a mono-O-sulfated HS 6-mer carrying a single internal IdoUA(2-OSO(3))-unit. However, a di-O-sulfated -IdoUA(2-OSO(3))-GlcNSO(3)-IdoUA(2-OSO(3))-trisaccharide sequence within a HS 8-mer gave stronger binding. These findings show that not only the number but also the positions of individual sulfate groups determine affinity of HS for FGFs. Our findings support the notion that FGF-dependent processes can be modulated in vivo by regulated expression of distinct HS sequences.     

Highly sulfated nonreducing end-derived heparan sulfate domains bind fibroblast growth factor-2 with high affinity and are enriched in biologically active fractions

Human fibroblast growth factor-2 (FGF2) regulates cellular processes including proliferation, adhesion, motility, and angiogenesis. FGF2 exerts its biological function by binding and dimerizing its receptor (FGFR), which activates signal transduction cascades. Effective binding of FGF2 to its receptor requires the presence of heparan sulfate (HS), a linear polysaccharide with N-sulfated domains (NS) localized at the cell surface and extracellular matrix. HS acts as a platform facilitating the formation of a functional FGF-FGFR-HS ternary complex. Crystal structures of the signaling ternary complex revealed two conflicting architectures. In the asymmetrical model, two FGFs and two FGFRs bind a single HS chain. In contrast, the symmetrical model postulates that one FGF and one FGFR bind to the free end of the HS chain and dimerization require these ends to join, bringing the two half-complexes together. In this study, we screened a hexasaccharide HS library for compositions that are able to bind FGF2. The library was composed primarily of NS domains internal to the HS chain with minor presence of non-reducing end (NRE) NS. The binders were categorized into low versus high affinity binders. The low affinity fraction contained primarily hexasaccharides with low degree of sulfation that were internal to the HS chains. In contrast, the high affinity bound fraction was enriched in NRE oligosaccharides that were considerably more sulfated and had the ability to promote FGFR-mediated cell proliferation. The results suggest a role of the NRE of HS in FGF2 signaling and favor the formation of the symmetrical architecture on short NS domains.