Broadly distributed nucleophilic reactivity of proteins coordinated with specific ligand binding activity

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We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures. Epitope mapping with synthetic peptides and electrophilic peptide analogs indicated binary recognition of two distinct gp120 regions by anti-E-gp120 mAbs, the 421-433 and 288 -306 peptide regions. Univalent Fab and single chain Fv fragments expressed the ability to recognize both peptides. X-ray crystallography of an anti-E-gp120 Fab fragment revealed two neighboring cavities, the typical antigen-binding cavity formed by the complementarity determining regions (CDRs) and another cavity dominated by antibody heavy chain variable (V H ) domain framework (FR) residues. Substitution of the FR cavity V H Lys-19 residue by an Ala residue resulted in attenuated binding of the 421-433 region peptide probe. The CDRs and V H FR replacement/silent mutation ratios exceeded the ratio for a random mutation process, suggesting adaptive development of both putative binding sites. All mAbs studied were derived from V H 1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp120. The conserved 421-433 region of gp120 is essential for HIV binding to host CD4 receptors. This region is recognized weakly by the FR of antibodies produced without exposure to HIV, but it usually fails to induce adaptive synthesis of neutralizing antibodies. We present models accounting for improved CD4-binding site recognition and broad HIV neutralizing activity of the mAbs, long sought goals in HIV vaccine development.

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Toward Effective HIV Vaccination

Nishiyama

Planque

Mitsuda

et al. 2009

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“…5 legend). Smaller signals for the following peptide products were also detected as follows: A␤-(1-15), A␤-(1-20), A␤- (16 -40), and A␤- (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). The corresponding scissile bonds in A␤40 are Gln 15 -Lys 16 and Phe 20 -Ala 21 .…”

Section: Figure 3 Structure and Hydrolytic Activity Of Igv L2 -T 2e6mentioning

confidence: 99%

“…We screened molecular models of IgV L2 -t 2E6 and IgV L -tЈ 5D3 for side chain hydroxyls located within 4 Å of atoms that can serve as general bases as described in Ref. 31. One triad and several dyads fulfilling this requirement were found in each of the catalysts (supplemental Fig.…”

Section: Table 2 Apparent Kinetic Parameters For Igv-catalyzed A␤40 Hmentioning

confidence: 99%

Exceptional Amyloid β Peptide Hydrolyzing Activity of Nonphysiological Immunoglobulin Variable Domain Scaffolds

Taguchi

Planque

Sapparapu

et al. 2008

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Following initial noncovalent binding of antigen, some Igs proceed to catalyze its chemical transformation. Examples of Ig-catalyzed reactions include hydrolysis of polypeptide antigens (2, 3), hydrolysis of nucleic acids (4, 5), and various acyl transfer reactions of other antigen classes (6). Proteolytic Igs that utilize serine protease-like covalent hydrolytic pathways have been described (7,8). Serine protease-like catalytic triads have been identified in the V domains of Igs by site-directed mutagenesis and crystallography (9, 10). The catalytic mechanism involves nucleophilic attack on the electrophilic carbonyl of peptide bonds. Electrophilic phosphonate diesters originally developed as covalent probes for the nucleophilic site of serine proteases bind catalytic Igs irreversibly and inhibit their catalytic activity (7,11,12). The strength of Igantigen noncovalent binding often exceeds that of enzyme-substrate binding. An important limitation holding back the application of catalytic Igs for clearance of undesirable antigens is that their catalytic rate constants (turnover number; k cat ) are small compared with enzymes. Evidently, Ig adaptive selection is geared toward noncovalent immune complexation (the ground state stabilization step), and the ability of Igs to recognize the high energy transition state complex that must be stabilized to accelerate chemical reactions is limited. This is supported by observations that IgMs, the first and least diversified Ig class produced during B cell differentiation, express superior catalytic rate constants than IgGs produced by the cells at later stages of their adaptive differentiation (12).Accumulation of amyloid ␤ peptide (A␤) 2 aggregates in the brain is thought to be a central contributor to neurodegenera-* This work was supported, in whole or in part, by National Institutes of Health Grant R01AG025304. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) FJ231714, FJ231715, FJ231716, FJ231717, FJ231718, FJ231719, FJ231720, FJ231721, FJ231722, and FJ231723

“…The models obtained from the two servers were mostly superimposable (root mean square deviation values of the models for the C-␣ position and side chains, respectively, 1.47 and 2.45 Å). Potential nucleophilic dyads in the RosettaAntibody model of scFV 1H2 were identified as described (34,35), defined as sites in which the hydroxyl oxygen atoms of Ser or Thr are located within 4 Å from a possible general base (the imidazole nitrogen atoms of His, the ␤/␥-carboxylate oxygen atoms of Glu/Asp, the ⑀-amino nitrogen atom of Lys, or the -amino/imino nitrogen atoms of Arg). Potential nucleophilic triads contained an additional second general base within hydrogen bonding distance of the first general base.…”

Section: Methodsmentioning

confidence: 99%

Constitutive Production of Catalytic Antibodies to a Staphylococcus aureus Virulence Factor and Effect of Infection

Brown

Nishiyama

Dunkle

et al. 2012

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Background:We examined the role of catalytic antibodies in immune defense against Staphylococcus aureus. Results: IgG from non-infected humans and mice hydrolyzed the S. aureus extracellular fibrinogen-binding protein (Efb), and the activity was reduced following infection. Conclusion: Constitutive but not adaptive production of catalytic antibodies suggests that Efb expresses B-lymphocyte superantigenic character. Significance: Efb superantigenic character may be a factor in S. aureus pathogenesis.