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An isotope-edited NMR study of the peptide hormone bradykinin (RPPGFSPFR) bound to the Fab fragment of a monoclonal antibody against bradykinin (MBK3) is reported. MBK3 was previously shown to provide a binding site model of the B2 bradykinin receptor [Haasemann, M., Buschko, J., Faussner, A., Roscher, A. A., Hoebeke, J., Burch, R. M. & Muller-Esterl, W. (1991) Anti-idiotypic antibodies bearing the internal image of a bradykinin epitope, J. Immunol. 147, 3882-3892]. Bradykinin was obtained in a uniformly 15N-labelled form using recombinant expression of a fusion protein consisting of the glutathione-binding domain of glutathione S-transferase fused to residues 354-375 of the high-molecular-mass kininogen from which bradykinin was released by proteolytic digestion with its natural protease plasma kallikrein. Bradykinin forms a complex with the Fab fragment of MBK3 which exchanges slowly on the NMR time scale. The 15N and 1H resonances of the tightly bound residues of bradykinin show appreciable changes in chemical shift with respect to the free form, while the 15N and 1H linewidths indicate that the hydrodynamic behaviour of bound bradykinin is dominated by the high-molecular-mass Fab fragment. The NMR data indicate that essentially the entire nonapeptide is involved in binding. The kinetics of the ligand-exchange process, together with resonance assignments obtained via exchange spectroscopy. indicate that bradykinin binds to MBK3 only in the all-trans conformation at all three Xaa-Pro amide bonds. NH-NH NOE connectivities suggest that bradykinin is bound in an extended conformation. The spectroscopic data obtained from this study are compared to recently proposed computational models of the conformation of bradykinin bound to the B2 receptor.

NMR spectroscopy has been used to obtain structural information on the bioactive conformation of the nonapeptide hormone bradykinin (Arg-Pro-Pro-Gly-Ser-Pro-Phe-Arg, BK) bound to the Fab-fragment of an antibody that mimics the hormone binding site of the natural bradykinin B2-receptor. Using 15N or 15N,13C, 60% 2H isotope labelled bradykinin, complete 1H, 13C and 15N assignments for bradykinin bound to the Fab-fragment have been obtained. Preliminary interpretation of 15N edited NOE spectra indicates that the conformation of bradykinin bound to the model receptor differs substantially from previous computer models of the bioactive conformation of bradykinin.

Many of the physiological functions of bradykinin are mediated via the B2 receptor. Little is known about binding sites for bradykinin on the receptor. Therefore, antisera against peptides derived from the putative extracellular domains of the B2 receptor were raised. The antibodies strongly reacted with their corresponding antigens and cross-reacted both with the denatured and the native B2 receptor. Affinity-purified antibodies to the various extracellular domains were used to probe the contact sites between the receptor and its agonist, bradykinin or its antagonist HOE140. Antibodies to extracellular domain 3 (second loop) efficiently interfered, in a concentration-dependent manner, with agonist and antagonist binding and vice versa. Antibodies to extracellular domain 4 (third loop) blocked binding of the agonist but not of the antagonist, whereas antibodies to extracellular domains 1 and 2 or to intracellular domains failed to block ligand binding. Antibodies to ectodomain 3 competed with agonistic anti-idiotypic antibodies for B2 receptor binding. Further, affinity-purified antibodies to the amino-terminal portion of extracellular domain 3 transiently increased intracellular free Ca2+ concentration and thus are agonists. The Ca2+ signal was specifically blocked by the B2 antagonist HOE140. By contrast, antibodies to the carboxyl-terminal segment of extracellular domain 4 failed to trigger Ca2+ release. The specific effects of antibodies to the amino-terminal portion of extracellular domain 3 suggest that this portion of the B2 receptor may be involved in ligand binding and in agonist function.

Based on the amino acid sequence deduced from the recently cloned human beta 3-adrenergic receptor (hu beta 3AR) gene, polyclonal antibodies were prepared against synthetic peptides, corresponding to regions of hu beta 3AR presumed to be exposed at the outer or the inner side of the membrane on the basis of the putative three-dimensional structure of the previously characterized beta 1 and beta 2 adrenergic receptors. Affinity-purified antibodies directed against N-terminal, extracellular or intracellular loops and C-terminal peptides reacted specifically with the hu beta 3AR and not with either the human beta 1 or beta 2 adrenergic receptor. Using these antibodies, it was demonstrated that the receptor is present at the surface of Chinese Hamster Ovary (CHO) cells transfected with the hu beta 3AR gene; in addition, the presence of the receptor protein was established in a human tissue (gall bladder). Immuno-affinity chromatography of solubilized CHO hu beta 3AR-containing cell membranes allowed the isolation of hu beta 3AR protein with an overall yield of 30%. The degree of purity of the receptor was more than 80%, as assessed by N-terminal sequencing of the protein eluted from the column. Sequence analysis demonstrated the absence of a methionine residue at the N-terminal position, and suggested that the side chain of the asparagine residue at position 7 is glycosylated.

Peptides corresponding to sequences derived from predicted extra- and intracellular loops of the rat bradykinin receptor were analyzed for interspecies homology as well as for matches within the present dataset of protein sequences to provide a theoretical basis for the specific recognition of the native cognate protein by antibodies raised against these antigens. Application of polyclonal antibodies raised against the selected peptides allowed the immunocytochemical localization of the native receptor protein in cells of rat and human origin. The detection of the molecule was achieved by different immunohisto- and immunocytochemical methods in combination with light, fluorescence, confocal optical laser and electron microscopy. These results were compared to localization studies by autoradiography. Distribution and subcellular localization were determined in human neutrophils, human epithelial carcinoma cells (A431) and in rat kidney tissue.

The X-lined gene for Norrie disease, which is characterized by blindness, deafness and mental retardation has been cloned recently. This gene has been thought to code for a putative extracellular factor; its predicted amino acid sequence is homologous to the C-terminal domain of diverse extracellular proteins. Sequence pattern searches and three-dimensional modelling now suggest that the Norrie disease protein (NDP) has a tertiary structure similar to that of transforming growth factor beta (TGF beta). Our model identifies NDP as a member of an emerging family of growth factors containing a cystine knot motif, with direct implications for the physiological role of NDP. The model also sheds light on sequence related domains such as the C-terminal domain of mucins and of von Willebrand factor.

The marine sponge Geodia cydonium contains several lectins. The main component, called lectin-1, is composed of three to four identical subunits. The subunits of the lectins were cloned from a cDNA library; two clones were obtained. From the deduced aa sequence of one clone, LECT-1, a mol. wt of 15,313 Da is calculated; this value is in good agreement with mass spectrometric analysis of 15,453 +/- 25 Da. The sequence of another clone, LECT-2, was analysed and the aa sequence was deduced (15,433 Da). The two subunits have a framework sequence of 38 conserved aa which are characteristic for the carbohydrate-binding site of vertebrate S-type lectins. Clustering of lectin sequences of various species following their pairwise comparison establishes a dendrogram, which reveals that the sponge lectin could be considered as the ancestor for vertebrate S-type lectins.

A candidate gene for Norrie disease, an X-linked disorder characterized by blindness, deafness and mental disturbances, was recently isolated and found to contain microdeletions in numerous patients. No strong homologies were identified. By studying the number and spacing of cysteine residues, we now detect homologies between the Norrie gene product and a C-terminal domain which is common to a group of proteins including mucins. Three newly-characterized missense mutations, replacing evolutionarily conserved cysteines or creating new cysteine codons, emphasize the functional importance of these sites. These findings and the clinical features of this disorder suggest a possible role for the Norrie gene in neuroectodermal cell-cell interaction.

Fetuins are among the major plasma proteins, yet their biological role has remained elusive. Here we report the molecular cloning of rat fetuin and the sequence analysis of a full-length clone, RF619 of 1456 bp with an open reading frame of 1056 bp encoding 352 amino acid residues. The coding part of RF619 was identical with the cDNA sequence of the natural inhibitor of the insulin receptor tyrosine kinase from rat (pp63) except for four substitutions and a single base insertion causing divergence of the predicted protein sequences. Partial amino acid sequences of rat plasma fetuin were in agreement with the predictions based on the RF619 cDNA. Purified rat fetuin inhibited the insulin receptor tyrosine kinase in vitro. Therefore, we conclude that RF619 and pp63 cDNA encode the same protein, i.e. authentic rat fetuin which is a functional tyrosine kinase inhibitor.

Three sets of monoclonal antibodies against bradykinin (MBK1, MBK2, MBK3) were generated by somatic cell fusion, characterized by their peptide specificity and compared to the known ligand specificity of the kinin receptor subtypes. By these criteria the paratope of MBK3 resembled the B2 receptor binding site whereas MBK1 shared principal binding characteristics with the B1 recrptor. Anti-idiotypic antibodies against MBK1, MBK2 and MBK3 were raised in rabbit and sheep. Specificity of the network components was verified by inhibition experiments on the level of peptide, idiotype and anti-idiotype. Anti-idiotypic antibodies against MBK3 recognized a conformation-dependent epitope which was binding site-related. Binding studies on human foreskin fibroblasts and guinea pig ileum showed mutual displacement of the anti-idiotypic antibody and bradykinin at the binding site pointing to a specific interaction of the antibody with the receptor from various species. An agonist activity of the antibodies, demonstrated in human (inositolphosphate pathway) and mouse (prostaglandin pathway) fibroblasts indicated that the anti-idiotypes bear an internal image of the ligand epitope. This molecular mimicry which was further substantiated by the detection of bradykinin specific anti-idiotypic antibodies, provides the structural basis for the observed cross-reactivity over species borders.

Human alpha 2-HS glycoprotein and bovine fetuin, abundant proteins of fetal plasma, are structural members of the fetuin family within the cystatin superfamily. They are characterized by the presence of two N-terminally located cystatin-like units and a unique C-terminal sequence segment not present in the other members of the cystatin superfamily. Search for related sequences revealed that the natural inhibitor of the insulin receptor tyrosine kinase [Auberger, Falquerho, Contreres, Pages, Le Cam, Rossi & Le Cam (1989) Cell (Cambridge, Mass.) 58, 631-640] shows sequence similarity to the mammalian fetuins. The sequence identity between rat tyrosine kinase inhibitor, human alpha 2-HS glycoprotein and bovine fetuin is 56 and 60% respectively (percentage of residues in identical positions). The sequence similarity extends over the entire protein structures, except the extreme C-terminal portions. In particular, the number and relative positions of the cysteine residues are invariant among the proteins, suggesting that the characteristic array of linearly arranged and tandemly repeated disulphide loops of the cystatin superfamily is also present in rat tyrosine kinase inhibitor. We conclude that rat tyrosine kinase inhibitor may be classified as a novel member of the mammalian fetuin family.