Q95163715
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We have designed, synthesized, and characterized peptides containing four repeats of the sequences VAALEKE (peptide E) or VAALKEK (peptide K). While the peptides alone adopt in aqueous solutions a random coil conformation, their equimolar mixture forms heterodimeric coiled coils as confirmed by CD spectroscopy. 5-Azidopentanoic acid was connected to the N-terminus of peptide E via a short poly(ethylene glycol) spacer. The terminal azide group enabled conjugation of the peptide with a synthetic drug carrier based on the N-(2-hydroxypropyl)methacrylamide copolymer containing propargyl groups using "click" chemistry. When incorporated into the polymer drug carrier, peptide E formed a stable noncovalent complex with peptide K belonging to a recombinant single-chain fragment (scFv) of the M75 antibody. The complex thereby mediates a noncovalent linkage between the polymer drug carrier and the protein. The recombinant scFv antibody fragment was selected as a targeting ligand against carbonic anhydrase IX-a marker overexpressed by tumor cells of various human carcinomas. The antigen binding affinity of the polymer-scFv complex was confirmed by ELISA. This approach offers a well-defined, specific, and nondestructive universal method for the preparation of protein (antibody)-targeted polymer drug and gene carriers designed for cell-specific delivery.
- MeSH
- akrylamidy chemie MeSH
- antigeny nádorové imunologie metabolismus MeSH
- bakteriální transformace MeSH
- cirkulární dichroismus MeSH
- click chemie metody MeSH
- dimerizace MeSH
- ELISA MeSH
- Escherichia coli MeSH
- imunokonjugáty chemie imunologie farmakologie MeSH
- karboanhydrasy imunologie metabolismus MeSH
- karcinom farmakoterapie enzymologie imunologie patologie MeSH
- klonování DNA MeSH
- lidé MeSH
- molekulární konformace MeSH
- monoklonální protilátky chemie genetika imunologie MeSH
- nádorové biomarkery imunologie metabolismus MeSH
- nosiče léků chemická syntéza farmakologie MeSH
- oligopeptidy chemická syntéza imunologie farmakologie MeSH
- plazmidy MeSH
- polyethylenglykoly chemie MeSH
- rekombinantní proteiny chemie genetika imunologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Galectin-4, a member of the tandem-repeat subfamily of galectins, participates in cell-membrane interactions and plays an important role in cell adhesion and modulation of immunity and malignity. The oligosaccharide specificity of the mouse galectin-4 carbohydrate-recognition domains (CRDs) has been reported previously. In this work, the structure and binding properties of the N-terminal domain CRD1 were further investigated and the crystal structure of CRD1 in complex with lactose was determined at 2.1 Å resolution. The lactose-binding affinity was characterized by fluorescence measurements and two lactose-binding sites were identified: a high-affinity site with a K(d) value in the micromolar range (K(d1) = 600 ± 70 µM) and a low-affinity site with K(d2) = 28 ± 10 mM.
- MeSH
- galektin 4 chemie metabolismus MeSH
- interakční proteinové domény a motivy MeSH
- krystalografie rentgenová MeSH
- laktosa chemie metabolismus MeSH
- ligandy MeSH
- molekulární modely MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Galectin-4 is thought to play a role in the process of tumour conversion of cells of the alimentary tract and the breast tissue; however, its exact function remains unknown. With the aim of elucidating the structural basis of mouse galectin-4 (mGal-4) binding specificity, we have undertaken X-ray analysis of the N-terminal domain, CRD1, of mGal-4 in complex with lactose (the basic building block of known galectin-4 carbohydrate ligands). Crystals of CRD1 in complex with lactose were obtained using vapour-diffusion techniques. The crystals belong to tetragonal space group P42(1)2 with unit-cell parameters a = 91.1, b = 91.16, c = 57.10 A and preliminary X-ray diffraction data were collected to 3.2 A resolution. An optimized crystallization procedure and cryocooling protocol allowed us to extend resolution to 2.1 A. Structure refinement is currently under way; the initial electron-density maps clearly show non-protein electron density in the vicinity of the carbohydrate binding site, indicating the presence of one lactose molecule. The structure will help to improve understanding of the binding specificity and function of the potential colon cancer marker galectin-4.
- MeSH
- aminokyselinové motivy MeSH
- difrakce rentgenového záření MeSH
- financování organizované MeSH
- galektin 4 chemie metabolismus MeSH
- krystalizace MeSH
- laktosa chemie metabolismus MeSH
- ligandy MeSH
- myši MeSH
- nádorové biomarkery chemie metabolismus MeSH
- nádory tračníku chemie metabolismus MeSH
- peptidové fragmenty chemie metabolismus MeSH
- terciární struktura proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
Specific antibodies interfere with the function of human tumor-associated carbonic anhydrase IX (CA IX), and show potential as tools for anticancer interventions. In this work, a correlation between structural elements and thermodynamic parameters of the association of antibody fragment Fab M75 to a peptide corresponding to its epitope in the proteoglycan-like domain of CA IX, is presented. Comparisons of the crystal structures of free Fab M75 and its complex with the epitope peptide reveal major readjustments of CDR-H1 and CDR-H3. In contrast, the overall conformations and positions of CDR-H2 and CDR-L2 remain unaltered, and their positively charged residues may thus present a fixed frame for epitope recognition. Adoption of the altered CDR-H3 conformation in the structure of the complex is accompanied by an apparent local stabilization. Analysis of domain mobility with translation-libration-screw (TLS) method shows that librations of the entire heavy chain variable domain (V(H)) decrease and reorient in the complex, which correlates well with participation of the heavy chain in ligand binding. Isothermal titration microcalorimetry (ITC) experiments revealed a highly unfavorable entropy term, which can be attributed mainly to the decrease in the degrees of freedom of the system, the loss of conformational freedom of peptide and partially to a local stabilization of CDR-H3. Moreover, it was observed that one proton is transferred from the environment to the protein-ligand complex upon binding. Molecular dynamics simulations followed by molecular mechanics/generalized Born surface area (MM-GBSA) calculations of the ligand (epitope peptide) binding energy yielded energy values that were in agreement with the ITC measurements and indicated that the charged residues play crucial role in the epitope binding. Theoretical arguments presented in this work indicate that two adjacent arginine residues (ArgH50 and ArgH52) are responsible for the observed proton transfer. 2007 Wiley-Liss, Inc.
- MeSH
- antigeny nádorové chemie imunologie MeSH
- epitopy chemie imunologie MeSH
- financování organizované MeSH
- imunoglobuliny - Fab fragmenty chemie MeSH
- izoenzymy chemie imunologie MeSH
- kalorimetrie MeSH
- karboanhydrasy chemie imunologie MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- monoklonální protilátky chemie MeSH
- nádorové buněčné linie MeSH
- počítačová simulace MeSH
- sekvence aminokyselin MeSH
- termodynamika MeSH
- vazebná místa protilátek MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- srovnávací studie MeSH
The monoclonal antibodies 1696 and F11.2.32 strongly inhibit the activity of wild-type HIV-1 protease (PR) by binding to epitopes at the enzyme N-terminus (residues 1-6) and flap residues 36-46, respectively. Here we demonstrate that these antibodies are also potent inhibitors of PR variants resistant to active-site inhibitors used as anti-AIDS drugs. Our in vitro experiments revealed that the inhibitory potency of single-chain fragments (scFv) of these antibodies is not significantly affected by the presence of mutations in PR; inhibition constants for drug-resistant protease variants are 5-11 nM and 13-169 nM for scFv1696 and for scFvF11.2.32, respectively. Tethered dimer of HIV-1 PR variant proved to be a model protease variant resistant to dissociative inhibition by 1696, and, strikingly, it also displayed resistance to inhibition by F11.2.32 suggesting that dimer dissociation also plays a role in the inhibitory action of F11.2.32.
- MeSH
- dimerizace MeSH
- financování organizované MeSH
- genetická variace MeSH
- HIV infekce farmakoterapie virologie MeSH
- HIV-1 enzymologie genetika účinky léků MeSH
- HIV-proteasa genetika imunologie účinky léků MeSH
- imunoglobuliny - fragmenty farmakologie imunologie MeSH
- inhibitory HIV-proteasy farmakologie MeSH
- lidé MeSH
- molekulární modely MeSH
- monoklonální protilátky farmakologie imunologie MeSH
- mutace MeSH
- rekombinantní proteiny farmakologie imunologie MeSH
- virová léková rezistence genetika MeSH
- vysoce aktivní antiretrovirová terapie MeSH
- Check Tag
- lidé MeSH
Peptidomimetic inhibitors of human immunodeficiency virus-1 protease are successful lead substances for the development of virostatic drugs against HIV as the causative agent of acquired immunodeficiency syndrome (AIDS). The hydroxyethylamine isostere of the proteolytic cleavage intermediate provides a suitable replacement for the peptide bond. A series of acyclic pseudopeptide inhibitors with the hydroxyethylamine isostere varying in chiral carbon configuration and P'2 residue type were structurally analysed by single-crystal X-ray crystallography. The compounds inhibit HIV protease with subnanomolar inhibition constants and block viral replication in tissue cultures. Here, the structure of such a complex with the R configuration of the isosteric group (PDB code 1zsf) is presented together with newly available synchrotron data for a complex with the S stereoisomer of the inhibitor (PDB code 1zsr). Comparison of the structure and binding with other complexes of HIV-1 protease and similar inhibitors contributes to the understanding of how these molecules bind to the wild-type form of this enzyme. The hydroxy group of the R stereoisomer interacts with one of the catalytic aspartic acids by a short hydrogen bond with rather extreme geometry. The change of configuration of the chiral carbon bearing the hydroxyl from S to R does not influence the inhibition efficiency in this case.
- MeSH
- ethanolaminy chemie MeSH
- financování organizované MeSH
- HIV-1 enzymologie MeSH
- HIV-proteasa chemie metabolismus MeSH
- inhibitory HIV-proteasy chemie MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- oligopeptidy chemie MeSH
- stereoizomerie MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- vodíková vazba MeSH
Galectin-4 and its homologue galectin-6 are members of the tandem-repeat subfamily of monomer divalent galectins. Expression of mouse galectin-4 and galectin-6 by RT-PCR using primers designed to distinguish both galectin transcripts indicates that both are expressed in the small intestine, colon, liver, kidney, spleen and heart and P19X1 cells while only galectin-4 is expressed in BW-5147 and 3T3 cell lines. In situ hybridization confirmed the presence of galectin-4/-6 transcripts in the liver and small intestine. Galectin-4 is expressed in spermatozoons and oocytes and its expression during early mouse emryogenesis appears in 8-cell embryos and remains in later stages, as tested by RT-PCR. To study the role of carbohydrate recognition domains (CRDs) in oligosaccharide binding and epitope recognition, we cloned mouse full-length galectin-4 and galectin-6 cDNA and constructed bacterial expression vectors producing histidin-tagged recombinant galectin-4 and its truncated CRD1 and CRD2 forms. Oligosaccharide binding profile for all recombinant forms was assessed using Glycan Array available through the Consortium for Functional Glycomics. Acquired data indicate that mGalectin-4 binds to alpha-GalNAc and alpha-Gal A and B type structures with or without fucose. While the CRD2 domain has a high specificity and affinity for A type-2 alpha-GalNAc structures, the CRD1 domain has a broader specificity in correlation to the total binding profile. These data suggest that CRD2 might be the dominant binding domain of mouse galectin-4. Mapping of epitopes reactive for biotinylated his-tagged CRD1, CRD2 and mGalectin-4 performed on mouse cryosections showed that all three forms bind to alveolar macrophages, macrophages of red pulp of the spleen and proximal tubuli of the kidney and this binding was inhibited by 5 mM lactose. Interestingly, mGalectin-4, but not CRD forms, binds to the suprabasal layer of squamous epithelium of the tongue, suggesting that the link region also plays an important role in ligand recognition.
- MeSH
- buňky 3T3 MeSH
- epitopy chemie metabolismus MeSH
- financování organizované MeSH
- galektin 4 genetika chemie MeSH
- galektiny genetika chemie MeSH
- hybridizace in situ metody MeSH
- imunohistochemie MeSH
- komplementární DNA genetika MeSH
- molekulární sekvence - údaje MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- oligosacharidy chemie metabolismus MeSH
- rekombinantní proteiny biosyntéza chemie MeSH
- sacharidové sekvence MeSH
- stanovení celkové genové exprese MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- MeSH
- aspartátové endopeptidasy genetika chemie imunologie MeSH
- finanční podpora výzkumu jako téma MeSH
- HIV protilátky chemie imunologie MeSH
- HIV-proteasa imunologie MeSH
- lidé MeSH
- monoklonální protilátky chemie MeSH
- peptidy chemie imunologie MeSH
- sekvence aminokyselin MeSH
- zkřížené reakce MeSH
- Check Tag
- lidé MeSH
