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1.
Post-translational changes in tertiary and quaternary structure of the insulin proreceptor. Correlation with acquisition of function 总被引:10,自引:0,他引:10
Tertiary and quaternary structural changes that occur during post-translational processing of the insulin proreceptor were examined in 3T3-L1 adipocytes. In pulse-chase experiments with [35S]methionine, labeled insulin receptor species, isolated by immuno- and insulin-affinity adsorption, were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under conditions where intra- and intermolecular disulfide bonds remained intact or were cleaved by reduction. Reducing SDS-polyacrylamide gel electrophoresis confirmed that the insulin receptor is synthesized as a long-lived (t1/2 = 3 h) proreceptor precursor of 210 kDa which undergoes proteolytic cleavage and carbohydrate maturation to form the alpha- and beta-subunits of the mature receptor. The proreceptor acquires insulin binding activity through a subtle structural change (t1/2 = 45 min) detected only by an autoimmune antibody specific for an epitope of the active insulin binding site. Analysis of insulin receptor species by nonreducing SDS-polyacrylamide gel electrophoresis revealed that the proreceptor undergoes two additional structural changes not detected by reducing SDS-polyacrylamide gel electrophoresis. The proreceptor is synthesized as a monomer (M1) with an apparent molecular mass of 170 kDa that is converted by disulfide rearrangement to another monomeric form of 190-kDa apparent molecular mass (M2). N-Linked glycosylation is required for this transition, since aglycoproreceptor, synthesized in the presence of tunicamycin, does not undergo any detectable tertiary or quaternary structural changes. M2 self-associates to form a disulfide-linked proreceptor dimer (D) which is subsequently proteolytically processed, forming the mature, disulfide-linked alpha 2 beta 2 receptor tetramer. The mature receptor was distinguished from the three proreceptor species (M1, M2, and D) by its cell surface location and its ability to bind tightly to wheat germ agglutinin-agarose, indicating the presence of complex oligosaccharide chains. Subcellular fractionation indicated that both the M1 to M2 and M2 to D conversions occur in the endoplasmic reticulum. Separation of the nonreduced proreceptor species into "active" and "inactive" forms by affinity chromatography on insulin-agarose revealed that neither the transition of M1 to M2, nor of M2 to D, is correlated with the acquisition of insulin binding function. Rather, during its life-time, the M2 species acquires insulin binding activity and an epitope recognized by a binding site specific autoimmune antibody through a subtle structural change not detected by reducing or nonreducing SDS-polyacrylamide gel electrophoresis. 相似文献
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The biogenesis, intracellular transport, and functional properties of the insulin proreceptor and modified insulin receptors were studied in hormone-responsive 3T3-L1 adipocytes. After control cells were labeled with [35S]Met for 7 min, the principal polypeptide that was precipitated by anti-insulin receptor antibodies had a molecular weight (Mr) of 180 000. This initial precursor was rapidly converted (t1/2 = 35 min) to a 200-kilodalton (kDa) polypeptide, designated the insulin proreceptor, by the apparent posttranslational addition of N-linked, high mannose core oligosaccharide units. Mature alpha (Mr 130 000) and beta (Mr 90 000) subunits were derived from sequences within the proreceptor by proteolytic cleavage and late processing steps, and these subunits appeared on the cell surface 2-3 h after synthesis of the 180-kDa precursor. The cation ionophore monensin was used in combination with metabolic labeling, affinity cross-linking, and external proteolysis to probe aspects of proreceptor function, transit, and the development of insulin sensitivity at the target cell surface. At 5 micrograms/mL, monensin potently inhibited the proteolytic cleavage step, and the 200-kDa polypeptide accumulated. Lower concentrations of the ionophore selectively blocked late processing steps in 3T3-L1 adipocytes so that apparently smaller alpha' (Mr 120 000) and beta' (Mr 85 000) subunits were produced. Proreceptor and alpha' and beta' subunits were translocated to the cell surface, indicating that the signal for intracellular transit occurs in the 200-kDa polypeptide and is independent of the posttranslational proteolysis and late processing steps. The alpha' subunit bound insulin both at the surface of intact cells and after solubilization with Triton X-100; the beta' subunit was phosphorylated in an insulin-stimulated manner. The detergent-solubilized 200-kDa proreceptor also exhibited both functional properties. However, the proreceptor that was transported to and exposed on the cell surface was incapable of binding insulin in intact adipocytes. Thus, late processing is not essential for the expression of functions associated with mature alpha and beta subunits. In contrast, it appears that the proteolytic generation of subunits is required for the correct orientation of the hormone binding site in the plasma membrane bilayer and the development of insulin responsiveness in 3T3-L1 adipocytes. 相似文献
4.
Summary During 70 days of dessicated storage at 32°C over CaSO4, rabbit IgG and rabbit antihorseradish peroxidase antibody remained adsorbed onto polyester cloth, retaining full immunoactivity both as an antibody and an antigen. After dessicated storage, the adsorbed antibody could not be released from the polyester cloth by agitated washing in any of the following anhydrous water-mixable organic solvents: methanol, glacial acetic acid, dimethyl sulfoxide, dimethylformamide, or 1,4-dioxane. 相似文献
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Post-translational processing of the epidermal growth factor receptor. Glycosylation-dependent acquisition of ligand-binding capacity 总被引:8,自引:0,他引:8
The post-translational processing of the epidermal growth factor receptor in human A431 epidermoid carcinoma cells has been investigated. By employing the affinity matrix epidermal growth factor Affi-Gel in conjunction with immunoprecipitation, it has been demonstrated that core oligosaccharide addition is essential for the acquisition of epidermal growth factor-binding activity. Furthermore, the initial 160-kDa translation product was observed to undergo a processing step by which ligand-binding activity was acquired with a half-time of approximately 30 min while exhibiting no apparent change in mobility on sodium dodecyl sulfate-polyacrylamide gels. This was shown not to involve the conversion of high-mannose chains to complex chains which have been capped with fucose and sialic acid. Possible explanations for this activation in terms of translocation of intermediates and/or formation of disulfide bonds are discussed. 相似文献
8.
Jhih-Wei Jian Hong-Sen Chen Yi-Kai Chiu Hung-Pin Peng Chao-Ping Tung Ing-Chien Chen 《MABS-AUSTIN》2019,11(2):373-387
Antibodies provide immune protection by recognizing antigens of diverse chemical properties, but elucidating the amino acid sequence-function relationships underlying the specificity and affinity of antibody-antigen interactions remains challenging. We designed and constructed phage-displayed synthetic antibody libraries with enriched protein antigen-recognition propensities calculated with machine learning predictors, which indicated that the designed single-chain variable fragment variants were encoded with enhanced distributions of complementarity-determining region (CDR) hot spot residues with high protein antigen recognition propensities in comparison with those in the human antibody germline sequences. Antibodies derived directly from the synthetic antibody libraries, without affinity maturation cycles comparable to those in in vivo immune systems, bound to the corresponding protein antigen through diverse conformational or linear epitopes with specificity and affinity comparable to those of the affinity-matured antibodies from in vivo immune systems. The results indicated that more densely populated CDR hot spot residues were sustainable by the antibody structural frameworks and could be accompanied by enhanced functionalities in recognizing protein antigens. Our study results suggest that synthetic antibody libraries, which are not limited by the sequences found in antibodies in nature, could be designed with the guidance of the computational machine learning algorithms that are programmed to predict interaction propensities to molecules of diverse chemical properties, leading to antibodies with optimal characteristics pertinent to their medical applications. 相似文献
9.
R F Arakaki R J Comi P Gorden 《Biochemical and biophysical research communications》1989,160(1):296-302
The insulin receptor is synthesized as a single chain, 190 kDa glycoprotein precursor, which undergoes proteolytic cleavage, carbohydrate processing, and fatty acylation to generate the mature receptor on the plasma membrane. The relationship of these post-translational modifications to the acquisition of receptor function, i.e. ligand binding and phosphokinase activity, is not fully understood. Therefore, the 190 kDa proreceptor and mature receptor kinase activities were separately examined in vitro, and their phosphorylation properties compared. The solubilized receptor precursor from IM-9 lymphocytes was purified by sequential lectin chromatography and, following site specific anti-receptor antibody immunoprecipitation, phosphokinase studies performed. The isolated proreceptor was activated by insulin and phosphorylated exogenous substrate alpha-casein, as similarly observed for the mature receptor. Structurally, the phosphorylated proreceptor was identified as a 360 kDa homodimer under non-reducing condition. 相似文献
10.
Yoram Shechter Dana Elias Ruth Maron Irun R. Cohen 《Journal of cellular biochemistry》1983,21(2):179-185
We immunized mice with insulin and found that those strains that develop insulin antibodies subsequently produce insulin-like activity in amount equivalent to 300–400 ng insulin per ml serum. The activity was due exclusively to IgG2 antibodies. Bioactivity could be blocked efficiently by insulin antibodies from guinea pigs and from mice. The active IgG2 also displaced labeled insulin from fat cells. Preliminary in vivo studies have indicated that the appearance of insulin-like antibodies in the mouse resulted in abnormal glucose homeostasis and “down regulation” of insulin receptors. These results indicate that immunization to insulin can initiate an idiotype-anti-idiotype network resulting in antibodies to the hormone receptor. 相似文献
11.
T Kieber-Emmons 《DNA and cell biology》1992,11(3):199-206
Sequence analysis of autoimmune-associated antibodies has suggested a structural relatedness between genes used to encode autoantibodies and those encoding unrelated antibodies without autoreactive specificities. Subsequently, the basis for cross-reactive idiotypes across germ-line lineages, as well as conserved interspecies cross-reactivities of autoantigens among serologically similar antibodies, may result from evolutionary duplication of particular types of recognition motifs. As a first step toward elucidating structural recognition principles underlying possible cross-reactive epitopes involved in autoimmune pathologies, structural features of selected motifs associated with native ligand binding are examined for their inherent occurrence in antibody and T-cell receptor repertoires. This analysis considers the putative recognition features representative of common motif subsets shared with loop structures in CDR2 and FR3 regions of antibodies such as charge-2x-charge-x-charge or hydrogen bond donor (acceptor)-2x-charge-x-hydrogen bond donor (acceptor) type motifs, where x is any residue that can participate in maintaining a loop conformation. Such tracts encoded in the CRD2 and FR3 regions of heavy chains of antibodies and T-cell receptors (TCRs) associated with autoimmune dysfunction, with non-autoreactive antibodies, and with native host proteins. Such evolutionarily conserved motifs may be targets for complementary interactions involving autoantibodies and receptors. 相似文献
12.
Post-translational modifications of the core-specific lectin. Relationship to assembly, ligand binding, and secretion 总被引:1,自引:0,他引:1
The rat core-specific lectin (CSL) or mannan-binding protein is synthesized and secreted by rat hepatocytes and H-4-II-E hepatoma cells. Prior to secretion proline and lysine residues with collagen-like sequences undergo hydroxylation and subsequent glycosylation of hydroxylysine to produce glucosylgalactosylhydroxylysine. Hydroxylation and subsequent glycosylation are inhibited by alpha,alpha'-dipyridyl (Colley, K. J., and Baenziger, U. U. (1987) J. Biol. Chem. 262, 10290-10295). We have used alpha,alpha'-dipyridyl to investigate the role of hydroxylation and glycosylation on interchain disulfide bond formation, assembly of subunits into high molecular weight complexes, attainment of carbohydrate and lipid binding ability, and secretion. Formation of disulfide-bonded dimers and trimers in the endoplasmic reticulum, assembly into high molecular weight complexes in the Golgi, and attainment of carbohydrate binding activity occur in either the presence or absence of these post-translational modifications. The mature fully processed form of the CSL binds hydrophobic matrices and is secreted at a slow, but linear, rate. Inhibition of proline and lysine hydroxylation and hydroxylysine glycosylation prevents CSL secretion and attainment of binding activity for hydrophobic matrices. Secretion of the lectin, although slow, appears to be an active process and may be related to the capacity to interact with membranes and/or lipids. Other proteins known to contain collagen-like sequences such as acetylcholinesterase, pulmonary surfactant apoproteins, and C1q also interact with lipids and/or membranes. The collagen-like domains of these proteins may also play a role in promoting such interactions. 相似文献
13.
IgE response specific to those molecular regions of casein that contain a major phosphorylation site was analyzed using native and modified caseins and derived peptides. This study included (i) the naturally occurring common variants A1 and A from beta- and alphas2-caseins, respectively, which were purified in the native form and then dephosphorylated, (ii) a purified rare variant D of alphas2-casein which lacks one major phosphorylation site, and (iii) the native and dephosphorylated tryptic fragment f(1-25) from beta-casein. Direct and indirect ELISA using sera from patients allergic to milk showed that the IgE response to caseins is affected by modifying or eliminating the major phosphorylation site. 相似文献
14.
The MPM-2 antibody inhibits mitogen-activated protein kinase activity by binding to an epitope containing phosphothreonine-183. 总被引:3,自引:2,他引:1
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S Taagepera P Dent J H Her T W Sturgill G J Gorbsky 《Molecular biology of the cell》1994,5(11):1243-1251
Mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases implicated in the control of cell proliferation and differentiation. We have found that activated p42mapk is a target for the phosphoepitope antibody MPM-2, a monoclonal antibody that recognizes a cell cycle-regulated phosphoepitope. We have determined that the MPM-2 antibody recognizes the regulatory region of p42mapk. Binding of the MPM-2 antibody to active p42mapk in vitro results in a decrease in p42mapk enzymatic activity. The MPM-2 phosphoepitope can be generated in vitro on bacterially expressed p42mapk by phosphorylation with either isoform of MAP kinase kinase (MKK), MKK1, or MKK2. Analysis of p42mapk proteins mutated in their regulatory sites shows that phosphorylated Thr-183 is essential for the binding of the MPM-2 antibody. MPM-2 binding to Thr-183 is affected by the amino acid present in the other regulatory site, Tyr-185. Substitution of Tyr-185 with phenylalanine results in strong binding of the MPM-2 antibody, whereas substitution with glutamic acid substantially diminishes MPM-2 antibody binding. The MPM-2 phosphoepitope antibody recognizes an amino acid domain incorporating the regulatory phosphothreonine on activated p42mapk in eggs during meiosis and in mammalian cultured cells during the G0 to G1 transition. 相似文献
15.
G W Bruse R Wollin S Oscarson P E Jansson A A Lindberg 《Journal of molecular recognition : JMR》1991,4(4):121-128
Phage G13 binds to the carbohydrate part of lipopolysaccharides from rough mutants of Salmonella and Escherichia coli as the first event of infection. Equilibrium dialysis inhibition studies with native and synthetic trisaccharides as inhibitors suggested that phage G13 recognizes branched oligosaccharides having 6-O-alpha- or 7-O-alpha-glycosyl groups with alpha-Man(1----3) [alpha-Man(1----6)]Man (Man[Man]Man) and alpha-Glc(1----3)-[alpha-Hep(1----7)] alpha-Hep(1----3) alpha-Hep(1----5)Kdo as the smallest saccharides with inhibitory activity (Wollin et al., 1989). Of four synthetic analogues to Man[Man]Man only Man(1----3)[alpha-Gal(1----6)]alpha-Man-OMe (Man[Gal]-Man) and alpha-Glc(1----3)[alpha-Hep(1----7)]alpha-Hep-OMe (Glc[Hep]Hep) inhibited the binding of labelled E. coli C core nonasaccharide ligand to G13 with activities which were 10- and 15-fold lower than Man[Man]Man. The trisaccharides alpha-Man(1----3)[alpha-Glc(1----6)[alpha-Man-OMe (Man[Glc]Mann) and alpha-Man(1---3)[alpha-Tal(1----6)]alpha-Man-OMe (Man[Tal]Man) showed no inhibition at concentrations 75-fold higher than Man[Man]Man. Minimum energy conformation calculations of the saccharides using the GESA method showed that the 6-O-alpha-Man group in Man[Man]Man and the 7-O-alpha-Hep group SL805 pentasaccharide expose their OH-2 and OH-3 groups in a similar way and these are postulated to be key structural features for binding activity. The importance of hydroxy groups at certain positions is implied from the fact that both manno- and galacto-isomers are active. We also conclude that the O6-C6-C5-O5-C1 region of the 3-O-alpha-glycosyl group in the Man[Man]Man trisaccharide, or part of it, is important for the G13 binding activity. 相似文献
16.
The 51-kDa telomere protein from Euplotes crassus binds to the extreme terminus of macronuclear telomeres, generating a very salt-stable telomeric DNA-protein complex. The protein recognizes both the sequence and the structure of the telomeric DNA. To explore how the telomere protein recognizes and binds telomeric DNA, we have examined the DNA-binding specificity of the purified protein using oligonucleotides that mimic natural and mutant versions of Euplotes telomeres. The protein binds very specifically to the 3' terminus of single-stranded oligonucleotides with the sequence (T4G4) > or = 3 T4G2; even slight modifications to this sequence reduce binding dramatically. The protein does not bind oligonucleotides corresponding to the complementary C4A4 strand of the telomere or to double-stranded C4A4.T4G4-containing sequences. Digestion of the telomere protein with trypsin generates an N-terminal protease-resistant fragment of approximately 35 kDa. This 35-kDa peptide appears to comprise the DNA-binding domain of the telomere protein as it retains most of the DNA-binding characteristics of the native 51-kDa protein. For example, the 35-kDa peptide remains bound to telomeric DNA in 2 M KCl. Additionally, the peptide binds well to single-stranded oligonucleotides that have the same sequence as the T4G4 strand of native telomeres but binds very poorly to mutant telomeric DNA sequences and double-stranded telomeric DNA. Removal of the C-terminal 15 kDa from the telomere protein does diminish the ability of the protein to bind only to the terminus of a telomeric DNA molecule. 相似文献
17.
G M Ward S Clark L C Harrison 《Biochemical and biophysical research communications》1984,123(2):849-853
We investigated whether insulin forms covalent bonds with its receptors on erythrocytes and reticulocytes, as it does in adipocytes (1). Of the [125I]-insulin specifically bound at 37 degrees C to human and rat erythrocytes and rat reticulocytes, only 1.5-2.3% was non-dissociable on extensive washing. When ghosts prepared from the washed cells were solubilized in Triton X-100, only 0.6-1.5% of the specifically bound radioactivity appeared in the void volume of a Sephadex G-50 column. Moreover in contrast to adipocytes, this high molecular weight radioactivity was not immunoprecipitable by antibodies to the insulin receptor and was dissociated during chromatography in sodium dodecyl sulphate. Thus we have been unable to demonstrate the formation of covalent bonds between insulin and its receptors on erythrocytes and reticulocytes. This finding is consistent with the hypothesis that covalent binding of insulin is a necessary receptor modification for insulin's metabolic effects. 相似文献
18.
Purification of insulin receptor with full binding activity 总被引:24,自引:0,他引:24
Y Fujita-Yamaguchi S Choi Y Sakamoto K Itakura 《The Journal of biological chemistry》1983,258(8):5045-5049
Insulin receptor was purified 2400-fold with an overall yield of 40% from human placental membranes by affinity chromatography on wheat germ agglutinin-Sepharose and insulin-Sepharose. The receptor was eluted from insulin-Sepharose using mild conditions, eliminating urea, so that it was stable and retained full insulin-binding activity. Chromatofocusing and gel filtration analysis indicated that the receptor preparation was apparently pure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three high molecular weight protein bands with Mr = 320,000, 300,000, and 270,000 under nonreducing conditions and two major protein bands with Mr = 135,000 and 90,000 under reducing conditions. The purified receptor showed a curvilinear Scatchard plot with maximum insulin binding of 28.5 micrograms per mg of protein. In comparison, the receptor eluted from insulin-Sepharose with previously used conditions in the presence of urea resulted in maximum insulin binding of only 6 micrograms per mg of protein. This indicates that a 4-to 5-fold increase in specific activity can be obtained by using the new elution conditions. 相似文献
19.
Two localized monovalent cation binding sites have been identified in cubic insulin from 2.8 A-resolution difference electron density maps comparing crystals in which the Na+ ions have been replaced by Tl+. One cation is buried in a closed cavity between insulin dimers and is stabilized by interaction with protein carbonyl dipoles in two juxtaposed alternate positions related by the crystal dyad. The second cation binding site, which also involves ligation with carbonyl dipoles, is competitively occupied by one position of two alternate His B10 side chain conformations. The cation occupancy in both sites depends on the net charge on the protein which was varied by equilibrating crystals in the pH range 7-10. Detailed structures of the cation binding sites were inferred from the refined 2-A resolution map of the sodium-insulin crystal at pH 9. At pH 9, the localized monovalent cations account for less than one of the three to four positive counterion charges necessary to neutralize the negative charge on each protein molecule. The majority of the monovalent counterions are too mobile to show up in the electron density maps calculated using data only at resolution higher than 10 A. Monovalent cations of ionic radius less than 1.5 A are required for crystal stability. Replacing Na+ with Cs+, Mg++, Ca++ or La+++ disrupts the lattice order, but crystals at pH 9 with 0.1 M Li+, K+, NH4+, Rb+ or Tl+ diffract to at least 2.8 A resolution. 相似文献
20.
A leucine to proline mutation at position 233 in the insulin receptor inhibits cleavage of the proreceptor and transport to the cell surface 总被引:3,自引:0,他引:3
J A Maassen E R Van der Vorm G C Van der Zon M P Klinkhamer H M Krans W M?ller 《Biochemistry》1991,30(44):10778-10783
We have previously shown that a homozygous mutation encoding a substitution of proline for leucine at position 233 in the insulin receptor is linked with the syndrome of leprechaunism, being a lethal form of insulin resistance in newborn children. Specific binding of insulin and insulin-stimulated autophosphorylation of the insulin receptor are nearly absent in fibroblasts from the leprechaun patient. To examine the molecular basis of the observed insulin receptor abnormalities, CHO cell lines overexpressing mutant insulin receptors were made by transfection. The results show that the mutation inhibits cleavage and transport of the proreceptor from intracellular sites to the cell surface. As the mutant receptor is poorly precipitated by two different monoclonal antibodies recognizing epitopes on undenatured wild-type alpha-subunits, the mutation probably affects overall folding of the alpha-subunit. The mutant proreceptor is unable to bind insulin and exhibits no insulin-stimulated autophosphorylation. These data explain the abnormalities seen in the patient's fibroblasts. Pulse-chase labeling experiments on transfected cells show that the mutant precursor has an extended half-life (approximately 5 h) compared to the precursor of wild-type insulin receptors (approximately 2 h). This mutation is the first example of a naturally occurring mutation in the insulin receptor which completely blocks cleavage of the proreceptor and transport to the cell surface. 相似文献