A V region mutation in a phosphocholine-binding monoclonal antibody results in loss of antigen binding

A V region mutant producing an antibody that had lost the ability to bind phosphocholine was isolated from a hybridoma producing a germline encoded T15 antibody. The mutation resulted in a single aspartic acid to asparagine substitution at residue 95 of the H chain V region. This confirms that the aspartic acid at residue 95 plays a major role in Ag binding. The results also suggest that somatic cell genetic techniques can be used to generate mAb with useful changes in Ag binding.     

Direct eIF2-eIF3 contact in the multifactor complex is important for translation initiation in vivo

Translation initiation factor 3 (eIF3) of Saccharo myces cerevisiae forms a multifactor complex (MFC) with eIFs 1, 2, 5 and Met-tRNA(i)(Met). We previously constructed a subunit interaction model for the MFC. Here we incorporated affinity tags into the three largest eIF3 subunits (eIF3a/TIF32, eIF3b/PRT1 and eIF3c/NIP1) and deleted predicted binding domains in each tagged protein. By characterizing the mutant subcomplexes, we confirmed all key predictions of our model and uncovered new interactions of NIP1 with PRT1 and of TIF32 with eIF1. In addition to the contact between eIF2 and the N-terminal domain (NTD) of NIP1 bridged by eIF5, the C-terminal domain (CTD) of TIF32 binds eIF2 directly and is required for eIF2-eIF3 association in vivo. Overexpressing a CTD-less form of TIF32 exacerbated the initiation defect of an eIF5 mutation that weakens the NIP1-eIF5-eIF2 connection. Thus, the two independent eIF2-eIF3 contacts have additive effects on translation in vivo. Overexpressing the NIP1-NTD sequestered eIF1-eIF5-eIF2 in a defective subcomplex that derepressed GCN4 translation, providing the first in vivo evidence that association with eIF3 promotes binding of eIF2 and Met-tRNA(i)(Met) to 40S ribosomes.     

C3, C4, and the terminal complement complex differ from C1q by binding predominantly to the antigenic part of solid phase immune complexes

The binding of the C components C1q, C4, C3, the terminal C5b-9 complement complex (TCC) and S protein to immune complexes was studied. The hapten 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) conjugated to BSA was adsorbed to polystyrene plates and reacted with a human IgG3-mouse chimeric anti-NIP antibody. After addition of serum a dose-dependent binding of C1q, C4, C3, and TCC to the immune complexes was found. An increase in the amount of NIP-BSA was associated with an increase in the binding of TCC and a decrease in the binding of S-protein. After addition of soluble NIP only 4 to 6% of the anti-NIP antibody remained bound to the Ag. C1q showed diminished binding after addition of NIP, whereas C4, C3, and TCC quantitatively remained bound to the Ag. Binding of TCC to the immune complexes was also found in an alternative assay, in which the anti-NIP antibody was adsorbed to the solid phase before NIP-BSA and an additional layer of anti-NIP antibody were added. The supernatants from the solid phase assay were tested for C3 activation and formation of the fluid phase TCC (SC5b-9). Activation of the C3 was reflected in the fluid phase by a dose-dependent increase in C3 activation products. This was not seen for TCC despite increased binding to the solid phase.     

Localized mutagenesis defines regions of the Bacillus thuringiensis delta-endotoxin involved in toxicity and specificity.

Bacillus thuringiensis produces a variety of delta-endotoxins which bind to specific receptors in insect larval midguts. Following insertion into the membrane there is an alteration of ion flux culminating in osmotic lysis. Mutagenic oligonucleotides were used to define regions in one of these toxins involved in specificity and toxicity. One region is highly conserved among all toxins sequenced to date and many mutations resulted in loss of toxicity for three test Lepidoptera. The mutant toxins had lost the capacity to inhibit K(+)-dependent amino acid transport into larval midgut vesicles, but there was no effect on their ability to compete with wild type toxin for binding. The results are consistent with this amphiphilic helical region of the toxin being essential for toxicity. A second mutagenized region overlapped a portion of another potential amphiphilic helix. Mutations of only 2 residues, Ala-92 and Arg-93, resulted in loss of toxicity for two lepidopteran larvae but some activity remained for a third. The A92D mutant toxin competed with the wild type toxin for binding to vesicles prepared from midguts from the sensitive but not from the insensitive larvae. Decreased toxicity was also found when this mutation was transferred to two other related protoxin genes. A number of mutations of each of these residues was analyzed and selective loss of toxicity correlated with the absence of a positive charge. Despite being distal from the presumptive specificity domain, 1 or both of these residues must have an important role in the specific binding of toxins.     

Computer analysis of mutations that affect antibody specificity

The mouse hybridoma cell line 40-150 secretes antibodies with high affinity toward the cardiac glycosides digoxin and digitoxin. A spontaneous mutant, 40-150 A2.4, produces an antibody which carries a single residue mutation, Ser----Arg, in its heavy chain (H94) and has an altered specificity. A second-order mutant, 40-150 A2.4 P.10, produces two antibody molecules, one the same as 40-150 A2.4, the other lacking two residues at the N-terminus of its H chain, and having a specificity profile approaching that of 40-150 antibody. The N-terminus and the position H94 are distant from the antigen-binding site of the antibody; thus, the structural basis of the specificity changes was not immediately clear. Approximate structures of the 40-150 antibody and its mutants were constructed in the computer, based on atomic coordinates of the homologous mouse antibody McPC 603. Using the program CONGEN, the torsional space of the polypeptide backbone and side chains around position H94 was uniformly sampled, and the lowest energy conformations were analyzed in detail. The results indicate that when Arg-H94 is substituted for Ser, Arg-H94 can hydrogen bond to side chains of Asp-H101, Arg-L46, and Asp-L55. This results in a change in the surface of the combining site which may account for the affinity changes. Deletion of the two N-terminal residues increases solvent accessibility of Arg-H94. The solvation may cause a hydrogen bond between Arg-H94 and Asp-H101 to be lost, restoring the structure to one similar to that of 40-150.     

Genes of pyelonephritogenic E. coli required for digalactoside-specific agglutination of human cells.

Most pyelonephritic Escherichia coli strains bind to digalactoside-containing glycolipids on uroepithelial cells. Purified Pap pili (pili associated with pyelonephritis) show the same binding specificity. A non-polar mutation early in the papA pilin gene abolishes formation of Pap pili but does not affect the degree of digalactoside-specific hemagglutination. Three novel pap genes, papE , papF and papG are defined in this report. The papF and papG gene products are both required for digalactoside-specific agglutination by whole bacteria cells as well as for agglutination by pilus preparations. Pili prepared from a papE mutant have lost their binding ability although whole cells from this mutant retain it, implying an adhesin anchoring role for the papE gene product. A mutant with lesions both in the papA and the papE genes does not mediate digalactoside-specific agglutination. The implications of this finding for pilus biogenesis are discussed.     

Molecular characterization of the β‐amyloid(4‐10) epitope of plaque specific Aβ antibodies by affinity‐mass spectrometry using alanine site mutation

Alzheimer disease is a neurodegenerative disease affecting an increasing number of patients worldwide. Current therapeutic strategies are directed to molecules capable to block the aggregation of the β‐amyloid(1‐42) (Aβ) peptide and its shorter naturally occurring peptide fragments into toxic oligomers and amyloid fibrils. Aβ‐specific antibodies have been recently developed as powerful antiaggregation tools. The identification and functional characterization of the epitope structures of Aβ antibodies contributes to the elucidation of their mechanism of action in the human organism. In previous studies, the Aβ(4‐10) peptide has been identified as an epitope for the polyclonal anti‐Aβ(1‐42) antibody that has been shown capable to reduce amyloid deposition in a transgenic Alzheimer disease mouse model. To determine the functional significance of the amino acid residues involved in binding to the antibody, we report here the effects of alanine single‐site mutations within the Aβ‐epitope sequence on the antigen‐antibody interaction. Specific identification of the essential affinity preserving mutant peptides was obtained by exposing a Sepharose‐immobilized antibody column to an equimolar mixture of mutant peptides, followed by analysis of bound peptides using high‐resolution MALDI‐Fourier transform‐Ion Cyclotron Resonance mass spectrometry. For the polyclonal antibody, affinity was preserved in the H6A, D7A, S8A, and G9A mutants but was lost in the F4, R5, and Y10 mutants, indicating these residues as essential amino acids for binding. Enzyme‐linked immunosorbent assays confirmed the binding differences of the mutant peptides to the polyclonal antibody. In contrast, the mass spectrometric analysis of the mutant Aβ(4‐10) peptides upon affinity binding to a monoclonal anti‐Aβ(1‐17) antibody showed complete loss of binding by Ala‐site mutation of any residue of the Aβ(4‐10) epitope. Surface plasmon resonance affinity determination of wild‐type Aβ(1‐17) to the monoclonal Aβ antibody provided a binding constant K_D in the low nanomolar range. These results provide valuable information in the elucidation of the binding mechanism and the development of Aβ‐specific antibodies with improved therapeutic efficacy.     

Interdependence of calcium and cobalamin binding by wild-type and mutant BtuB protein in the outer membrane of Escherichia coli.

The binding of calcium and cobalamin to outer membranes from cells of Escherichia coli that contained amplified levels of wild-type or mutant btuB was studied. The mutant (BBam50) had an aspartyl-prolyl dipeptide inserted after the original 50th amino acid residue of the mature BtuB protein, which is within a region that shows extensive homology with the ferric siderophore receptors. This insertion resulted in cleavage of the BtuB in two places. The larger form retained the insertion but had lost 11 amino acid residues from the amino terminus. The smaller form was cut at the insertion site. Both the wild-type protein and the larger form of mutant BtuB showed calcium-dependent cobalamin binding with the same affinity for cobalamin, although the mutant had a much lower affinity for calcium. The smaller form of the mutant BtuB protein had a greatly reduced affinity for cobalamin, which was probably the result of inactivation of the cobalamin-dependent calcium-binding site. Cobalamin-dependent calcium binding was measured in wild-type BtuB preparations and was found to have the same corrinoid specificity and response to various corrinoid concentrations as shown previously for cobalamin binding. The results are consistent with a role for calcium in the cobalamin pump of the outer membrane of E. coli and show that a conserved part of the BtuB protein is required for the cobalamin-dependent binding of calcium.     

Conversion of cyclodextrin glycosyltransferase into a starch hydrolase by directed evolution: the role of alanine 230 in acceptor subsite +1

Cyclodextrin glycosyltransferase (CGTase) preferably catalyzes transglycosylation reactions, whereas many other alpha-amylase family enzymes are hydrolases. Despite the availability of three-dimensional structures of several transglycosylases and hydrolases of this family, the factors that determine the hydrolysis and transglycosylation specificity are far from understood. To identify the amino acid residues that are critical for the transglycosylation reaction specificity, we carried out error-prone PCR mutagenesis and screened for Bacillus circulans strain 251 CGTase mutants with increased hydrolytic activity. After three rounds of mutagenesis the hydrolytic activity had increased 90-fold, reaching the highest hydrolytic activity ever reported for a CGTase. The single mutation with the largest effect (A230V) occurred in a residue not studied before. The structure of this A230V mutant suggests that the larger valine side chain hinders substrate binding at acceptor subsite +1, although not to the extent that catalysis is impossible. The much higher hydrolytic than transglycosylation activity of this mutant indicates that the use of sugar acceptors is hindered especially. This observation is in favor of a proposed induced-fit mechanism, in which sugar acceptor binding at acceptor subsite +1 activates the enzyme in transglycosylation [Uitdehaag et al. (2000) Biochemistry 39, 7772-7780]. As the A230V mutation introduces steric hindrance at subsite +1, this mutation is expected to negatively affect the use of sugar acceptors. Thus, the characteristics of mutant A230V strongly support the existence of the proposed induced-fit mechanism in which sugar acceptor binding activates CGTase in a transglycosylation reaction.     

A Two-in-One antibody engineered from a humanized interleukin 4 antibody through mutation in heavy chain complementarity-determining regions

A mono-specific antibody may recruit a second antigen binding specificity, thus converting to a dual-specific Two-in-One antibody through mutation at the light chain complementarity-determining regions (CDRs). It is, however, unknown whether mutation at the heavy chain CDRs may evolve such dual specificity. Herein, we examined the CDRs of a humanized interleukin 4 (IL4) antibody using alanine scanning and structural modeling, designed libraries of mutants in regions that tolerate mutation, and isolated dual specific antibodies harboring mutation at the heavy chain CDRs only. We then affinity improved an IL4/IL5 dual specific antibody to variants with dissociation constants in the low nanomolar range for both antigens. The results demonstrate the full capacity of antibodies to evolve dual binding specificity.     

A Two-in-One antibody engineered from a humanized interleukin 4 antibody through mutation in heavy chain complementarity-determining regions

A mono-specific antibody may recruit a second antigen binding specificity, thus converting to a dual-specific Two-in-One antibody through mutation at the light chain complementarity-determining regions (CDRs). It is, however, unknown whether mutation at the heavy chain CDRs may evolve such dual specificity. Herein, we examined the CDRs of a humanized interleukin 4 (IL4) antibody using alanine scanning and structural modeling, designed libraries of mutants in regions that tolerate mutation, and isolated dual specific antibodies harboring mutation at the heavy chain CDRs only. We then affinity improved an IL4/IL5 dual specific antibody to variants with dissociation constants in the low nanomolar range for both antigens. The results demonstrate the full capacity of antibodies to evolve dual binding specificity.     

Contribution of a single heavy chain residue to specificity of an anti-digoxin monoclonal antibody.

Two distinct spontaneous variants of the murine anti-digoxin hybridoma 26-10 were isolated by fluorescence-activated cell sorting for reduced affinity of surface antibody for antigen. Nucleotide and partial amino acid sequencing of the variant antibody variable regions revealed that 1 variant had a single amino acid substitution: Lys for Asn at heavy chain position 35. The second variant antibody had 2 heavy chain substitutions: Tyr for Asn at position 35, and Met for Arg at position 38. Mutagenesis experiments confirmed that the position 35 substitutions were solely responsible for the markedly reduced affinity of both variant antibodies. Several mutants with more conservative position 35 substitutions were engineered to ascertain the contribution of Asn 35 to the binding of digoxin to antibody 26-10. Replacement of Asn with Gln reduced affinity for digoxin 10-fold relative to the wild-type antibody, but maintained wild-type fine specificity for cardiac glycoside analogues. All other substitutions (Val, Thr, Leu, Ala, and Asp) reduced affinity by at least 90-fold and caused distinct shifts in fine specificity. The Ala mutant demonstrated greatly increased relative affinities for 16-acetylated haptens and haptens with a saturated lactone. The X-ray crystal structure of the 26-10 Fab in complex with digoxin (Jeffrey PD et al., 1993, Proc Natl Acad Sci USA 90:10310-10314) reveals that the position 35 Asn contacts hapten and forms hydrogen bonds with 2 other contact residues. The reductions in affinity of the position 35 mutants for digoxin are greater than expected based upon the small hapten contact area provided by the wild-type Asn. We therefore performed molecular modeling experiments which suggested that substitution of Gln or Asp can maintain these hydrogen bonds whereas the other substituted side chains cannot. The altered binding of the Asp mutant may be due to the introduction of a negative charge. The similarities in binding of the wild-type and Gln-mutant antibodies, however, suggest that these hydrogen bonds are important for maintaining the architecture of the binding site and therefore the affinity and specificity of this antibody. The Ala mutant eliminates the wild-type hydrogen bonding, and molecular modeling suggests that the reduced side-chain volume also provides space that can accommodate a congener with a 16-acetyl group or saturated lactone, accounting for the altered fine specificity of this antibody.     

Identification of critical contact residues in the NC41 epitope of a subtype N9 influenza virus neuraminidase

We have examined amino acids on influenza virus neuraminidase (NA) subtype N9 (A/tern/Australia/G70c/75) which are in contact with monoclonal antibody NC41 to analyze individual interactions important for antibody recognition. The crystal structure of NA complexed with NC41 Fab¹ shows antibody contacts at 19 amino acid residues on the NA surface which are localized on five polypeptide loops surrounding the enzyme active site. Fifteen mutant NA genes were constructed to encode a protein which contained a single amino acid substitution and these were tested for effects of the replacement on NC41 binding. Our data revealed that NAs with changes at 368, 400, and 434 completely lost NC41 recognition. NAs with side chains replaced at residues 346 and 373 exhibited binding reduced to less than 50% of wild-type binding. Changes in seven other contacting residues, including substituted side chains which differed considerably from wild-type NA in size and charge, had no significant effect on NC41 binding. These results indicate that only a few of the many residues which make up an epitope are crucial for interaction and provide the critical contacts required for antibody recognition. This implies that antibody escape mutants are selected only if they contain changes at these crucial sites, or changes which introduce bulky side chains that sterically prevent antibody attachment. © 1993 Wiley-Liss, Inc.     

A single binding site mediates resistance- and disease-associated activities of the effector protein NIP1 from the barley pathogen Rhynchosporium secalis

The effector protein NIP1 from the barley (Hordeum vulgare) pathogen Rhynchosporium secalis specifically induces the synthesis of defense-related proteins in cultivars of barley expressing the complementary resistance gene, Rrs1. In addition, it stimulates the activity of the barley plasma membrane H(+)-ATPase in a genotype-unspecific manner and it induces necrotic lesions in leaf tissues of barley and other cereal plant species. NIP1 variants type I and II, which display quantitative differences in their activities as elicitor and H(+)-ATPase stimulator, and the inactive mutant variants type III* and type IV*, were produced in Escherichia coli. Binding studies using (125)I-NIP1 type I revealed a single class of binding sites with identical binding characteristics in microsomes from near-isogenic resistant (Rrs1) and susceptible (rrs1) barley. Binding was specific, reversible, and saturable, and saturation ligand-binding experiments yielded a K(d) of 5.6 nm. A binding site was also found in rye (Secale cereale) and the nonhost species wheat (Triticum aestivum), oat (Avena sativa), and maize (Zea mays), but not in Arabidopsis (Arabidopsis thaliana). For NIP1 types I and II, equilibrium competition-binding experiments revealed a correlation between the difference in their affinities to the binding site and the differences in their elicitor activity and H(+)-ATPase stimulation, indicating a single target molecule to mediate both activities. In contrast, the inactive proteins type III* and type IV* are both characterized by high affinities similar to type I, suggesting that binding of NIP1 to this target is not sufficient for its activities.     

Interactions of eukaryotic translation initiation factor 3 (eIF3) subunit NIP1/c with eIF1 and eIF5 promote preinitiation complex assembly and regulate start codon selection

The N-terminal domain (NTD) of NIP1/eIF3c interacts directly with eIF1 and eIF5 and indirectly through eIF5 with the eIF2-GTP-Met-tRNA(i)(Met) ternary complex (TC) to form the multifactor complex (MFC). We investigated the physiological importance of these interactions by mutating 16 segments spanning the NIP1-NTD. Mutations in multiple segments reduced the binding of eIF1 or eIF5 to the NIP1-NTD. Mutating a C-terminal segment of the NIP1-NTD increased utilization of UUG start codons (Sui(-) phenotype) and was lethal in cells expressing eIF5-G31R that is hyperactive in stimulating GTP hydrolysis by the TC at AUG codons. Both effects of this NIP1 mutation were suppressed by eIF1 overexpression, as was the Sui(-) phenotype conferred by eIF5-G31R. Mutations in two N-terminal segments of the NIP1-NTD suppressed the Sui(-) phenotypes produced by the eIF1-D83G and eIF5-G31R mutations. From these and other findings, we propose that the NIP1-NTD coordinates an interaction between eIF1 and eIF5 that inhibits GTP hydrolysis at non-AUG codons. Two NIP1-NTD mutations were found to derepress GCN4 translation in a manner suppressed by overexpressing the TC, indicating that MFC formation stimulates TC recruitment to 40S ribosomes. Thus, the NIP1-NTD is required for efficient assembly of preinitiation complexes and also regulates the selection of AUG start codons in vivo.     

Tuning the specificity of a Two-in-One Fab against three angiogenic antigens by fully utilizing the information of deep mutational scanning

Monoclonal antibodies developed for therapeutic or diagnostic purposes need to demonstrate highly defined binding specificity profiles. Engineering of an antibody to enhance or reduce binding to related antigens is often needed to achieve the desired biologic activity without safety concern. Here, we describe a deep sequencing-aided engineering strategy to fine-tune the specificity of an angiopoietin-2 (Ang2)/vascular endothelial growth factor (VEGF) dual action Fab, 5A12.1 for the treatment of age-related macular degeneration. This antibody utilizes overlapping complementarity-determining region (CDR) sites for dual Ang2/VEGF interaction with K_D in the sub-nanomolar range. However, it also exhibits significant (K_D of 4 nM) binding to angiopoietin-1, which has high sequence identity with Ang2. We generated a large phage-displayed library of 5A12.1 Fab variants with all possible single mutations in the 6 CDRs. By tracking the change of prevalence of each mutation during various selection conditions, we identified 35 mutations predicted to decrease the affinity for Ang1 while maintaining the affinity for Ang2 and VEGF. We confirmed the specificity profiles for 25 of these single mutations as Fab protein. Structural analysis showed that some of the Fab mutations cluster near a potential Ang1/2 epitope residue that differs in the 2 proteins, while others are up to 15 Å away from the antigen-binding site and likely influence the binding interaction remotely. The approach presented here provides a robust and efficient method for specificity engineering that does not require prior knowledge of the antigen antibody interaction and can be broadly applied to antibody specificity engineering projects.     

Isolation, characterization, and physical mapping of a pseudorabies virus mutant containing antigenically altered gp50.

A pseudorabies virus variant ( mar197 -1) containing a mutation in a viral glycoprotein with a molecular weight of 50,000 ( gp50 ) was isolated by selecting for resistance to a neurtralizing monoclonal antibody ( MCA50 -1) directed against gp50 . This mutant was completely resistant to neutralization with MCA50 -1 in the presence or absence of complement, and was therefore defined as a mar (monoclonal-antibody-resistant) mutant. The mutation did not affect neutralization with polyvalent immune serum. The mar197 -1 mutant synthesized and processed gp50 normally, but the mutation prevented the binding and immunoprecipitation of gp50 by MCA50 -1. Thus, the mutation was within the structural portion of the gp50 gene affecting the epitope of the monoclonal antibody. The mutation was mapped by marker rescue with cloned pseudorabies restriction enzyme fragments to the short region of the pseudorabies genome between 0.813 and 0.832 map units. This is equivalent to a 2.1-kilobase-pair region.     

Identification of the amino acid residues of the platelet glycoprotein Ib (GPIb) essential for the von Willebrand factor binding by clustered charged-to-alanine scanning mutagenesis

At the site of vascular injury, von Willebrand factor (VWF) mediates platelet adhesion to subendothelial connective tissue through binding to the N-terminal domain of the alpha chain of platelet glycoprotein Ib (GPIbalpha). To elucidate the molecular mechanisms of the binding, we have employed charged-to-alanine scanning mutagenesis of the soluble fragment containing the N-terminal 287 amino acids of GPIbalpha. Sixty-two charged amino acids were changed singly or in small clusters, and 38 mutant constructs were expressed in the supernatant of 293T cells. Each mutant was assayed for binding to several monoclonal antibodies for human GPIbalpha and for ristocetin-induced and botrocetin-induced binding of 125I-labeled human VWF. Mutations at Glu128, Glu172, and Asp175 specifically decreased both ristocetin- and botrocetin-induced VWF binding, suggesting that these sites are important for VWF binding of platelet GPIb. Monoclonal antibody 6D1 inhibited ristocetin- and botrocetin-induced VWF binding, and a mutation at Glu125 specifically reduced the binding to 6D1. In contrast, antibody HPL7 had no effect for VWF binding, and mutant E121A reduced the HPL7 binding. Mutations at His12 and Glu14 decreased the ristocetin-induced VWF binding with normal botrocetin-induced binding. Crystallographic modeling of the VWF-GPIbalpha complex indicated that Glu128 and Asp175 form VWF binding sites; the binding of 6D1 to Glu125 interrupts the VWF binding of Glu128, but HPL7 binding to Glu121 has no effect on VWF binding. Moreover, His12 and Glu14 contact with Glu613 and Arg571 of VWF A1 domain, whose mutations had shown similar phenotype. These findings indicated the novel binding sites required for VWF binding of human GPIbalpha.