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B Mulloy

Publications and source records attributed to B Mulloy.

At least 19 recordsLinked to original sources
PubMed2006

Cytokines and proteoglycans: an introductory overview.

The defining characteristic of the glycoproteins known as proteoglycans is the presence of O-linked acidic polysaccharides known as GAGs (glycosaminoglycans). The backbone of these linear polysaccharides is a repeating disaccharide, comprising N-acetyl hexosamine alternating with beta-D-glucuronic acid, alpha-L-iduronic acid, or galactose. For some GAGs, partial deacetylation, epimerization of glucuronic acid, and substitution with N- and O-sulphates result in highly complex, heterogeneous structures. The interactions with proteins through which GAGs exert their biological effects depend on the resulting sequences. Some proteins, for example antithrombin, have highly specific sequence requirements for their GAG ligand [in this case heparin or HS (heparan sulphate)]; others, for example the fibroblast growth factors, are less demanding. GAGs, in particular HS, play a role as co-receptors for some cytokines. In addition, HS is thought to be important for the localization of cytokines, acting both as a tissue store and as a mediator of morphogen gradient formation in development. The structural determinants of GAG-cytokine interactions are therefore clearly important to understanding the biology of development, wound healing and the immune system. No single paradigm has been identified for such interactions, and the search for general principles underlying involvement of GAGs in cytokine function is at an early stage.

Animals↗
PubMed2006

Signalling by HGF/SF and Met: the role of heparan sulphate co-receptors.

The receptor tyrosine kinase Met and its ligand HGF/SF (hepatocyte growth factor/scatter factor) are essential in the signalling pathways required for embryogenesis and tissue regeneration. Aberrant signalling of this complex is also a feature of many tumours and appears to contribute to the growth, invasiveness and metastasis of both carcinomas and sarcomas. HGF/SF, like many other angiogenic growth factors, employs heparan sulphate as co-receptor. The role of this interaction has not been completely defined but appears to be physiologically relevant. Thus the presence of heparin increases the potency of HGF/SF in experiments with cells in culture leading to elevated downstream signalling effects and, although not vital for the Met-HGF/SF interaction, heparin or heparan sulphate is essential for the activity of certain isoforms of HGF/SF, such as NK1 and NK2. Here, we summarize the progress made in understanding the interaction between heparin and heparan sulphate and NK1, NK2 and HGF/SF and we discuss their role in HGF/SF-Met signalling.

Animals↗
PubMed2006

Computational approaches to the identification of heparin-binding sites on the surfaces of proteins.

The identification of heparin-binding sites is important for understanding the physiological function of many secreted proteins. Most of the experimental techniques for mapping these sites do not define them to atomic resolution. The use of automated docking methods can aid this process by facilitating both the design of experiments and visualization of their results. A method designed for a systematic search over the whole protein surface for heparin-binding sites, using heparin oligosaccharide structures as ligands, is described, with its validation and details of several published applications. The scope and limitations of this crude but effective computational chemistry method are discussed.

Animals↗
PubMed2001

Crystal structures of NK1-heparin complexes reveal the basis for NK1 activity and enable engineering of potent agonists of the MET receptor.

NK1 is a splice variant of the polypeptide growth factor HGF/SF, which consists of the N-terminal (N) and first kringle (K) domain and requires heparan sulfate or soluble heparin for activity. We describe two X-ray crystal structures of NK1-heparin complexes that define a heparin-binding site in the N domain, in which a major role is played by R73, with further contributions from main chain atoms of T61, K63 and G79 and the side chains of K60, T61, R76, K62 and K58. Mutagenesis experiments demonstrate that heparin binding to this site is essential for dimerization in solution and biological activity of NK1. Heparin also comes into contact with a patch of positively charged residues (K132, R134, K170 and R181) in the K domain. Mutation of these residues yields NK1 variants with increased biological activity. Thus, we uncover a complex role for heparan sulfate in which binding to the primary site in the N domain is essential for biological activity whereas binding to the K domain reduces activity. We exploit the interaction between heparin and the K domain site in order to engineer NK1 as a potent receptor agonist and suggest that dual (positive and negative) control may be a general mechanism of heparan sulfate-dependent regulation of growth factor activity.

Animals↗
PubMed2001

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.

Anticoagulants↗
PubMed2001

Order out of complexity--protein structures that interact with heparin.

Many proteins of widely differing functionality and structure are capable of binding heparin. Structural characterisations of the many types of such complexes are being reported in ever-increasing number and at improved resolution. Several crystal structures of complexes formed through the interaction of heparin-derived oligosaccharides with one or more protein partners have been described.

Annexin A5↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

Molecular modelling study of HIV p17gag (MA) protein shell utilising data from electron microscopy and X-ray crystallography.

The matrix protein p17gag (MA) is a product of proteolytic cleavage of the gag gene encoded polyprotein (pr55gag) and is formed when HIV particles undergo the process of maturation. The MA protein is associated with the inner surface of the viral membrane and determines the overall shape of the virion. Previous studies have shown the existence of trimers of MA in solution and in the crystalline state. Here, we used molecular modelling methods to identify feasible interactions between pairs of MA trimers and have related this to structural data from electron microscopy. A systematic search docking procedure was able to identify many energetically favourable conformations for a pair of trimers, including some which have been previously reported. These conformations were used to generate several networks of MA trimers, which were then evaluated against structural observations of the MA network. The model suggested here provides a good match with experimental data such as the spacing between gag protein rings, the number and disposition of glycoprotein (gp41-gp120) knobs and the number of copies of MA in a virus particle. It also rationalizes the observed distribution of sizes of virus particles and is consistent with the presence of icosahedral organisation in mature HIV. Energy minimisation performed with explicit water and counter ions, was used to identify residues participating in inter-trimer interactions. The nature of these interactions is discussed in relation to the conservation of these residues in reported variants of the HIV and SIV MA protein sequences.

Computer Simulation↗
PubMed2000

Structure/function studies of anticoagulant sulphated polysaccharides using NMR.

Sulphated polysaccharides have many biological functions, which depend on binding of highly specific carbohydrate structures to proteins. NMR spectroscopy is a technique capable of detailed structural elucidation of these polysaccharides, and can be used in applications ranging from routine analysis to research into covalent and conformational aspects of polysaccharide structure. This technique can be used to characterise sequence variations in heparin samples. The NMR-determined solution conformation of heparin has been used to predict binding sites on the surface of heparin-binding proteins. Sulphation patterns for dermatan sulphates of marine invertebrates have been determined. Their anticoagulant effects depend on an exact pattern of sulphate substitution. A small alteration in dermatan sulphate structure, from 4-O-sulphated to 6-O-sulphated galactosamine, leads to almost complete loss of anticoagulant activity in spite of an overall high level of sulphation. A fucosylated chondroitin sulphate isolated from sea cucumber has anticoagulant and antithrombotic activity depending on its sulphated fucose branches. The anticoagulant activity of algal fucans has been compared with that of regular, linear sulphated fucans from marine echinoderms; again high activity appears to correlate with the presence of sulphated fucose branches.

Animals↗
PubMed2000

Conformation and dynamics of heparin and heparan sulfate.

The glycosaminoglycans heparin and heparan sulfate contain similar structural units in varying proportions providing considerable diversity in sequence and biological function. Both compounds are alternating copolymers of glucosamine with both iduronate- and glucuronate-containing sequences bearing N-sulfate, N-acetyl, and O-sulfate substitution. Protein recognition of these structurally-diverse compounds depends upon substitution pattern, overall molecular shape, and on internal mobility. In this review particular attention is paid to the dynamic aspects of heparin/heparan sulfate conformation. The iduronate residue possesses an unusually flexible pyranose ring conformation. This extra source of internal mobility creates special problems in rationalization of experimental data for these compounds. We present herein the solution-state NMR parameters, fiber diffraction data, crystallographic data, and molecular modeling methods employed in the investigation of heparin and heparan sulfate. Heparin is a useful model compound for the sulfated, protein-binding regions of heparan sulfate. The literature contains a number of solution and solid-state studies of heparin oligo- and polysaccharides for both isolated heparin species and those bound to protein receptors. These studies indicate a diversity of iduronate ring conformations, but a limited range of glycosidic linkage geometries in the repeating disaccharides. In this sense, heparin exhibits a well-defined overall shape within which iduronate ring forms can freely interconvert. Recent work suggests that computational modeling could potentially identify heparin binding sites on protein surfaces.

Animals↗
PubMed2000

A collaborative study to establish the 5th International Standard for Unfractionated Heparin.

Twenty-four laboratories participated in a collaborative study to calibrate a replacement for the 4th International Standard for Unfractionated Heparin (82/502). Both candidate materials A and B, gave excellent intra- and inter-laboratory variations (majority of mean %gcv <10%) when assayed against the 4th International Standard. No major differences of potency estimates were found between methods, although the USP method generally gave lower potencies than the other methods and candidate B gave a greater variation between methods than A. Overall, this study showed that the differences between the candidates are marginal. Based on its narrower molecular weight profile, higher specific activity and slightly lower inter-method variation, candidate A, 97/578, was proposed and accepted in October, 1998, by the Expert Committee on Biological Standardisation of the World Health Organisation to be the 5th International Standard for Unfractionated Heparin with an assigned potency of 2031 IU/ampoule.

Blood Coagulation Tests↗
PubMed2000

Characterization of unfractionated heparin: comparison of materials from the last 50 years.

Physicochemical and anticoagulant characteristics of 27 samples from recent batches of commercially produced unfractionated heparin have been determined as part of the process of establishment of the 5th International Standard Unfractionated Heparin. They have been compared with current heparin standards (European Pharmacopoeia, United States Pharmacopoeia, Chinese), with the 4th International Standard Unfractionated Heparin. and with the three predecessor International Standards. The results indicate that the 4th International Standard Unfractionated Heparin, established in 1982, has significantly lower molecular weight and specific activity than recently produced heparin; this is also true of all preceding International Standard Heparins and of the United States Pharmacopoeial standard. The composition of commercial unfractionated heparin may therefore have changed over time; reasons for this are discussed.

Blood Coagulation Tests↗
PubMed1999

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.

Animals↗
PubMed1998

Highly sulfated dermatan sulfates from Ascidians. Structure versus anticoagulant activity of these glycosaminoglycans.

Dermatan sulfates with the same backbone structure [4-alpha-L-IdceA-1-->3-beta-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 alpha-L-iduronic acid residues but differ in the pattern of sulfation of the N-acetyl-beta-D-galactosamine units. Styela plicata and Halocynthia pyriformis have 4-O-sulfated units, but in Ascidian 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 the N-acetyl-beta-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 approximately 10-fold and approximately 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.

Acetylgalactosamine↗
PubMed1998

Oligosaccharide sequence of human breast cancer cell heparan sulfate with high affinity for laminin.

Laminin-1 is a basement membrane glycoprotein implicated in tumor-host adhesion, which involves the cell-binding domain(s) of laminin-1 and tumor cell surface heparan sulfate (HS). The specific tumor cell surface HS oligosaccharide sequences that are necessary for binding to laminin-1 have not been characterized. To identify this laminin-binding oligosaccharide sequence, GlcNSO4-rich oligosaccharides terminating with [3H]2,5-anhydromannitol (AManR) residues were isolated from human breast cancer cell (MCF-7)-derived HS through hydrazinolysis/high pH (4.0) nitrous acid treatment/[3H]NaBH4 reduction. These oligosaccharides were chromatographed on a laminin-1 affinity column. A high affinity dodecasaccharide was isolated and characterized. Disaccharide analysis yielded IdoA(2-SO4) --> AManR(6-SO4) as the only disaccharide upon treatment of this dodecasaccharide with nitrous acid at low pH (1.5). The sequence of laminin-binding high affinity oligosaccharide is therefore [IdoA(2-SO4) --> GlcNSO4(6-SO4)]5[IdoA(2-SO4) --> AManR(6-SO4)]. Low affinity dodecasaccharides composed of [IdoA(2-SO4) --> GlcNSO4(6-SO4)]5, [IdoA(2-SO4) --> GlcNSO4] were also isolated by laminin-1 affinity chromatography. Molecular modeling studies indicate that a heparin-binding peptide sequence corresponding to amino acid residues 3010-3031 (KQNCLSSRASFRGCVRNLRLSR) in the G domain of laminin-1, modeled as a right-handed alpha-helix, carries an array of basic residues well placed to bind to clusters of sulfate groups on the high affinity dodecasaccharide.

Amino Acid Sequence↗
PubMed1998

Antithrombotic activity of a fucosylated chondroitin sulphate from echinoderm: sulphated fucose branches on the polysaccharide account for its antithrombotic action.

The antithrombotic activity of a fucosylated, chondroitin-sulphate-like polysaccharide extracted from the body wall of sea cucumber, and of chemically modified derivatives of the same polysaccharide, have been assessed using a stasis thrombosis model in rabbits. Intravenous administration of the native polysaccharide reduced thrombosis in a dose-dependent manner and, at a dose of 1.5 mg/kg (60 IU/kg) body weight, completely prevented thrombosis after 10 min stasis. Removal of the sulphated fucose branches of the polysaccharide abolished antithrombotic effectiveness. After intravenous injection of an antithrombotic dose of radioactively labelled polysaccharide, a correlation was observed between removal of radioactivity from the plasma and decrease in ex vivo APTT values, demonstrating that antithrombotic effectiveness depends on the level of circulating polysaccharide rather than on an indirect effect of the polysaccharide on the vascular endothelium. Reduction of the glucuronic acid carboxyl groups in the polysaccharide did not affect its in vitro and in vivo activities. Both partial defucosylation and desulphation of the polysaccharide abolished all its anticoagulant or antithrombotic action.

Animals↗
PubMed1998

Further characterization of the binding of human recombinant interleukin 2 to heparin and identification of putative binding sites.

We have previously provided compelling evidence that human recombinant interleukin 2 (IL-2) binds to the sulfated polysaccharides heparin, highly sulfated heparan sulfate and fucoidan. Here we show that IL-2 binding is dependent on heparin chain length, but with fragments as small as 15-mers retaining binding activity. The addition of exogenous heparin has no effect on the in vitro biological activity of IL-2. In addition soluble IL-2 receptor alpha and beta polypeptides do not compete with heparin for the binding of IL-2. IL-2 bound by heparin is still recognized by two IL-2 specific monoclonal antibodies, 3H9 and H2-8, whose epitopes lie in the amino terminal region. Murine IL-2 unlike its human counterpart fails to bind to heparin. Human IL-2 analogs with single amino acid substitutions at positions Lys43, Thr51, and Gln126 analogs no longer bind to heparin. By contrast the Arg38Ala analog retains heparin full heparin binding activity. These experimental findings together with molecular modeling studies suggest two putative heparin binding sites on human IL-2, one involving four basic residues, Lys48, Lys49, Lys54, and His55, and the other being a discontinuous site comprising Lys43, Lys64, Arg81, and Arg83. Neither of these two clusters is completely conserved in murine IL-2. Overall our data suggest that the binding of human IL-2 to heparin and heparan sulfate does not interfere with IL-2/IL-2 receptor interactions. Therefore, binding to glycosaminoglycan may be a mechanism for retaining the cytokine in an active form close to its site of secretion in the tissue, thus favoring a paracrine role for IL-2.

Animals↗