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Biomedical subjects

Eva M Muñoz

Publications and source records attributed to Eva M Muñoz.

6 recordsLinked to original sources
PubMed2005

Enzymatic redesigning of biologically active heparan sulfate.

Heparan sulfate carries a wide range of biological activities, regulating blood coagulation, cell differentiation, and inflammatory responses. The sulfation patterns of the polysaccharide are essential for the biological activities. In this study, we report an enzymatic method for the sulfation of multimilligram amounts of heparan sulfate with specific functions using immobilized sulfotransferases combined with a 3'-phosphoadenosine 5'-phosphosulfate regeneration system. By selecting appropriate enzymatic modification steps, an inactive precursor has been converted to the heparan sulfate having three distinct biological activities, associated with binding to antithrombin, fibroblast growth factor-2, and herpes simplex virus envelope glycoprotein D. Because the recombinant sulfotransferases are expressed in bacteria, and the method uses a low cost sulfo donor, it can be readily utilized to synthesize large quantities of anticoagulant heparin drug or other biologically active heparan sulfates.

Animals↗
PubMed2005

Synthesis of double C-glycoside analogue of sTn.

A sTn double C-glycoside, sTn analogue 2, was synthesized using samarium chemistry developed in our laboratory. Complications in the oxidation reaction affording aldehyde acceptor were overcome by double protection of amide and the use of a room-temperature ionic liquid as solvent. Studies are underway to conjugate the sTn double C-glycoside hapten 2 to KLH carrier protein for biological evaluation as a vaccine.

Carbohydrate Conformation↗
PubMed2005

Kinetic studies on the interactions of heparin and complement proteins using surface plasmon resonance.

Heparin is a naturally occurring polysaccharide known to interact with complement proteins and regulate multiple steps in the complement cascade. Quantitative information, in the form of affinity constants for heparin-complement interactions, is not generally available and there are no reports of a comprehensive analysis using the same interaction method. Such information should improve our understanding of how exogenously administered pharmaceutical heparin and the related endogenous polysaccharide, heparan sulfate, regulate complement activation. The current study provides the first comprehensively analysis of the binding of various complement proteins to heparin using surface plasmon resonance (SPR). Complement proteins C1, C2, C3, C4, C5, C6, C7, C8, C9, C1INH, factor I, factor H, factor B and factor P all bind heparin but exhibit different binding kinetics and dissociation constants (Kd) ranging from 2 to 320 nM. By taking into account these Kd values and the serum concentrations of these complement proteins, the percentage of each binding to exogenously administered heparin was calculated and found to range from 2% to 41%. This study provides essential information required for the rational design of new therapeutic agents capable of regulating the complement activation.

Complement Activation↗
PubMed2005

Carbohydrate-based DNA ligands: sugar-oligoamides as a tool to study carbohydrate-nucleic acid interactions.

Sugar-oligoamides have been designed and synthesized as structurally simple carbohydrate-based ligands to study carbohydrate-DNA interactions. The general design of the ligands 1-3 has been done as to favor the bound conformation of Distamycin-type gamma-linked covalent dimers which is a hairpin conformation. Indeed, NMR analysis of the sugar-oligoamides in the free state has indicated the presence of a percentage of a hairpin conformation in aqueous solution. The DNA binding activity of compounds 1-3 was confirmed by calf thymus DNA (ct-DNA) NMR titration. Interestingly, the binding of the different sugar-oligoamides seems to be modulated by the sugar configuration. Semiquantitative structural information about the DNA ligand complexes has been derived from NMR data. A competition experiment with Netropsin suggested that the sugar-oligoamide 3 bind to DNA in the minor groove. The NMR titrations of 1-3 with poly(dA-dT) and poly(dG-dC) suggested preferential binding to the ATAT sequence. TR-NOE NMR experiments for the sugar-oligoamide 3-ct-DNA complex both in D(2)O and H(2)O have confirmed the complex formation and given information on the conformation of the ligand in the bound state. The data confirmed that the sugar-oligoamide ligand is a hairpin in the bound state. Even more relevant to our goal, structural information on the conformation around the N-glycosidic linkage has been accessed. Thus, the sugar asymmetric centers pointing to the NH-amide and N-methyl rims of the molecule have been characterized.

Amides↗
PubMed2004

Heparin-binding domains in vascular biology.

Heparin is a major anticoagulant with activity mediated primarily through its interaction with antithrombin (AT). Heparan sulfate (HS), structurally related to heparin, binds a wide range of proteins of different functionality, taking part in various physiological and pathological processes. The heparin-AT complex, the most well understood facet of anticoagulation, serves as a prototypical example of the important role of heparin/HS in vascular biology. Extensive studies have identified common structural features in heparin/HS-binding sites of proteins. These include the elucidation of consensus sequences in proteins, patterns of clusters of basic and nonbasic residues, and common spatial arrangements of basic amino acids in the heparin-binding sites. Although these studies have provided valuable information, heparin/HS-binding proteins differ widely in structure. The prediction of heparin/HS-binding proteins from sequence information is not currently possible, and elucidation of protein-binding sites requires the individual study of each glycosaminoglycan-protein complex. Thus, x-ray crystallography and site-directed mutagenesis experiments are among the most powerful tools, providing accurate structural information, facilitating the characterization of heparin-protein complexes. Heparin and structurally related heparan sulfate bind a large number of proteins, taking part in a wide range of biological processes, particularly ones involved in vascular biology. Heparin-binding domains share certain common structural features, but there is no absolute dependency on specific sequences or protein folds.

Amino Acid Sequence↗