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PubMed2003

The substrate specificity-determining amino acid code of 4-coumarate:CoA ligase.

To reveal the structural principles determining substrate specificity of 4-coumarate:CoA ligase (4CL), the crystal structure of the phenylalanine activation domain of gramicidin S synthetase was used as a template for homology modeling. According to our model, 12 amino acid residues lining the Arabidopsis 4CL isoform 2 (At4CL2) substrate binding pocket (SBP) function as a signature motif generally determining 4CL substrate specificity. We used this substrate specificity code to create At4CL2 gain-of-function mutants. By increasing the space within the SBP we generated ferulic- and sinapic acid-activating At4CL2 variants. Increasing the hydrophobicity of the SBP resulted in At4CL2 variants with strongly enhanced conversion of cinnamic acid. These enzyme variants are suitable tools for investigating and influencing metabolic channeling mediated by 4CL. Knowledge of the 4CL specificity code will facilitate the prediction of substrate preference of numerous, still uncharacterized 4CL-like proteins.

Amino Acid Motifs↗
PubMed1988

Non-determinant specificity of feedback immunosuppression by IgG antibodies injected after the antigen.

The determinant specificity of the IgG-mediated suppression of the humoral immune response in mice was studied. One hour before or 2.5, 6, 12, or 24 h after the injection of sheep erythrocytes (SRBC) or SRBC-TNP, CBA/Ca mice received SRBC-specific monoclonal IgG antibodies. The antibodies did not cross-react with TNP or goat erythrocytes, the latter an antigen which shows 30% cross-reactivity with SRBC. Five days later the determinant-specific plaque-forming cell response against SRBC and the non-determinant-specific response against goat erythrocytes and TNP were determined. Regardless of whether the antibodies were injected before or after the antigen, they suppressed not only the response to the antigenic determinant they bound to, but also the response to other epitopes on the same antigen. This shows that Fc parts of the IgG molecules play a crucial part in suppression of the in vivo antibody response even when, as in a natural situation, the antigen is presented to the immune system before the antibody.

Animals↗
PubMed2000

Docking domains and substrate-specificity determination for MAP kinases.

Signalling specificity in eukaryotic cells is maintained by several mechanisms. One mechanism by which mitogen-activated protein (MAP) kinases ensure their specificity of action is by interacting with their substrates through docking domains. These docking domains recruit the kinases to the correct substrates and enhance their fidelity and efficiency of action. Additional specificity determinants in the substrates serve to enhance the specificity of substrate phosphorylation by MAP kinases further.

Amino Acid Sequence↗
PubMed2000

Bacillus thuringiensis delta-endotoxin Cry1C domain III can function as a specificity determinant for Spodoptera exigua in different, but not all, Cry1-Cry1C hybrids.

In order to test our hypothesis that Bacillus thuringiensis delta-endotoxin Cry1Ca domain III functions as a determinant of specificity for Spodoptera exigua, regardless of the origins of domains I and II, we have constructed by cloning and in vivo recombination a collection of hybrid proteins containing domains I and II of various Cry1 toxins combined with domain III of Cry1Ca. Cry1Ab, Cry1Ac, Cry1Ba, Cry1Ea, and Cry1Fa all become more active against S. exigua when their domain III is replaced by (part of) that of Cry1Ca. This result shows that domain III of Cry1Ca is an important and versatile determinant of S. exigua specificity. The toxicity of the hybrids varied by a factor of 40, indicating that domain I and/or II modulate the activity as well. Cry1Da-Cry1Ca hybrids were an exception in that they were not significantly active against S. exigua or Manduca sexta, whereas both parental proteins were highly toxic. Incidentally, in a Cry1Ba-Cry1Ca hybrid, Cry1Ca domain III can also strongly increase toxicity for M. sexta.

Amino Acid Sequence↗
PubMed1990

Autoantigenic determinants on human thyroglobulin. I. Determinant specificities of murine monoclonal antibodies.

To map the antigenic determinants, a panel of 20 mouse monoclonal antibodies (mAbs) to native human thyroglobulin (Tg) was generated. Four criteria were established for distinguishing the determinants recognized by the various mAbs: (i) reactivity to Tgs from eight different species; (ii) reactivity to oxidized and reduced human Tg; (iii) the ability of thyroxine to inhibit binding of mAbs; and (iv) the pattern of antigenic determinant reactivity as determined in reciprocal competitive inhibition binding assays. Of 20 mAbs examined, 12 bound only to human Tg, 3 bound to all eight species tested, and 5 bound to human Tg and to Tg from at least one of the other seven species. Eight mAbs bound oxidized/reduced and native human Tg equally well, 2 bound to the oxidized/reduced protein better than to the native, 7 showed virtually no reactivity to oxidized/reduced human Tg, and 3 others reacted to the oxidized/reduced protein significantly less than they bound to the native. The binding of 5 mAbs to native human Tg was inhibited by thyroxine. The pattern of shared determinant specificities revealed that at least 12 determinant clusters were defined by the panel of mAbs. Among the 12 determinant clusters were 3 designated as immunodominant; 8 of 20 mAbs defined these immunodominant clusters. The four criteria taken together indicate that at least 19 different epitopes on human Tg could be distinguished by this panel of mAbs. These mAbs are useful for the study of determinant specificities of Tg autoantibodies in humans.

Animals↗
PubMedDate not supplied

Identification of H-specific determinants in flagellin of four Escherichia coli strains.

The H serogroup of Escherichia coli is determined by the flagellar antigen, flagellin. Sequence analysis of the flagellin gene, fliC, reveals a central variable region and the highly conserved N- and C-termini. This variable region has been shown to encode both H-specific and cross-reactive epitopes. Using polyclonal antibodies, we mapped the linear H-specific determinants in flagellin from four E. coli serotypes O157:H10, 0138:H14, O157:H42 and O157:H43. The specificity of all potential fragments was verified with 52 ECRC (Escherichia coli Reference Center) H-specific antisera. Our results indicated that: (a) a specific determinant of H10 flagellin (1263 bp long) maps to the region covering amino acid residues 305-331; (b) a specific determinant of H14 flagellin (1653 bp long) maps to the region covering amino acid residues 430-461; (c) a specific determinant of H42 flagellin (1281 bp long) maps to a region covering amino acid residues 171-201; and (d) a specific determinant of H43 flagellin (1506 bp long) maps to a region covering amino acid residues 200-260.

Amino Acid Sequence↗
PubMed1998

Statistical analysis of protein kinase specificity determinants.

The site and sequence specificity of protein kinases, as well as the role of the secondary structure and surface accessibility of the phosphorylation sites on substrate proteins, was statistically analyzed. The experimental data were collected from the literature and are available on the World Wide Web at http://www.cbs.dtu.dk/databases/PhosphoBase/. The set of data involved 1008 phosphorylatable sites in 406 proteins, which were phosphorylated by 58 protein kinases. It was found that there exists almost absolute Ser/Thr or Tyr specificity, with rare exceptions. The sequence specificity determinants were less strict and were located between positions -4 and +4 relative to the phosphorylation site. Secondary structure and surface accessibility predictions revealed that most of the phosphorylation sites were located on the surface of the target proteins.

Amino Acids↗
PubMed2003

Three distinct epitopes on the extracellular face of the glucagon receptor determine specificity for the glucagon amino terminus.

The glucagon and glucagon-like peptide-1 (GLP-1) receptors are homologous family B seven-transmembrane (7TM) G protein-coupled receptors, and they selectively recognize the homologous peptide hormones glucagon (29 amino acids) and GLP-1 (30-31 amino acids), respectively. The amino-terminal extracellular domain of the glucagon and GLP-1 receptors (140-150 amino acids) determines specificity for the carboxyl terminus of glucagon and GLP-1, respectively. In addition, the glucagon receptor core domain (7TM helices and connecting loops) strongly determines specificity for the glucagon amino terminus. Only 4 of 15 residues are divergent in the glucagon and GLP-1 amino termini; Ser2, Gln3, Tyr10, and Lys12 in glucagon and the corresponding Ala8, Glu9, Val16, and Ser18 in GLP-1. In this study, individual substitution of these four residues of glucagon with the corresponding residues of GLP-1 decreased the affinity and potency at the glucagon receptor relative to glucagon. Substitution of distinct segments of the glucagon receptor core domain with the corresponding segments of the GLP-1 receptor rescued the affinity and potency of specific glucagon analogs. Site-directed mutagenesis identified the Asp385 --> Glu glucagon receptor mutant that specifically rescued Ala2-glucagon. The results show that three distinct epitopes of the glucagon receptor core domain determine specificity for the N terminus of glucagon. We suggest a glucagon receptor binding model in which the extracellular ends of TM2 and TM7 are close to and determine specificity for Gln3 and Ser2 of glucagon, respectively. Furthermore, the second extracellular loop and/or proximal segments of TM4 and/or TM5 are close to and determine specificity for Lys12 of glucagon.

Amino Acid Sequence↗
PubMed2000

Mutational investigation of the specificity determining region of the Src SH2 domain.

SH2 domains are protein modules which bind tyrosine phosphorylated sequences in many signaling pathways. These domains contain two regions with specialized functions: residues in one region form a deep pocket into which the phosphotyrosine of the target inserts, while the other region contains the so-called "specificity determining residues" which interact with the three residues C-terminal to the phosphotyrosine in the target. Here, titration calorimetry and site-directed mutagenesis have been used to probe the importance of eight specificity determining residues of the SH2 domain of the Src kinase involved in contacts with its tyrosine phosphorylated consensus peptide target (sequence pYEEI where pY indicates a phosphotyrosine). Mutating six of these eight residues to Ala individually, resulted in a threefold or less loss in binding affinity; hence the majority of the residues in the specificity determining region are by themselves of minimal importance for binding. Two residues were found to have significant effects on binding: Tyr betaD5 and Lys betaD3. Tyr betaD5 was the most crucial residue as evidenced by the 30-fold loss in affinity when Tyr betaD5 is mutated to Ile. However, while this mutation eliminated the specificity of the Src SH2 domain for the pYEEI peptide sequence, it was not sufficient to switch the specificity of the Src SH2 domain to that of a related SH2 domain which has an Ile at the betaD5 position. Mutation of Lys betaD3 to an Ala residue resulted in a modest reduction in binding affinity (sevenfold). It is interesting that this mutation resulted in a change of specificity affecting the selection of the +1 position residue C-terminal to the phosphotyrosine. Except for the Lys betaD3-+1 Glu interaction which is significantly coupled, only weak energetic coupling was observed across the binding interface, as assessed using double mutant cycles. The results of this study suggest that interactions involving the specificity determining region of SH2 domains may be insufficient by themselves to target single SH2 domains to particular phosphorylated sites.

Amino Acid Substitution↗
PubMed1983

Antigenic determinants of the HLA-B7 molecule; Bw6- and B7-specific determinants are spatially separate.

Monoclonal antibodies that bind HLA-B7 were used to show that the B7-specific determinant is at a topologically different site from that of the broad polymorphic, Bw6 determinant. The relationship to other antigenic determinants defined by monoclonal antibodies was also assessed. These results were independently obtained in four ways: (1) by cellular blocking assays, in which there was no inhibition of 125I-B7 antibody binding in the presence of Bw6 antibody and no inhibition of 125I-Bw6 antibody binding in the presence of B7 antibody; (2) cellular binding assays under conditions of antibody saturation showed the binding of B7-specific and Bw6 antibodies were additive; (3) solid-phase radioimmune assays demonstrated enhancement between B7-specific and Bw6 antibodies; (4) analysis of antigen antibody complexes by size-exclusion high pressure liquid chromatography showed Bw6 and B7 antibodies could form tetramolecular complexes with papain-solubilized HLA-B7. Limitations were encountered in using cellular blocking assays to map antigenic determinants of HLA-B7. These assays can produce blocking in cases where two antibodies are not competing for an antigenic determinant. Mapping antigenic determinants with assays using purified HLA-B7 as the antigenic target, in addition to cell-based assays, provided a more accurate picture.

Antibodies, Monoclonal↗
PubMed1995

Sequence context of antisense RelA/NF-kappa B phosphorothioates determines specificity.

The use of antisense oligomers to achieve inhibition of gene expression is complicated by frequent non-specific effects, and even the control oligomers often exhibit sequence-specific effects. We have recently shown that in diverse tumor-derived cell lines, a 24mer phosphorothioate oligomer antisense to the relA subunit of NF-kappa B transcription factor causes a block of cellular adhesion, inhibition of nuclear NF-kappa B and Sp1 DNA-binding activity and inhibition of tumor cell growth in vitro and in vivo. In this study we use the same model to attempt to define the limits of antisense specificity. We demonstrate that single base pair substitution can virtually abolish the antisense activity. The relative position of mismatches within the antisense sequence is critical to the loss of activity. Our results further indicate that antisense specificity is determined not only by the content of the sequence but also by its occurrence with reference to the surrounding sequences.

Animals↗
PubMed1997

[Specific determination of deltamethrin and tralomethrin by preparative HPLC and GC-ECD].

Tralomethrin quickly changes to deltamethrin in gas chromatograph by debromination. Therefore deltamethrin and tralomethrin are not able to be distinguished by gas chromatographic determination. A method for specific determination of deltamethrin and tralomethrin was established. The method consists of fractionation by high performance liquid chromatography and determination by gas chromatography. Recoveries of deltamethrin and tralomethrin spiked to three agricultural products were from 42 to 78% and from 18 to 76%, respectively with relative standard deviations ranging from 0.3 to 15%. From the analysis of samples spiked with tralomethrin, it was found that a part of tralomethrin quickly changed to deltamethrin in homogenate of agricultural products. The addition of phosphoric acid to the homogenate did not prevent the change of tralomethrin.

Chromatography, Gas↗
PubMedDate not supplied

Length of the antibody heavy chain complementarity determining region 3 as a specificity-determining factor.

The antigen binding site of an antibody is made up of residues residing in six hypervariable loops of the heavy and light chains. In most cases several or all of these loops are required for the establishment of the antigen-binding surface. Five of these loops display a limited diversity in length and sequence while the third complementarity determining region (CDR) of the heavy chain is highly different between antibodies not only with respect to sequence but also with respect to length. Its extensive diversity is a key component in the establishment of binding sites allowing for the recognition of essentially any antigen by humoral immunity. The relative importance of its sequence vs its length diversity in this context is however, not very well established. To investigate this matter further we have used an approach employing combinatorial antibody libraries and antigen-specific selection in the search for CDRH3 length and sequence diversity compatible with a given antigen specificity, the major antigenic determinant on the tumour-associated antigen mucin-1. In this way we have now defined heavy chain CDR3 length as a critical parameter in the creation of an antigen-specific binding site. We also propose that this may reflect a dependence of a particular structure of this hypervariable loop, the major carrier of diversity in the binding site, for establishment of a given specificity.

Antibody Specificity↗
PubMed2001

Characterizing Class I WW domains defines key specificity determinants and generates mutant domains with novel specificities.

INTRODUCTION: WW domains are small protein interaction modules found in a wide range of eukaryotic signaling and structural proteins. Five classes of WW domains have been annotated to date, where each class is largely defined by the type of peptide ligand selected, rather than by similarities within WW domains. Class I WW domains bind Pro-Pro-Xxx-Tyr containing ligands, and it would be of interest to determine residues within the domains that determine this specificity. RESULTS: Fourteen WW domains selected Leu/Pro-Pro-Xxx-Tyr containing peptides ligands via phage display and were thus designated as Class 1 WW domains. These domains include those present in human YAP (hYAP) and WWP3, as well as those found in ubiquitin protein ligases of the Nedd4 family, including mouse Nedd4 (mNedd4), WWP1, WWP2 and Rsp5. Comparing the primary structures of these WW domains highlighted a set of highly conserved residues, in addition to those originally noted to occur within WW domains. Substitutions at two of these conserved positions completely inhibited ligand binding, whereas substitution at a non-conserved position did not. Moreover, mutant WW domains containing substitutions at conserved positions bound novel peptide ligands. CONCLUSIONS: Class I WW domains contain a highly conserved set of residues that are important in selecting Pro-Xxx-Tyr containing peptide ligands. The presence of these residues within an uncharacterized WW domain can be used to predict its ability to bind Pro-Xxx-Tyr containing peptide ligands.

Amino Acid Sequence↗
PubMed1984

Monoclonal antibodies reactive with idiotypic and variable-region specific determinants on human immunoglobulins.

Monoclonal antibody JG-B1, specific for the human VKIIIb sub-subgroup of L chains, and JG-B4 specific for an idiotypic determinant on Glo, a monoclonal human IgM-VKIIIb anti-IgG, were produced and characterized. The VKIIIb determinant was detected on L chains alone and intact immunoglobulins with VKIIIb L chains. However, the idiotypic determinant was expressed only on IgM-Glo and required association of H and L chains. Binding of the immunogen Glo, to its antigen-IgG partially inhibited anti-idiotype and anti-VKIIIb binding. Cross-inhibition experiments demonstrated that intact pentameric IgM-Glo expressed one-half the number of idiotypic sites as VKIIIb determinants. However, Glo half-molecules expressed equal numbers of idiotypic and VKIIIb determinants. This is the first described monoclonal antibody produced by hybridoma technology which recognizes an antigenic determinant specific for a single variable region in intact immunoglobulin.

Animals↗
PubMed1985

Antibody-mediated suppression of the immune response is determinant specific.

It was studied whether antibody-mediated suppression of the immune response was determinant specific. Fluorescein isothiocyanate (FITC)-conjugated sheep red cells were used as the immunogen and antibodies were raised both against the carrier sheep red cells and the hapten FITC. When these antibodies were injected 1-3 h after the immunogen they only suppressed the immune response to the corresponding determinant. Anti-carrier antibodies usually enhanced the response to the hapten. Therefore, antibody-mediated suppression of the immune response is determinant specific and cannot be mediated in vivo to a detectable extent by the Fc part of the antibodies.

Animals↗
PubMed1998

Predominant recognition of species-specific determinants of the GroES homologues from Mycobacterium leprae and M. tuberculosis.

The Mycobacterium leprae and M. tuberculosis 10,000 MW heat-shock protein homologues of GroES have previously been identified as major immunogens for human T cells. We used synthetic peptides to characterize the determinants recognized by murine T cells. The findings suggest that, despite 90% sequence identity between these two proteins, T cells recognize prominently the species-specific determinants localized within amino acid residues 21-40 and 49-72. Analysis of the molecular determinants of species-specificity for the M. leprae GroES sequence 25-40, using T-cell hybridomas and major histocompatibility complex (MHC)-binding assays, led to the identification of epitope cores and critical residues. Interestingly, closely overlapping epitope cores were found to be restricted by either H-2Ad (24-34) or H-2Ed (28-34). Furthermore, the site recognized by the M. leprae-specific monoclonal antibodies ML06 and ML10 was also localized in the overlapping sequences 25-31 and 25-29. In conclusion, we demonstrated that immunodominant species-specific T- and B-cell epitopes can be found in a mycobacterial heat-shock protein despite its highly conserved amino acid sequence. This finding suggests the feasibility of identifying a sufficient number of M. leprae-specific determinants for a composite T-cell immunodiagnostic reagent for tuberculoid leprosy.

Amino Acid Sequence↗
PubMed2006

Protease specificity determination by using cellular libraries of peptide substrates (CLiPS).

We report a general combinatorial approach to identify optimal substrates of a given protease by using quantitative kinetic screening of cellular libraries of peptide substrates (CLiPS). A whole-cell protease activity assay was developed by displaying fluorescent reporter substrates on the surface of Escherichia coli as N-terminal fusions. This approach enabled generation of substrate libraries of arbitrary amino acid composition and length that are self-renewing. Substrate hydrolysis by a target protease was measured quantitatively via changes in whole-cell fluorescence by using FACS. FACS enabled efficient screening to identify optimal substrates for a given protease and characterize their cleavage kinetics. The utility of CLiPS was demonstrated by determining the substrate specificity of two unrelated proteases, caspase-3 and enteropeptidase (or enterokinase). CLiPS unambiguously identified the caspase-3 consensus cleavage sequence DXVDG. Enteropeptidase was unexpectedly promiscuous, but exhibited a preference for substrates with the motif (D/E)RM, which were cleaved substantially faster than the canonical DDDDK recognition sequence, widely used for protein purification. CLiPS provides a straightforward and versatile approach to determine protease specificity and discover optimal substrates on the basis of cleavage kinetics.

Amino Acid Sequence↗