Epitope Of mAb Research Articles

Antigenic variants with amino acid deletions clarify a neutralizing epitope specific for influenza B virus Victoria group strains.

To study the neutralizing epitopes of influenza B virus Victoria group strains, two monoclonal antibodies (MAbs) were used to select antigenic variants of the virus. MAbs 10B8 and 8E6 were found to react with B/Victoria group strains in three tests, peroxidase-antiperoxidase staining, haemagglutination inhibition and neutralization tests; no reactivity with B/Yamagata group strains was observed. Analysis of the deduced amino acid sequences of 10B8-induced variants identified a single amino acid deletion at residue 165 or 170, as well as a single amino acid substitution at residues 164 (Asp-->Tyr), 165 (Asn-->Ser or Thr) or 203 (Lys-->Thr or Asn). A single amino acid substitution at residue 241 (Pro-->Ser) was observed in 8E6-induced variants. Three-dimensional analysis showed that the epitopes for both MAbs were situated in close proximity to each other. Since B/Yamagata group strains are characterized by amino acid deletions at residues 164-166, the epitope for MAb 10B8 is strictly specific for B/Victoria group strains.

Locus of a virus neutralization epitope on the Japanese encephalitis virus envelope protein determined by use of long PCR-based region-specific random mutagenesis.

We prepared recombinant Japanese encephalitis (JE) virus populations possessing random mutations at the envelope (E) protein region by a long PCR-based method. Neutralization-resistant mutants were selected from these populations by application of JE-specific virus neutralizing monoclonal antibody (mAb) 503, which possessed a 51,200-fold neutralization titer. We classified the mutants into three groups, each bearing two amino acid alterations at the E protein region: 52, Gln-Arg, and 136, Lys-Glu; 136, Lys-Glu, and 275, Ser-Pro; and 126, Ile-Thr, and 136, Lys-Glu, respectively. Three different genetically engineered variants, each bearing a single mutation, 126, Ile-Thr; 136, Lys-Glu; and 275, Ser-Pro, respectively, showed partial but not complete recovery of reactivity to mAb 503. Our results indicate that the amino acid substitutions at amino acid positions 52, 126, 136, and 275 altered the structure of the neutralization epitope for mAb 503 on the E protein. All these mutations were clustered at the junction of domains I and II of the E protein and it is likely that the epitope for mAb 503 is composed of at least E(0)-e, D(0)-a, and k strands of the E protein. We also demonstrated the efficacy of the long PCR-based recombinant virus technique as a useful tool for the creation of a variety of mutants bearing random mutations at targeted areas of the virus genome.

A Method for Defining Binding Sites Involved in Protein–Protein Interactions: Analysis of the Binding of Plasminogen Activator Inhibitor 1 to the Somatomedin Domain of Vitronectin

Plasminogen activator inhibitor type-1 (PAI-1) is bound to vitronectin (VN) in plasma and in the extracellular matrix. We previously employed a domain-swapping approach to show that the high-affinity binding site for PAI-1 in VN is contained within residues 12–30 in the amino-terminal somatomedin B (SMB) domain. In this study, we attempt to further delineate the location of this site by employing a novel approach that is based on the use of monoclonal antibodies (Mabs) together with site-directed mutagenesis. Six separate Mabs were identified that bound to the SMB domain and competed with PAI-1 for binding to VN. The relative affinity of each of the Mabs, and of PAI-1 itself, for binding to individual variants of SMB (prepared by alanine scanning mutagenesis), was then determined and compared in competitive binding experiments. Three separate, partially overlapping Mab epitopes within SMB were defined by these studies, and the PAI-1 binding site was localized to the region between residues 24 and 37. When considered together with the domain swapping data, these studies suggest that the PAI-1 binding site is contained within a common seven-residue region (i.e., residues 24–30) in the SMB domain.

Localization of epitopes for monoclonal antibodies to urokinase-type plasminogen activator: relationship between epitope localization and effects of antibodies on molecular interactions of the enzyme.

We localized the epitopes for several murine mAbs to human urokinase-type plasminogen activator (uPA) by Ala scanning mutagenesis and related the localization to the effects of the mAbs on the molecular interactions of uPA. Several antibodies against the serine proteinase domain (SPD) were found to have overlapping epitopes composed of variable combinations of Arg178, Arg179, His180, Arg181, Tyr209, Lys211, and Asp214 in the so-called 37-loop and 60-loop, located near the active site and taking part in the binding of uPA to plasminogen activator inhibitor-1 (PAI-1). Besides inhibiting uPA-catalysed plasminogen activation, all antibodies to SPD strongly delayed the binding of uPA to PAI-1, decreasing the second-order rate constant 15- to 6500-fold. There was no correlation between the relative effects of the 37-loop and 60-loop substitutions on the second-order rate constant and on the binding of the antibodies, indicating that the antibodies did not delay complex formation by blocking residues of specific importance for the uPA-PAI-1 reaction, but rather by steric hindrance of the access of PAI-1 to the active site. The affinity of the SPD antibodies for the uPA-PAI-1 complex was only slightly lower than that for free uPA, indicating that the 37-loop and 60-loop are exposed in the complex. The epitopes for two antibodies to the kringle included Arg108, Arg109, and Arg110. The ability of these antibodies to block the binding of uPA to polyanions correlated with a reduced uPA-polyanion affinity after substitution of the three Arg residues.

EWI-2 is a major CD9 and CD81 partner and member of a novel Ig protein subfamily.

A novel Ig superfamily protein, EWI-2, was co-purified with tetraspanin protein CD81 under relatively stringent Brij 96 detergent conditions and identified by mass spectrometric protein sequencing. EWI-2 associated specifically with CD9 and CD81 but not with other tetraspanins or with integrins. Immunodepletion experiments indicated that EWI-2-CD9/CD81 interactions are highly stoichiometric, with approximately 70% of CD9 and CD81 associated with EWI-2 in an embryonic kidney cell line. The EWI-2 molecule was covalently cross-linked (in separate complexes) to both CD81 and CD9, suggesting that association is direct. EWI-2 is part of a novel Ig subfamily that includes EWI-F (F2alpha receptor regulatory protein (FPRP), CD9P-1), EWI-3 (IgSF3), and EWI-101 (CD101). All four members of this Ig subfamily contain a Glu-Trp-Ile (EWI) motif not seen in other Ig proteins. As shown previously, the EWI-F molecule likewise forms highly proximal, specific, and stoichiometric complexes with CD9 and CD81. Human and murine EWI-2 protein sequences are 91% identical, and transcripts in the two species are expressed in virtually every tissue tested. Thus, EWI-2 potentially contributes to a variety of CD9 and CD81 functions seen in different cell and tissue types.

Cellular Activation of Leukocyte Function-Associated Antigen-1 and Its Affinity Are Regulated at the I Domain Allosteric Site

The I domain of the integrin LFA-1 possesses a ligand binding interface that includes the metal ion-dependent adhesion site. Binding of the LFA-1 ligand, ICAM-1 to the metal ion-dependent adhesion site is regulated by the I domain allosteric site (IDAS). We demonstrate here that intracellular signaling leading to activation of LFA-1 binding to ICAM-1 is regulated at the IDAS. Inhibitory mutations in or proximal to the IDAS are dominant to cytoplasmic signals that activate binding to ICAM-1. In addition, mutational activation at the IDAS greatly increases the binding of lymphocyte-expressed LFA-1 to ICAM-1 in response to PMA, but does not result in constitutive binding. Binding of a novel CD18 activation epitope mAb to LFA-1 in response to soluble ICAM-1 binding was also blocked by inhibitory and was enhanced by activating IDAS mutations. Surface plasmon resonance using soluble wild-type LFA-1 and an IDAS mutant of LFA-1 indicate that the IDAS can regulate a 6-fold change in the K(d) of ICAM-1 binding. The K(d) of wild-type LFA-1 (1.2 x 10(-1) s(-1)) differed with that of the activating IDAS mutant (1.9 x 10(-2) s(-1)), but their K(a) values were identical (2.2 x 10(5) M(-1)s(-1)). We propose that IDAS regulates the binding of LFA-1 to ICAM-1 activated by intracellular signals. IDAS can control the affinity state of LFA-1 with concomitant I domain and CD18 conformational changes.

Structure-Function of the Putative I-domain within the Integrin β2 Subunit

The central region (residues 125-385) of the integrin beta(2) subunit is postulated to adopt an I-domain-like fold (the beta(2)I-domain) and to play a critical role in ligand binding and heterodimer formation. To understand structure-function relationships of this region of beta(2), a homolog-scanning mutagenesis approach, which entails substitution of nonconserved hydrophilic sequences within the beta(2)I-domain with their homologous counterparts of the beta(1)I-domain, has been deployed. This approach is based on the premise that beta(1) and beta(2) are highly homologous, yet recognize different ligands. Altogether, 16 segments were switched to cover the predicted outer surface of the beta(2)I-domain. When these mutant beta(2) subunits were transfected together with wild-type alpha(M) in human 293 cells, all 16 beta(2) mutants were expressed on the cell surface as heterodimers, suggesting that these 16 sequences within the beta(2)I-domain are not critically involved in heterodimer formation between the alpha(M) and beta(2) subunits. Using these mutant alpha(M)beta(2) receptors, we have mapped the epitopes of nine beta(2)I-domain specific mAbs, and found that they all recognized at least two noncontiguous segments within this domain. The requisite spatial proximity among these non-linear sequences to form the mAb epitopes supports a model of an I-domain-like fold for this region. In addition, none of the mutations that abolish the epitopes of the nine function-blocking mAbs, including segment Pro(192)-Glu(197), destroyed ligand binding of the alpha(M)beta(2) receptor, suggesting that these function-blocking mAbs inhibit alpha(M)beta(2) function allosterically. Given the recent reports implicating the segment equivalent to Pro(192)-Glu(197) in ligand binding by beta(3) integrins, these data suggest that ligand binding by the beta(2) integrins occurs via a different mechanism than beta(3). Finally, both the conformation of the beta(2)I-domain and C3bi binding activity of alpha(M)beta(2) were dependent on a high affinity Ca(2+) binding site (K(d) = 105 microm), which is most likely located within this region of beta(2).

Analysis of the functional domains of Arcanobacterium pyogenes pyolysin using monoclonal antibodies

Pyolysin (PLO), secreted by Arcanobacterium pyogenes, is a novel member of the thiol-activated cytolysin (TACY) family of bacterial toxins. Four monoclonal antibodies (mAbs) to PLO were prepared for the analysis of functional domains of this toxin. Two (mAbs S and H) of these markedly inhibited the hemolytic activity of PLO, but the inhibiting activity of the other two antibodies (mAbs C and G) was weaker. Subsequently, nine truncated PLOs were derived from recombinant Escherichia coli by various deletions from the N-terminus. Strong hemolytic activity was recognized in truncates of PLO following the deletion of 30 or 55 amino acids, but not in the truncate with deletion of 74 residues. Truncated PLOs were used in immunoblotting experiments to locate the epitopes for the mAbs. The epitope for mAbs C and G lies within the undecapeptide region (amino acids 487–505) of the C-terminus of PLO, which seems to be the binding site to erythrocytes. In contrast, the epitopes for mAbs S and H, which showed strong neutralizing activity, were found to lie in the N-terminal regions of the PLO ranging from 55 to 73 and 123 to 166 amino acids, respectively. From these results, it seems that the N-terminal region of PLO, in particular, the region of amino acids 55–74 is important for hemolytic activity.

Inhibitory and blocking monoclonal antibody epitopes on merozoite surface protein 1 of the malaria parasite Plasmodium falciparum

Merozoite surface protein 1 (MSP-1) is a precursor to major antigens on the surface of Plasmodium spp. merozoites, which are involved in erythrocyte binding and invasion. MSP-1 is initially processed into smaller fragments; and at the time of erythrocyte invasion one of these of 42 kDa (MSP-142) is subjected to a second processing, producing 33 kDa and 19 kDa fragments (MSP-133 and MSP-119). Certain MSP-1-specific monoclonal antibodies (mAbs) react with conformational epitopes contained within the two epidermal growth factor domains that comprise MSP-119, and are classified as either inhibitory (inhibit processing of MSP-142 and erythrocyte invasion), blocking (block the binding and function of the inhibitory mAb), or neutral (neither inhibitory nor blocking). We have mapped the epitopes for inhibitory mAbs 12.8 and 12.10, and blocking mAbs such as 1E1 and 7.5 by using site-directed mutagenesis to change specific amino acid residues in MSP-119 and abolish antibody binding, and by using PEPSCAN to measure the reaction of the antibodies with every octapeptide within MSP-142. Twenty-six individual amino acid residue changes were made and the effect of each on the binding of mAbs was assessed by Western blotting and BIAcore analysis. Individual changes had either no effect, or reduced, or completely abolished the binding of individual mAbs. No two antibodies had an identical pattern of reactivity with the modified proteins. Using PEPSCAN each mAb reacted with a number of octapeptides, most of which were derived from within the first epidermal growth factor domain, although 1E1 also reacted with peptides spanning the processing site. When the single amino acid changes and the reactive peptides were mapped onto the three-dimensional structure of MSP-119, it was apparent that the epitopes for the mAbs could be defined more fully by using a combination of both mutagenesis and PEPSCAN than by either method alone, and differences in the fine specificity of binding for all the different antibodies could be distinguished. The incorporation of several specific amino acid changes enabled the design of proteins that bound inhibitory but not blocking antibodies. These may be suitable for the development of MSP-1-based vaccines against malaria.

Conformational alterations during biosynthesis of HLA-DR3 molecules controlled by invariant chain and HLA-DM.

HLA-DM is known to catalyze the exchange of class II-associated invariant chain (Ii) peptide (CLIP) for cognate peptide during biosynthesis. In DM-negative cells HLA-DR3 molecules have been shown to predominantly present CLIP and to lack the DR3-specific mAb epitope 16.23, which has led to the assumption that CLIP prevents binding of mAb 16.23. In the present study we show that CLIP does not prohibit 16.23 epitope expression, but that the formation of this epitope is directly influenced by interactions of the DR molecule with Ii and DM. Detergent solubilized DR3 from wild-type as well as DM(-) cells bound CLIP in a 16.23(+) mode. On cells, however, neither CLIP nor antigenic peptide bound to DR3 in a 16.23(+) conformation, unless HLA-DM was expressed. Thus, HLA-DM appears to alter the conformation of DR3 in a peptide-independent fashion. Since in DM-deficient cells that also lack Ii, DR3 molecules assembled in a 16.23(+) conformation, we conclude that during biosynthesis Ii and DM exert opposing conformational constraints, characterized by suppressing or releasing 16.23 epitope expression. These results imply that DR3/peptide complexes, including DR3/ CLIP, can exist in two conformations depending on previous interaction with DM, but independent of the nature of the peptide bound. We show that these naturally occurring class II conformers can be selectively recognized by T cells.

Disappearance of an epithelial cell surface‐specific glycoprotein (Epith‐1) associated with epithelial–mesenchymal conversion in sea urchin embryogenesis

Cell surface modification during mesenchyme ingression was examined using a monoclonal antibody (mAb), anti-Epith-1 mAb, raised against a protein (Epith-1) that was confined to the lateral surface of the epithelial cells in embryo of the sea urchin, Temnopleurus hardwicki. The mAb epitope was N-glycosylated oligosaccharides of 160 kDa monomeric Epith-1 protein. The glycoprotein was negatively charged, and its isoelectric point (IP) was 4.98. The mAb, however, is not immunologically cross-reactive with other sea urchin embryos including Hemicentrotus pulcherrimus, Strongylocentrotus nudus, and Scaphechinus mirabilis. Epith-1 is present initially in the cytoplasm of unfertilized eggs. Cytoplasmic Epith-1 shifted to the cell surface to be integrated in plasma membrane during the first cleavage, and remained there during early embryogenesis by retaining the same relative molecular mass (Mr). During primary and secondary mesenchyme ingression periods, however, Epith-1 disappears from the presumptive mesenchyme cell surface that was associated with internalization of the protein. In plutei, an additional anti-Epith-1 mAb-positive protein appears at the 142 kDa region, which was not associated with any visible alteration of the histologic localization of the protein in larvae. Anti-Epith-1 mAb IgG did not inhibit the reaggregation of epithelial cells in vitro, which suggests that either the protein is not involved in cell-cell adhesion or that the mAb is not recognizing the active site of the protein.

Analysis and quantification of the secretory products of the subcommissural organ by use of monoclonal antibodies.

Bovine Reissner's fiber (RF) glycoproteins were used as antigen for the production of polyclonal and monoclonal antibodies (Mabs). We also produced Mabs against intracellular secretory glycoproteins of the bovine subcommissural organ (SCO). These Mabs were used for immunodetection of secretory proteins in situ (structural and ultrastructural immunocytochemistry), in blots, and in solutions. Three different antigen-mediated ELISA were designed to evaluate the affinity of the Mabs, to study the nature of the epitopes, and for competition test among Mabs. Two double antibody sandwich ELISA were designed to detect and quantify soluble secretory materials in different samples, to study coexistence of epitopes, and to elucidate whether epitopes for Mabs are repeated or not in the RF-glycoproteins. Twenty-three Mabs recognizing the bovine RF- and SCO-glycoproteins in solutions (ELISA) as well as in tissue sections, were obtained. Nineteen of these Mabs also recognized the pig SCO, 11 the rabbit SCO, 6 the dog SCO, and 5 the rat SCO. None of the Mabs recognized the SCO of non-mammalian species. The different types of ELISA demonstrated that: (1) the epitopes reside in the proteinaceous moiety of the secretion, (2) they coexist in the same molecular forms and, with few exceptions, they did not overlap, (3) they were not repeated in the secretory molecule(s). Three Mabs were used for immunoblotting of RF; one of them revealed the same band pattern as that shown by an anti-RF serum. It is concluded that all Mabs raised in our laboratory are directed against non-repeated sequences of RF-glycoproteins that have not been conserved in vertebrate phylogeny.

Identification of protective epitopes by sequencing of the major outer membrane protein gene of a variant strain of Chlamydia psittaci serotype 1 (Chlamydophila abortus).

Protective monoclonal antibodies (MAbs) to the major outer membrane protein (MOMP) of species of the family Chlamydiaceae, which is the primary vaccine candidate antigen, recognize nonlinear epitopes conferred by the oligomeric conformation of the molecule. Protective MAbs failed to recognize oligomeric MOMP of the variant strain LLG, which bears amino acid substitutions in variable segments (VSs) 1, 2, and 4, and competed with monomer-specific MAbs mapping to these VSs in reference strain 577. The results suggest that multiple sites located in the three VSs contribute to the epitope of protective MAbs.

ANALYSIS OF PROSTATE SPECIFIC ANTIGEN AND α1-ANTICHYMOTRYPSIN INTERACTION USING ANTIPEPTIDE MONOCLONAL ANTIBODIES

The synthetic peptides E30D and D10P that correspond to prostate specific antigen (PSA) sequences 60-91 and 78-89, respectively, and contain the kallikrein loop were used to immunize mice to obtain anti-PSA monoclonal antibodies (mAbs). Antipeptide mAb characteristics were studied using biosensor technology and enzyme-linked immunosorbent assay, and analyzing the mAb effects on PSA-alpha1-antichymotrypsin (ACT) complex formation and PSA enzymatic activity. Epitope mapping of these mAbs was performed using overlapping peptide synthesis on nitrocellulose membrane. Anti-E30D mAbs bound PSA coated on the solid phase only, whereas anti-D10P mAbs recognized PSA in detection as well as in capture. However, these mAbs appeared to be anti-total PSA mAbs. Anti-E30D and anti-D10P mAbs were directed against linear epitopes corresponding to residues H74-Y77 and N84-R88, respectively, of the PSA sequence. Anti-D10P mAb recognition of PSA and PSA-ACT complex was equimolar, although an existing molecular model suggested that the sequence corresponding to anti-D10P mAb epitope was involved in the interaction site of PSA with ACT. Furthermore, we were unable to inhibit the enzymatic activity of PSA as well as PSA-ACT complex formation. Finally, the epitope N84-R88 overlapped the cleavage site R85-F86 of PSA. The linear anti-D10P mAb epitope is located outside of the PSA-ACT binding site. However, these mAbs may be of value for evaluating the presence of different molecular PSA forms in sera.

Reversible immunoneutralization of human follitropin receptor

Investigations of the human Follitropin receptor (hFSHR) have failed to identify the tertiary structure that forms the active hormone-receptor interaction site which is essential to develop an immunocontraceptive based upon the receptor. To identify such a domain of hFSHR, an immunoneutralizing monoclonal antibody (mAb) 106-105 (IgG2b) was generated. Flow cytometry tested whether mAb 106-105 recognized native hFSHR. The epitope of mAb 106-105 was mapped by Western blot and by peptide ELISA. Inhibition of hFSH binding and bioactivity was determined by radioreceptor assay and by cAMP production, respectively. MAb 106-105 bound native hFSHR through an epitope including residues 300–315. MAb 106-105 completely blocked hormone binding to receptor and cAMP production by Y1-R cells expressing hFSHR. These effects were completely reversible by increasing the concentration of hFSH. Coincubation of this antibody with peptide D300-F315 blocked antibody activity. These data demonstrate that a discrete linear hFSHR epitope is a target for interference with hormone activity. These results further demonstrate that antibody binding to the extracellular domain (ECD) of hFSHR and subsequent bioactivation can be modulated through a domain specific hindrance, offering a reversible immunoneutralizing target.

Epitope mapping of the transforming growth factor-beta superfamily protein, macrophage inhibitory cytokine-1 (MIC-1): identification of at least five distinct epitope specificities.

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the transforming growth factor-beta (TGF-beta) superfamily whose increased expression is associated with macrophage activation and which is expressed highly in placenta as compared to other tissues. There are two known allelic forms of human MIC-1 due an amino acid substitution at position 6 of the mature protein. We have raised four monoclonal antibodies (MAbs) and one polyclonal antiserum to the mature protein region of human MIC-1 and have used an extensive panel of MIC-1 relatives, mutants, and chimeras to map their epitopes. None of the MAbs were able to cross-react with either the murine homologue of MIC-1 or with hTGF-beta1, and all of the MAb epitopes were conformation-dependent. A distinct cross-reactivity pattern with the various antigens was observed for each of the monoclonal and polyclonal antibodies suggesting the presence of at least five immunogenic regions on the MIC-1 surface. One of the MAbs is directed against the amino terminus of the protein and can distinguish between the two allelic forms of MIC-1. The epitopes for the other three MAbs were located near the tips of the so-called "fingers" of the protein and appeared to be partially overlapping as each involved amino acids in the region 24-37. In one case, it was possible to mutate murine MIC-1 so that it could be recognized by one of the MAbs. Finally, the use of another mutant in which Cys 77 was replaced by serine enabled confirmation of the location of the MIC-1 interchain disulfide bond.

A role for serine-175 in modulating the molecular conformation of calponin

Calponin is an actin filament-associated protein found in smooth muscle and non-muscle cells. Calponin inhibits actin–myosin interaction in a manner that is prevented by protein kinase C (PKC)-catalysed phosphorylation of serine-175. To investigate the molecular basis of serine-175-mediated regulation, we examined the effect of phosphorylation on the conformation of calponin using monoclonal antibody (mAb) epitope analysis. Eight mAbs against different epitopes on chicken gizzard calponin were developed to monitor the conformational changes in calponin induced by PKC-mediated phosphorylation or serine-175 → alanine (S175A) substitution. The relative affinities of the mAbs for calponins immobilized on microtitre plates or bound to actin–tropomyosin thin filaments were determined, and epitope competitions between free and immobilized calponins were carried out. The changes in binding affinity between mAb paratopes and calponin epitopes demonstrate several serine-175 modification-induced conformational effects: (a) structures of calponin are reconfigured by serine-175 modification, supporting the regulatory function of serine-175; (b) there are submolecular structures unaffected by modification of serine-175 in both free and thin filament-associated calponins, suggesting that the serine-175-based conformational modulation is a targeted allosteric effect; (c) significant conformational changes are detected between free and thin filament-associated calponins, indicating two functional states of the molecular conformation; and (d) the different epitope characteristics between thin filament-bound and free calponins suggest that calponin is a flexible molecule, and the modifications of serine-175 may also determine the structural flexibility to increase the epitope accessibility. These results provide novel information concerning the structure–function relationships of calponin and its regulation by phosphorylation.

A role for serine-175 in modulating the molecular conformation of calponin

Calponin is an actin filament-associated protein found in smooth muscle and non-muscle cells. Calponin inhibits actin-myosin interaction in a manner that is prevented by protein kinase C (PKC)-catalysed phosphorylation of serine-175. To investigate the molecular basis of serine-175-mediated regulation, we examined the effect of phosphorylation on the conformation of calponin using monoclonal antibody (mAb) epitope analysis. Eight mAbs against different epitopes on chicken gizzard calponin were developed to monitor the conformational changes in calponin induced by PKC-mediated phosphorylation or serine-175-->alanine (S175A) substitution. The relative affinities of the mAbs for calponins immobilized on microtitre plates or bound to actin-tropomyosin thin filaments were determined, and epitope competitions between free and immobilized calponins were carried out. The changes in binding affinity between mAb paratopes and calponin epitopes demonstrate several serine-175 modification-induced conformational effects: (a) structures of calponin are reconfigured by serine-175 modification, supporting the regulatory function of serine-175; (b) there are submolecular structures unaffected by modification of serine-175 in both free and thin filament-associated calponins, suggesting that the serine-175-based conformational modulation is a targeted allosteric effect; (c) significant conformational changes are detected between free and thin filament-associated calponins, indicating two functional states of the molecular conformation; and (d) the different epitope characteristics between thin filament-bound and free calponins suggest that calponin is a flexible molecule, and the modifications of serine-175 may also determine the structural flexibility to increase the epitope accessibility. These results provide novel information concerning the structure-function relationships of calponin and its regulation by phosphorylation.

Molecular Basis of Ligand Recognition by Integrin α5β1: II. SPECIFICITY OF Arg-Gly-Asp BINDING IS DETERMINED BY Trp157 OF THE α SUBUNIT

Different beta(1) integrins bind Arg-Gly-Asp (RGD) peptides with differing specificities, suggesting a role for residues in the alpha subunit in determining ligand specificity. Integrin alpha(5)beta(1) has been shown to bind with high affinity to peptides containing an Arg-Gly-Asp-Gly-Trp (RGDGW) sequence but with relatively low affinity to other RGD peptides. The residues within the ligand-binding pocket that determine this specificity are currently unknown. A cyclic peptide containing the RGDGW sequence was found to strongly perturb the binding of the anti-alpha(5) monoclonal antibody (mAb) 16 to alpha(5)beta(1). In contrast, RGD peptides lacking the tryptophan residue acted as weak inhibitors of mAb 16 binding. The epitope of mAb 16 has previously been localized to a region of the alpha(5) subunit that contains Ser(156)-Trp(157). Mutation of Trp(157) (but not of Ser(156) or surrounding residues) to alanine blocked recognition of mAb 16 and perturbed the high affinity binding of RGDGW-containing peptides to alpha(5)beta(1). The same mutation also abrogated recognition of the alpha(5)beta(1)-specific ligand peptide Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Based on these findings, we propose that Trp(157) of alpha(5) participates in a hydrophobic interaction with the tryptophan residue in RGDGW, and that this interaction determines the specificity of alpha(5)beta(1) for RGDGW-containing peptides. Since the RGD sequence is recognized predominantly by amino acid residues on the beta(1) subunit, our results suggest that Trp(157) of alpha(5) must lie very close to these residues. Our findings therefore provide new insights into the structure of the ligand-binding pocket of alpha(5)beta(1).

Failure of membrane targeting causes the functional defect of two mutant sodium iodide symporters.

Molecular cloning of the sodium/iodide symporter (NIS) allowed identification of NIS gene mutations in patients with iodide trapping defect. Whereas various mutant human (h) NIS molecules display loss of function when expressed by transfection in mammalian cells, the precise mechanism(s) responsible for the functional abnormality of these proteins remains unknown. With the aim to explore these mechanisms in three natural hNIS mutants identified previously in patients with iodide trapping defect (Q267E, S515X, and C272X), we have prepared tools allowing direct measurement of the protein at its normal location in the plasma membrane. A COS-7 cell line was made by transfection that stably expressed high levels of wild-type hNIS. It was used to screen by flow cytometry monoclonal antibodies (mAbs) prepared from mice immunized against hNIS. Genetic immunization was performed by im injection of a wild-type hNIS complementary DNA construct, because this procedure has demonstrated the ability to produce antibodies recognizing native membrane proteins. One mAb that recognized an epitope of hNIS exposed on the extracellular side of the plasma membrane was selected for further studies. The epitope was localized on the sixth putative extracellular loop of the protein on the basis that the mAb did not recognize rat NIS, which exhibits major sequence differences in this segment. When this mAb was used to test by flow cytometry the expression of the three mutant hNIS proteins in transfected COS-7 cells, it detected similar amounts of wild-type, Q267E, and the S515X hNIS molecules in permeabilized cells. In contrast, only the wild-type hNIS was detected at the surface of nonpermeabilized cells. The C272X hNIS truncation mutant was not detected in intact or permeabilized cells. This is consistent with the absence of the mAb epitope from this mutant, which is expected to lack the sixth extracellular loop. Our data demonstrate that faulty membrane targeting is implicated in the mechanisms causing iodide trapping defect in the Q267E and S515X natural hNIS mutants.