Ishan Capila

Heparin–Protein Interactions

Heparin, a sulfated polysaccharide belonging to the family of glycosaminoglycans, has numerous important biological activities, associated with its interaction with diverse proteins. Heparin is widely used as an anticoagulant drug based on its ability to accelerate the rate at which antithrombin inhibits serine proteases in the blood coagulation cascade. Heparin and the structurally related heparan sulfate are complex linear polymers comprised of a mixture of chains of different length, having variable sequences. Heparan sulfate is ubiquitously distributed on the surfaces of animal cells and in the extracellular matrix. It also mediates various physiologic and pathophysiologic processes. Difficulties in evaluating the role of heparin and heparan sulfate in vivo may be partly ascribed to ignorance of the detailed structure and sequence of these polysaccharides. In addition, the understanding of carbohydrate-protein interactions has lagged behind that of the more thoroughly studied protein-protein and protein-nucleic acid interactions. The recent extensive studies on the structural, kinetic, and thermodynamic aspects of the protein binding of heparin and heparan sulfate have led to an improved understanding of heparin-protein interactions. A high degree of specificity could be identified in many of these interactions. An understanding of these interactions at the molecular level is of fundamental importance in the design of new highly specific therapeutic agents. This review focuses on aspects of heparin structure and conformation, which are important for its interactions with proteins. It also describes the interaction of heparin and heparan sulfate with selected families of heparin-binding proteins.

Temporal targeting of tumour cells and neovasculature with a nanoscale delivery system.

In the continuing search for effective treatments for cancer, the emerging model is the combination of traditional chemotherapy with anti-angiogenesis agents that inhibit blood vessel growth. However, the implementation of this strategy has faced two major obstacles. First, the long-term shutdown of tumour blood vessels by the anti-angiogenesis agent can prevent the tumour from receiving a therapeutic concentration of the chemotherapy agent. Second, inhibiting blood supply drives the intra-tumoural accumulation of hypoxia-inducible factor-1α (HIF1-α); overexpression of HIF1-α is correlated with increased tumour invasiveness and resistance to chemotherapy. Here we report the disease-driven engineering of a drug delivery system, a ‘nanocell’, which overcomes these barriers unique to solid tumours. The nanocell comprises a nuclear nanoparticle within an extranuclear pegylated-lipid envelope, and is preferentially taken up by the tumour. The nanocell enables a temporal release of two drugs: the outer envelope first releases an anti-angiogenesis agent, causing a vascular shutdown; the inner nanoparticle, which is trapped inside the tumour, then releases a chemotherapy agent. This focal release within a tumour results in improved therapeutic index with reduced toxicity. The technology can be extended to additional agents, so as to target multiple signalling pathways or distinct tumour compartments, enabling the model of an ‘integrative’ approach in cancer therapy.

Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events

Recently, certain lots of heparin have been associated with an acute, rapid onset of serious side effects indicative of an allergic-type reaction. To identify potential causes for this sudden rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high-resolution analytical techniques. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked β1→3 to a β-N-acetylgalactosamine. The disaccharide unit has an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. Given the nature of this contaminant, traditional screening tests cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be used to determine whether or not heparin lots contain the contaminant reported here.

Annexin V–Heparin Oligosaccharide Complex Suggests Heparan Sulfate–Mediated Assembly on Cell Surfaces

BACKGROUND Annexin V, an abundant anticoagulant protein, has been proposed to exert its effects by self-assembling into highly ordered arrays on phospholipid membranes to form a protective anti-thrombotic shield at the cell surface. The protein exhibits very high-affinity calcium-dependent interactions with acidic phospholipid membranes, as well as specific binding to glycosaminoglycans (GAGs) such as heparin and heparan sulfate, a major component of cell surface proteoglycans. At present, there is no structural information to elucidate this interaction or the role it may play in annexin V function at the cell surface. RESULTS We report the 1.9 A crystal structure of annexin V in complex with heparin-derived tetrasaccharides. This structure represents the first of a heparin oligosaccharide binding to a protein where calcium ions are essential for the interaction. Two distinct GAG binding sites are situated on opposite protein surfaces. Basic residues at each site were identified from the structure and site-directed mutants were prepared. The heparin binding properties of these mutants were measured by surface plasmon resonance. The results confirm the roles of these mutated residues in heparin binding, and the kinetic and thermodynamic data define the functionally distinct character of each distal binding surface. CONCLUSION The annexin V molecule, as it self-assembles into an organized array on the membrane surface, can bind the heparan sulfate components of cell surface proteoglycans. A novel model is presented in which proteoglycan heparan sulfate could assist in the localization of annexin V to the cell surface membrane and/or stabilization of the entire molecular assembly to promote anticoagulation.

Heparin-Protein-Wechselwirkungen

Heparin, ein zu den Glycosaminoglycanen gehorendes sulfatiertes Polysaccharid, zeigt eine Reihe wichtiger biologischer Wirkungen, die aus der Wechselwirkung mit Proteinen resultieren. Da Heparin die Geschwindigkeit erhoht, mit der Antithrombin die Serinproteasen in der Blutgerinnungskaskade inhibiert, findet es breite Anwendung als Antikoagulans (Antiblutgerinnungsmittel). Heparin und das strukturverwandte Heparansulfat sind komplizierte lineare Polymere, die aus Ketten unterschiedlicher Lange und Sequenz bestehen. Heparansulfat, das an den Oberflachen tierischer Zellen und in der extrazellularen Matrix ubiquitar ist, vermittelt ebenfalls eine Reihe physiologischer und pathophysiologischer Prozesse. Die Schwierigkeiten bei der Beurteilung der Rolle von Heparin und Heparansulfat in vivo konnen zum Teil auf die Unkenntnis der genauen Struktur und Sequenz dieser Polysaccharide zuruckgefuhrt werden. Abgesehen davon weis man uber Kohlenhydrat-Protein-Wechselwirkungen viel weniger als uber die eingehend untersuchten Protein-Protein- und Protein-Nucleinsaure-Wechselwirkungen. Die neuere umfangreiche Forschung uber strukturelle, kinetische und thermodynamische Aspekte der Proteinbindung von Heparin und Heparansulfat fuhrte zu neuen Erkenntnissen uber die Heparin-Protein-Wechselwirkungen. Viele dieser Wechselwirkungen sind hochspezifisch, deshalb ist es fur die Entwicklung neuer Therapeutika von grundlegender Bedeutung, sie auf molekularer Ebene zu verstehen. Dieser Aufsatz konzentriert sich auf die strukturellen und konformativen Eigenschaften von Heparin, die fur die Wechselwirkungen mit Proteinen wichtig sind, auserdem wird die Wechselwirkung von Heparin und Heparansulfat mit ausgewahlten Heparin-bindenden Proteingruppen behandelt.

Chondroitinase ABC I from Proteus vulgaris: cloning, recombinant expression and active site identification

GalAGs (galactosaminoglycans) are one subset of the GAG (glycosaminoglycan) family of chemically heterogeneous polysaccharides that are involved in a wide range of biological processes. These complex biomacromolecules are believed to be responsible for the inhibition of nerve regeneration following injury to the central nervous system. The enzymic degradation of GAG chains in damaged nervous tissue by cABC I (chondroitinase ABC I), a broad-specificity lyase that degrades GalAGs, promotes neural recovery. In the present paper, we report the subcloning of cABC I from Proteus vulgaris, and discuss a simple methodology for the recombinant expression and purification of this enzyme. The originally expressed cABC I clone resulted in an enzyme with negligible activity against a variety of GalAG substrates. Sequencing of the cABC I clone revealed four point mutations at issue with the electron-density data of the cABC I crystal structure. Site-directed mutagenesis produced a clone with restored GalAG-degrading function. We have characterized this enzyme biochemically, including an analysis of its substrate specificity. By coupling structural inspections of cABC I and an evaluation of sequence homology against other GAG-degrading lyases, a set of amino acids was chosen for further study. Mutagenesis studies of these residues resulted in the first experimental evidence of cABC Is active site. This work will facilitate the structure-function characterization of biomedically relevant GalAGs and further the development of therapeutics for nerve regeneration.

Interaction of the N-terminal domain of apolipoprotein E4 with heparin.

Apolipoprotein E (apoE) is an important lipid-transport protein in human plasma and brain. It has three common isoforms (apoE2, apoE3, and apoE4). ApoE is a major genetic risk factor in heart disease and in neurodegenerative disease, including Alzheimers disease. The interaction of apoE with heparan sulfate proteoglycans plays an important role in lipoprotein remnant uptake and likely in atherogenesis and Alzheimers disease. Here we report our studies of the interaction of the N-terminal domain of apoE4 (residues 1-191), which contains the major heparin-binding site, with an enzymatically prepared heparin oligosaccharide. Identified by its high affinity for the N-terminal domain of apoE4, this oligosaccharide was determined to be an octasaccharide of the structure DeltaUAp2S(1-->[4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S(1-->](3)4)-alpha-D-GlcNpS6S by nuclear magnetic resonance spectroscopy, capillary electrophoresis, and polyacrylamide gel electrophoresis. Kinetic analysis of the interaction between the N-terminal apoE4 fragment and immobilized heparin by surface plasmon resonance yielded a K(d) of 150 nM. A similar binding constant (K(d) = 140 nM) was observed for the interaction between immobilized N-terminal apoE4 and the octasaccharide. Isothermal titration calorimetry revealed a K(d) of 75 nM for the interaction of the N-terminal apoE fragment and the octasaccharide with a binding stoichiometry of approximately 1:1. Using previous studies and molecular modeling, we propose a binding site for this octasaccharide in a basic residue-rich region of helix 4 of the N-terminal fragment. From the X-ray crystal structure of the N-terminal apoE4, we predicted that binding of the octasaccharide at this site would result in a change in intrinsic fluorescence. This prediction was confirmed experimentally by an observed increase in fluorescence intensity with octasaccharide binding corresponding to a K(d) of approximately 1 microM.

Interaction of heparin with annexin V

The energetics and kinetics of the interaction of heparin with the Ca+2 and phospholipid binding protein annexin V, was examined and the minimum oligosaccharide sequence within heparin that binds annexin V was identified. Affinity chromatography studies confirmed the Ca+2 dependence of this binding interaction. Analysis of the data obtained from surface plasmon resonance afforded a K d of ∼21 nM for the interaction of annexin V with end‐chain immobilized heparin and a K d of ∼49 nM for the interaction with end‐chain immobilized heparan sulfate. Isothermal titration calorimetry showed the minimum annexin V binding oligosaccharide sequence within heparin corresponds to an octasaccharide sequence. The K d of a heparin octasaccharide binding to annexin V was ∼1 μM with a binding stoichiometry of 1:1.

Adjuvanted influenza-H1N1 vaccination reveals lymphoid signatures of age-dependent early responses and of clinical adverse events

Adjuvanted vaccines afford invaluable protection against disease, and the molecular and cellular changes they induce offer direct insight into human immunobiology. Here we show that within 24 h of receiving adjuvanted swine flu vaccine, healthy individuals made expansive, complex molecular and cellular responses that included overt lymphoid as well as myeloid contributions. Unexpectedly, this early response was subtly but significantly different in people older than ∼35 years. Wide-ranging adverse clinical events can seriously confound vaccine adoption, but whether there are immunological correlates of these is unknown. Here we identify a molecular signature of adverse events that was commonly associated with an existing B cell phenotype. Thus immunophenotypic variation among healthy humans may be manifest in complex pathophysiological responses.

Biochemical characterization of the chondroitinase ABC I active site

cABC I (chondroitinase ABC I) from Proteus vulgaris is a GalAG (galactosaminoglycan) depolymerizing lyase that cleaves its substrates at the glycosidic bond via beta-elimination. cABC I cleaves a particularly broad range of GalAG substrates, including CS (chondroitin sulphate), DS (dermatan sulphate) and hyaluronic acid. We recently cloned and recombinantly expressed cABC I in Escherichia coli, and completed a preliminary biochemical characterization of the enzyme. In the present study, we have coupled site-directed mutagenesis of the recombinant cABC I with a structural model of the enzyme-substrate complex in order to investigate in detail the roles of active site amino acids in the catalytic action of the enzyme. The putative catalytic residues His-501, Tyr-508, Arg-560 and Glu-653 were probed systematically via mutagenesis. Assessment of these mutants in kinetic and end-point assays provided direct evidence on the catalytic roles of these active-site residues. The crystal structure of the native enzyme provided a framework for molecular docking of representative CS and DS substrates. This enabled us to construct recombinant enzyme-substrate structural complexes. These studies together provided structural insights into the effects of the mutations on the catalytic mechanism of cABC I and the differences in its processing of CS and DS substrates. All His-501 mutants were essentially inactive and thereby implicating this amino acid to play the critical role of proton abstraction during catalysis. The kinetic data for Glu-653 mutants indicated that it is involved in a hydrogen bonding network in the active site. The proximity of Tyr-508 to the glycosidic oxygen of the substrate at the site of cleavage suggested its potential role in protonating the leaving group. Arg-560 was proximal to the uronic acid C-5 proton, suggesting its possible role in the stabilization of the carbanion intermediate formed during catalysis.