1,721,135 research outputs found
Structural studies of DNP-binding immunoglobulins
Full text linkThe importance of tryptophan in the combining sites of anti-DNP antibodies is evaluated from a series of model studies. The thermodynamic parameters characterizing the formation of a DNP/tryptophan complex are determined. The contribution of this interaction to the affinity and specificity of anti-DNP antibodies is discussed. The accuracy of antibody combining site structures generated by model-building is examined, using available crystallographic data. The average error of alpha-carbon atom positions is estimated to be 1-2 Å. The binding of nitrophenyl compounds to the VL dimer of the DNP-binding mouse myeloma protein 315 is investigated by 1H n.m.r. It is concluded that any corformational changes are small, and that the physical basis for the DNP-binding specificity of the VL dimer is the conservation of structural features which are important in determining the specificity of the intact protein 315. A model of the combining site of the VL dimer is described. The proposed site is a large cavity bounded by the aromatic side-chains of the Trp-93L and Tyr-34L residues. The structure is able to explain the large upfield chemical shift changes of the ligand resonances observed on binding. The kinetic parameters and structural extent of the pH-dependent conformational change of the VL dimer are investigated by fluorescence and 1H n.m.r. The transition does not obey a reversible one-step mechanism, and is limited in extent. The involvement of two tyrosine residues in the hypervariable regions of protein 315 is investigated by specific nitration. Nitration of Tyr-34L has no effect on the affinity of protein 315 or of the VL dimer for several ligands. It is concluded that no hydrogen bond is formed between the phenolic group of Tyr-34L and the 2-nitro group of the ligand. From measurements of the perturbation of the visible absorption spectrum of protein 315 nitrated at Tyr-33H, it is concluded that this residue is in proximity to the side-chains, but not the nitrophenyl rings, of bound ligands. Several aspects of the interaction of a homogeneous mouse anti-DNP antibody, protein A3, with nitrophenyl and similar ligands are described. The findings are discussed in relation to the heterogeneity and cross-reactivity of antisera
Directing stem cell differentiation with antibodies.
Full text linkStem cells are highly specialized cells endowed with unlimited replicative self-renewing potential. These cells are capable of either limited multipotent (adult stem cells, ASC) or unlimited pluripotent (embryonic stem cells, ESC) differentiation to somatic cell lineages. Control of this differentiation process holds great promise in areas such as tissue regeneration and the treatment of chronic degenerative diseases. The medical exploitation of this phenomenon is carried out using stem cells derived from different sources (e.g., ASC/ESC), developmental stages (e.g., mesenchymal cells), cellular reprogramming (e.g., induced pluripotent stem cells), or even nonstem cells in a process known as transdifferentiation (Fig. 1). Despite this wide diversity, all of the differentiation steps that drive these cells to the desired somatic cell lineage rely on the directed manipulation of endogenous pathways. This process is achieved primarily by direct ligand signaling to cell-surface receptors, or use of distinct combinations of transcription factors derived from these signals. Now in PNAS, Melidoni et al. (1), building upon earlier reports (2, 3), raise the exciting possibility of using combinatorial library-derived antibodies intimately linked to stem-cell phenotype as a powerful tool in controlling these differentiation events
Identification of high-mannose and multiantennary complex-type N-linked glycans containing α-galactose epitopes from Nurse shark IgM heavy chain
No full textMALDI-TOF mass spectrometry, negative ion nano-electrospray MS/MS and exoglycosidase digestion were used to identify 36 N-linked glycans from 19S IgM heavy chain derived from the nurse shark (Ginglymostoma cirratum). The major glycan was the high-mannose compound, Man6GlcNAc2 accompanied by small amounts of Man5GlcNAc2, Man 7GlcNAc2 and Man8GlcNAc2. Bi- and tri-antennary (isomer with a branched 3-antenna) complex-type glycans were also abundant, most contained a bisecting GlcNAc residue (β1→4-linked to the central mannose) and with varying numbers of α-galactose residues capping the antennae. Small amounts of monosialylated glycans were also found. This appears to be the first comprehensive study of glycosylation in this species of animal. The glycosylation pattern has implications for the mechanism of activation of the complement system by nurse shark IgM.</p
Direct deprotected glycosyl-asparagine ligation
No full textA simple and efficient synthesis of N-linked glycoamino acids and glycopeptides from deprotected sugars using the Staudinger reactio
Glycosylation and the immune system
No full textAlmost all of the key molecules involved in the innate and adaptive immune response are glycoproteins. In the cellular immune system, specific glycoforms are involved in the folding, quality control, and assembly of peptide-loaded major histocompatibility complex (MHC) antigens and the T cell receptor complex. Although some glycopeptide antigens are presented by the MHC, the generation of peptide antigens from glycoproteins may require enzymatic removal of sugars before the protein can be cleaved. Oligosaccharides attached to glycoproteins in the junction between T cells and antigen-presenting cells help to orient binding faces, provide protease protection, and restrict nonspecific lateral protein-protein interactions. In the humoral immune system, all of the immunoglobulins and most of the complement components are glycosylated. Although a major function for sugars is to contribute to the stability of the proteins to which they are attached, specific glycoforms are involved in recognition events. For example, in rheumatoid arthritis, an autoimmune disease, agalactosylated glycoforms of aggregated immunoglobulin G may induce association with the mannose-binding lectin and contribute to the pathology
Emerging principles for the therapeutic exploitation of glycosylation.
No full textGlycosylation plays a key role in a wide range of biological processes. Specific modification to a glycan's structure can directly modulate its biological function. Glycans are not only essential to glycoprotein folding, cellular homeostasis, and immune regulation but are involved in multiple disease conditions. An increased molecular and structural understanding of the mechanistic role that glycans play in these pathological processes has driven the development of therapeutics and illuminated novel targets for drug design. This knowledge has enabled the treatment of metabolic disorders and the development of antivirals and shaped cancer and viral vaccine strategies. Furthermore, an understanding of glycosylation has led to the development of specific drug glycoforms, for example, monoclonal antibodies, with enhanced potency
Chromophore structure in the photocycle of the cyanobacterial phytochrome Cph1
No full textThe chromophore conformations of the red and far red light induced product states “Pfr” and “Pr” of the N-terminal photoreceptor domain Cph1-N515 from Synechocystis 6803 have been investigated by NMR spectroscopy, using specific 13C isotope substitutions in the chromophore. 13C-NMR spectroscopy in the Pfr and Pr states indicated reversible chemical shift differences predominantly of the C4 carbon in ring A of the phycocyanobilin chromophore, in contrast to differences of C15 and C5, which were much less pronounced. Ab initio calculations of the isotropic shielding and optical transition energies identify a region for C4-C5-C6-N2 dihedral angle changes where deshielding of C4 is correlated with red-shifted absorption. These could occur during thermal reactions on microsecond and millisecond timescales after excitation of Pr which are associated with red-shifted absorption. A reaction pathway involving a hula-twist at C5 could satisfy the observed NMR and visible absorption changes. Alternatively, C15 Z-E photoisomerization, although expected to lead to a small change of the chemical shift of C15, in addition to changes of the C4-C5-C6-N2 dihedral angle could be consistent with visible absorption changes and the chemical shift difference at C4. NMR spectroscopy of a 13C-labeled chromopeptide provided indication for broadening due to conformational exchange reactions in the intact photoreceptor domain, which is more pronounced for the C- and D-rings of the chromophore. This broadening was also evident in the F2 hydrogen dimension from heteronuclear 1H-13C HSQC spectroscopy, which did not detect resonances for the 13C5-H, 13C10-H, and 13C15-H hydrogen atoms whereas strong signals were detected for the 13C-labeled chromopeptide. The most pronounced 13C-chemical shift difference between chromopeptide and intact receptor domain was that of the 13C4-resonance, which could be consistent with an increased conformational energy of the C4-C5-C6-N2 dihedral angle in the intact protein in the Pr state. Nuclear Overhauser effect spectroscopy experiments of the 13C-labeled chromopeptide, where chromophore-protein interactions are expected to be reduced, were consistent with a ZZZssa conformation, which has also been found for the biliverdin chromophore in the x-ray structure of a fragment of Deinococcus radiodurans bacteriophytochrome in the Pr form
Structural studies on antibodies
Full text linkData from ¹H nuclear magnetic resonance studies on the Fv fragment of protein 315, a Dnp-binding BALB/c mouse lgA(λ2) myeloma protein, have been used to refine a predicted structure of the combining site of the protein. The Dnp-binding subsite in the modified structure is composed of the side chains of three aromatic amino acids Trp 93L, Tyr 34L and 34H. A fourth aromatic amino acid residue is close to the side chain-NH-CH 2 -group, this is Tyr 33 H - The antibody-hapten binding is a simple encounter process, which causes no extensive conformation change in the Fv fragment. A method for paramagnetic structural studies has been devised using Dnp derivatives with cllgophosphate side chains, which create a specific manganese binding site on the Fv fragment-hapten complex. The distances from the bound metal ion to the imidazole side chains of two of the three hlstldine residues of protein 315 have been determined. ¹H nuclear magnetic resonance has been used to study the histidine residues of the Fv fragment of protein 315. It has been shown that one of the three histidine residues (102H) is close to the combining site, but that this residue does not participate directly in binding haptens. 31 P nuclear magnetic resonance studies have shown the presence of a positively charged amino acid side chain near the entrance of the combining site of the Fv fragment. This residue has been identified as Arg 95L. The mode of binding of trini trophenyl derivatives to the Fv fragment has been studied by 1H nuclear magnetic resonance. It is concluded that these haptens, when bound to the Kv fragment, make contacts with the same amino acid side chains as Dnp derivatives
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