María J. Hernáiz

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.

Green Solvents in Carbohydrate Chemistry: From Raw Materials to Fine Chemicals

Fine Chemicals Angeles Farrań,† Chao Cai,‡ Manuel Sandoval, Yongmei Xu, Jian Liu, María J. Hernaíz,* and Robert J. Linhardt* †Departamento de Química Orgańica y Bio-Orgańica, Facultad de Ciencias, Universidad Nacional de Educacioń a Distancia, Paseo Senda del Rey 4, 28040 Madrid, Spain ‡Key Laboratory of Marine Drugs of Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China Escuela de Química, Universidad Nacional of Costa Rica, Post Office Box 86, 3000 Heredia, Costa Rica Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, United States Center for Biotechnology & Interdisciplinary Studies and Department of Chemistry & Chemical Biology, Rensselaer Polytechnic Institute, Biotechnology Center 4005, Troy, New York 12180, United States Departamento de Química Orgańica y Farmaceútica, Facultad de Farmacia, Universidad Complutense de Madrid, Pz/Ramoń y Cajal s/n, 28040 Madrid, Spain

Applied Biotransformations in Green Solvents

The definite interest in implementing sustainable industrial technologies has impelled the use of biocatalysts (enzymes or cells), leading to high chemo-, regio- and stereoselectivities under mild conditions. As usual substrates are not soluble in water, the employ of organic solvents is mandatory. We will focus on different attempts to combine the valuable properties of green solvents with the advantages of using biocatalysts for developing cleaner synthetic processes.

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.

Modification of purified lipases from Candida rugosa with polyethylene glycol: a systematic study

Abstract Semipurified lipase and pure isoenzymes [lipase A (CRLA) and lipase B (CRLB)] of Candida rugosa were chemically modified using pNPCF-PEG. The modified enzymes can be stored at 4°C for 6 months without losing activity. The chemically modified lipases were more stable than the native enzymes and were stored at 50°C in isooctane. The chemically modified enzymes were used in i) hydrolysis of triolein; ii) esterification of oleic acid; and iii) enantioselective esterification of ( r,s ) ibuprofen. Lipase activity was less than esterase activity as a result of the chemical modification of the lipase. The influence of purification and chemical modification degrees in the i) storage stability; ii) catalytic activity; iii) stability with respect to isooctane; and iv) stereoselectivity is discussed. We modulated the hydrophobicity of the biocatalyst by changing the modification degree of the lipase. This effect allowed us to select the optimum biocatalyst to achieve the maximum yield for esterification in different organic solvents. Only the purification of C. rugosa lipase increased the activity and enantioselectivity. Purification and chemical modification did not change the enantiopreference of the lipase.

Covalent immobilization of pure lipases A and B from Candida rugosa

Abstract Covalent immobilization on agarose and SiO2 of pure lipase A (LIP.A) and B (LIP.B) from C. rugosa is described. The results obtained are compared with the data obtained in the covalent binding of commercial lipase (CL) to the same supports. The immobilization of LIP.A and LIP.B on agarose affords more stable biocatalysts than on SiO2. Kinetic studies of all these lipases and derivatives in the hydrolysis of (R)-(+) and (S)-(−) methyl 2-chloropropionates were performed and their enantiomeric ratios ( E = ( V max K m ) fast ( V max K m ) slow ) calculated. The results show that (i) purification of the commercial lipase increased the E value 2.5-fold; (ii) both isoenzymes have similar E values, and (iii) in general, the E value increases with the immobilization process.

Efficient and selective enzymatic synthesis of N-acetyl-lactosamine in ionic liquid: a rational explanation

Room temperature ionic liquids (ILs) can affect enzyme activity in some enzyme-catalyzed reactions, however the effects of these cosolvents on the enzymes are not clearly understood. Using β-galactosidase from Thermus thermophilus HB27 (TTP0042), we found an important change from the classical regioselectivity of the transglycosylation reaction with this enzyme. The enzyme increases N-acetyl-D-lactosamine synthesis (Galβ[1→4]GlcNAc) when RTILs are used instead of the traditional self-condensated products. To understand the possible effect of these liquids on the synthetic behavior of the enzyme, we performed a molecular interaction study by surface plasmon resonance. The KD value obtained for this interaction could mean that ILs bind to β-galactosidase through non specific interactions characterized by very fast kinetics and millimolar affinity. Then, several reactions were performed, increasing the concentration of the IL. As a result, a dependence on the ILs concentration was found for transglycosylation products. We hypothesize that ILs might induce conformational changes in the enzyme, which would modify the enzymatic activity and regioselectivity. These structural modifications were confirmed in the secondary and tertiary structures of the protein by circular dichroism and fluorescence studies, respectively. Molecular modeling confirms this hypothesis and shows that the enzyme becomes more flexible in an IL–water mixture and that it allows stabilization of the GlcNAc molecule in the active centre of the enzyme, in order to develop a new product according to the original regioselectivity of the reaction.

Hydrolysis of (R,S)2-aryl propionic esters by pure lipase B from Candida cylindracea

Abstract Purified lipase B from Candida cylindracea (LB) has been obtained in large amounts. LB exhibits greater esterase and lipase activities than commercial lipase. The presence of divalent and/or monovalent cations increases the lipase activity with respect to the absence of external cations, using olive oil as substrate. LB is more active than commercial and semipurified lipases in the hydrolysis of ( R,S )2-arylpropionic ethyl esters. The presence of Na(I) or Ca(II) diminishes the enzymatic activity in the hydrolysis of these esters compared to that obtained in the absence of the external ions. LB is stereospecific in the hydrolysis of S (+)2-arylpropionate.

Solvents derived from glycerol modify classical regioselectivity in the enzymatic synthesis of disaccharides with Biolacta β-galactosidase

Green solvents made from glycerol change the classical regioselectivity of Biolacta No 5 β-galactosidase, from β(1→4) to β(1→6) linkages when a 2 M concentration was used. In order to explain these results, the non-proteic compounds present in the Biolacta preparation were separated by precipitation with ammonium sulfate and the remaining protein extract was used to set reactions with appropriate organic solvents to find that the regioselectivity towards the β(1→6) isomer is retained. According to proteomic analysis, a 98% homology between Streptococcus pneumoniae and Biolacta β-galactosidase preparation was found. With these data, molecular modelling was done which predicts a tridimensional interaction in the enzyme active site with the donor (GlcNAc) and the water-solvent mixture which explains this phenomenon.

Versatile strategy for the synthesis of biotin-labelled glycans, their immobilization to establish a bioactive surface and interaction studies with a lectin on a biochip.

The emerging role of glycans as versatile biochemical signals in diverse aspects of cellular sociology calls for establishment of sensitive methods to monitor carbohydrate recognition by receptors such as lectins. Most of these techniques involve the immobilization of one of the binding partners on a surface, e.g. atomic force microscopy, glycan array and Surface Plasmon Resonance (SPR), hereby simulating cell surface presentation. Here, we report the synthesis of fluorescent glycoconjugates, with a functionalization strategy which avoids the frequently occurring ring opening at the reducing end for further immobilization on a surface or derivatization with biotin. In order to improve the versatility of these derivatized glycans for biological studies, a new approach for the synthesis of biotinylated and fluorescent glycans has also been realized. Finally, to illustrate their usefulness the neoglycoconjugates were immobilized on different surfaces, and the interaction analysis with a model lectin, the toxin from mistletoe, proved them to act as potent ligands, underscoring the merit of the presented synthetic approach.