Barbara Mulloy

Crystal Structure of Fibroblast Growth Factor Receptor Ectodomain Bound to Ligand and Heparin

Fibroblast growth factors (FGFs) are a large family of structurally related proteins with a wide range of physiological and pathological activities. Signal transduction requires association of FGF with its receptor tyrosine kinase (FGFR) and heparan sulphate proteoglycan in a specific complex on the cell surface. Direct involvement of the heparan sulphate glycosaminoglycan polysaccharide in the molecular association between FGF and its receptor is essential for biological activity. Although crystal structures of binary complexes of FGF–heparin and FGF–FGFR have been described, the molecular architecture of the FGF signalling complex has not been elucidated. Here we report the crystal structure of the FGFR2 ectodomain in a dimeric form that is induced by simultaneous binding to FGF1 and a heparin decasaccharide. The complex is assembled around a central heparin molecule linking two FGF1 ligands into a dimer that bridges between two receptor chains. The asymmetric heparin binding involves contacts with both FGF1 molecules but only one receptor chain. The structure of the FGF1–FGFR2–heparin ternary complex provides a structural basis for the essential role of heparan sulphate in FGF signalling.

Structure and anticoagulant activity of sulfated fucans. Comparison between the regular, repetitive, and linear fucans from echinoderms with the more heterogeneous and branched polymers from brown algae

Sulfated fucans are among the most widely studied of all the sulfated polysaccharides of non-mammalian origin that exhibit biological activities in mammalian systems. Examples of these polysaccharides extracted from echinoderms have simple structures, composed of oligosaccharide repeating units within which the residues differ by specific patterns of sulfation among different species. In contrast the algal fucans may have some regular repeating structure but are clearly more heterogeneous when compared with the echinoderm fucans. The structures of the sulfated fucans from brown algae also vary from species to species. We compared the anticoagulant activity of the regular and repetitive fucans from echinoderms with that of the more heterogeneous fucans from three species of brown algae. Our results indicate that different structural features determine not only the anticoagulant potency of the sulfated fucans but also the mechanism by which they exert this activity. Thus, the branched fucans from brown algae are direct inhibitors of thrombin, whereas the linear fucans from echinoderms require the presence of antithrombin or heparin cofactor II for inhibition of thrombin, as reported for mammalian glycosaminoglycans. The linear sulfated fucans from echinoderms have an anticoagulant action resembling that of mammalian dermatan sulfate and a modest action through antithrombin. A single difference of one sulfate ester per tetrasaccharide repeating unit modifies the anticoagulant activity of the polysaccharide markedly. Possibly the spatial arrangements of sulfate esters in the repeating tetrasaccharide unit of the echinoderm fucan mimics the site in dermatan sulfate with high affinity for heparin cofactor II.

A disaccharide repeat unit is the major structure in fucoidans from two species of brown algae

The predominant repeating structure of a fraction of the fucoidan from Ascophyllum nodosum prepared by acid hydrolysis and centrifugal partition chromatography (LMWF) was established as: [-->3)-alpha-L-Fuc(2SO3-)-(1-->4)-alpha-L-Fuc(2,3diSO3-)-(1]n by NMR spectroscopy and methylation analysis. The proton and carbon NMR spectra of this unit have been assigned and found to correspond with features in the spectra of the whole purified fucan from A. nodosum which account for most of the integrated intensity. The same structure has also been recognised in the fucoidan of Fucus vesiculosus. The fraction LMWF has in vitro anticoagulant activity, indicating that the above structure may be partly responsible for biological activity in the native fucoidan.

Characterization of the Structural Features and Interactions of Sclerostin MOLECULAR INSIGHT INTO A KEY REGULATOR OF Wnt-MEDIATED BONE FORMATION

The secreted glycoprotein sclerostin has recently emerged as a key negative regulator of Wnt signaling in bone and has stimulated considerable interest as a potential target for therapeutics designed to treat conditions associated with low bone mass, such as osteoporosis. We have determined the structure of sclerostin, which resulted in the identification of a previously unknown binding site for heparin, suggestive of a functional role in localizing sclerostin to the surface of target cells. We have also mapped the interaction site for an antibody that blocks the inhibition of Wnt signaling by sclerostin. This shows minimal overlap with the heparin binding site and highlights a key role for this region of sclerostin in protein interactions associated with the inhibition of Wnt signaling. The conserved N- and C-terminal arms of sclerostin were found to be unstructured, highly flexible, and unaffected by heparin binding, which suggests a role in stabilizing interactions with target proteins.

Sulfated Polysaccharides from the Egg Jelly Layer Are Species-specific Inducers of Acrosomal Reaction in Sperms of Sea Urchins

We have characterized the fine structure of sulfated polysaccharides from the egg jelly layer of three species of sea urchins and tested the ability of these purified polysaccharides to induce the acrosome reaction in spermatozoa. The sea urchin Echinometra lucunter contains a homopolymer of 2-sulfated, 3-linked α-L-galactan. The species Arbacia lixula and Lytechinus variegatus contain linear sulfated α-L-fucans with regular tetrasaccharide repeating units. Each of these sulfated polysaccharides induces the acrosome reaction in conspecific but not in heterospecific spermatozoa. These results demonstrate that species specificity of fertilization in sea urchins depends in part on the fine structure of egg jelly sulfated polysaccharide.

Bone morphogenetic protein and growth differentiation factor cytokine families and their protein antagonists.

The BMPs (bone morphogenetic proteins) and the GDFs (growth and differentiation factors) together form a single family of cystine-knot cytokines, sharing the characteristic fold of the TGFbeta (transforming growth factor-beta) superfamily. Besides the ability to induce bone formation, which gave the BMPs their name, the BMP/GDFs display morphogenetic activities in the development of a wide range of tissues. BMP/GDF homo- and hetero-dimers interact with combinations of type I and type II receptor dimers to produce multiple possible signalling complexes, leading to the activation of one of two competing sets of SMAD transcription factors. BMP/GDFs have highly specific and localized functions. These are regulated in a number of ways, including the developmental restriction of BMP/GDF expression and through the secretion of several specific BMP antagonist proteins that bind with high affinity to the cytokines. Curiously, a number of these antagonists are also members of the TGF-beta superfamily. Finally a number of both the BMP/GDFs and their antagonists interact with the heparan sulphate side chains of cell-surface and extracellular-matrix proteoglycans.

Highly Sulfated Dermatan Sulfates from Ascidians STRUCTURE VERSUS ANTICOAGULANT ACTIVITY OF THESE GLYCOSAMINOGLYCANS

Dermatan sulfates with the same backbone structure [4-α-l-IdceA-1→3-β-d-GalNAc-1] n but with different patterns of sulfation substitutions have been isolated from the ascidian body. All the ascidian dermatan sulfates have a high content of 2-O-sulfated α-l-iduronic acid residues but differ in the pattern of sulfation of the N-acetyl-β-d-galactosamine units. Styela plicata and Halocynthia pyriformis have 4-O-sulfated units, but inAscidian nigra they are 6-O-sulfated. This collection of ascidian dermatan sulfates (together with native and oversulfated mammalian dermatan sulfate), where the extent and position of sulfate substitution have been fully characterized, were tested in anticoagulant assays. Dermatan sulfate from A. nigra has no discernible anticoagulant activity, which indicates that 4-O-sulfation of theN-acetyl-β-d-galactosamine is essential for the anticoagulant activity of this glycosaminoglycan. In contrast dermatan sulfates from S. plicata and H. pyriformis are potent anticoagulants due to potentiation of thrombin inhibition by heparin cofactor II. These ascidian dermatan sulfates have ∼10-fold and ∼6-fold higher activity with heparin cofactor II than native and an oversulfated mammalian dermatan sulfate, respectively. They have no effect on thrombin or factor Xa inhibition by antithrombin. These naturally oversulfated ascidian dermatan sulfates are sulfated at selected sites required for interaction with heparin cofactor II and thus have specific and potent anticoagulant activity.

His-384 Allotypic Variant of Factor H Associated with Age-related Macular Degeneration Has Different Heparin Binding Properties from the Non-disease-associated Form

A polymorphism in complement factor H has recently been associated with age-related macular degeneration (AMD), the leading cause of blindness in the elderly. A histidine rather than a tyrosine at residue position 384 in the mature protein increases the risk of AMD. Here, using a recombinant construct, we show that amino acid 384 is adjacent to a heparin-binding site in CCP7 of factor H and demonstrate that the allotypic variants differentially recognize heparin. This functional alteration may affect binding of factor H to polyanionic patterns on host surfaces, potentially influencing complement activation, immune complex clearance, and inflammation in the macula of AMD patients.

Heparin and low-molecular-weight heparin

Heparin is one of the oldest biological medicines, and has an established place in the prevention and treatment of venous thrombosis. Low-molecular-weight heparins (LMWH) have been developed by several manufacturers and have advantages in terms of pharmacokinetics and convenience of administration. They have been shown to be at least as effective and safe as unfractionated heparin and have replaced the latter in many indications. In this article the chemistry, mechanisms of action, measurement of anticoagulant activities, and clinical status of heparin and LMWH are reviewed.

The molecular-weight range of mucosal-heparin preparations.

A recently reported method describes the determination of the molecular-weight range of heparins by using an electrofocusing procedure to isolate individual molecular species. Commercially available heparins were fractionated on a column of polyacrylamide-agarose gel to give fractions whose molecular weights were estimated by viscometry. Fractions with mutually exclusive molecllar-weight ranges gave an appreciable number of common bands when subjected to the electrofocusing procedure; therefore, each of these bands cannot be formed from a single molecular species of heparin. Other mucopolysaccharides also gave band sequences indistinguishable from those of heparin; they differed in position and intensity with different ampholyte batches, and probably arose from individual molecular species of the ampholyte rather than the mucopolysaccharide. The molecular-weight range of the heparin was observed to be broader than that usually reported.