Cleavage specificity of a proteolytic antibody light chain and effects of the heavy chain variable domain 1 1Edited by A.R.Fersht

Sun, Mei; Gao, Qing; Kirnarskiy, Leonid; Rees, Anthony R.; Paul, Sudhir

doi:10.1006/jmbi.1997.1196

Cited by 68 publications

(72 citation statements)

References 32 publications

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“…The average K m of patients' IgG for PFR-MCA (794.2 Ϯ 300.8 M) was 80-fold greater than that scored previously when using radiolabeled FVIII as a substrate, i.e., 9.46 Ϯ 5.62 M (10), indicating that the affinity of anti-FVIII IgG is higher for FVIII than for PFR-MCA. This is reminiscent of previous observations that the active and Ag-binding sites are spatially separated in the V region of hydrolytic IgG (26). The estimated mean catalytic efficiencies ranged from 68.3 to 2682.8/M/ min.…”

Section: Discussionsupporting

confidence: 62%

Catalytic IgG from Patients with Hemophilia A Inactivate Therapeutic Factor VIII

Lacroix‐Desmazes

Wootla

Dasgupta

et al. 2006

The Journal of Immunology

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Factor VIII (FVIII) inhibitors are anti-FVIII IgG that arise in up to 50% of the patients with hemophilia A, upon therapeutic administration of exogenous FVIII. Factor VIII inhibitors neutralize the activity of the administered FVIII by sterically hindering its interaction with molecules of the coagulation cascade, or by forming immune complexes with FVIII and accelerating its clearance from the circulation. We have shown previously that a subset of anti-factor VIII IgG hydrolyzes FVIII. FVIII-hydrolyzing IgG are detected in over 50% of inhibitor-positive patients with severe hemophilia A, and are not found in inhibitor-negative patients. Although human proficient catalytic Abs have been described in a number of inflammatory and autoimmune disorders, their pathological relevance remains elusive. We demonstrate here that the kinetics of FVIII degradation by FVIII-hydrolyzing IgG are compatible with a pathogenic role for IgG catalysts. We also report that FVIII-hydrolyzing IgG from each patient exhibit multiple cleavage sites on FVIII and that, while the specificity of cleavage varies from one patient to another, catalytic IgG preferentially hydrolyze peptide bonds containing basic amino acids.

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Section: Discussionsupporting

confidence: 62%

Catalytic IgG from Patients with Hemophilia A Inactivate Therapeutic Factor VIII

Lacroix‐Desmazes

Wootla

Dasgupta

et al. 2006

The Journal of Immunology

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“…It may be concluded that the light chain nucleophile is in the immediate vicinity of the Ab noncovalent binding site and that the noncovalent binding interactions facilitate covalent binding. This statement is consistent with observations that the purified light chain of this Ab is capable of specifically catalyzing the cleavage of VIP (25). Previously, the purified light and heavy chain subunits of the Ab were reported to bind VIP independently determined by a conventional assay for noncovalent Ab-antigen complexes (K d for light chain, heavy chain, and intact IgG, respectively, 10.1, 6.8, and 1.9 nM) (33).…”

Section: Discussionsupporting

confidence: 76%

Toward Selective Covalent Inactivation of Pathogenic Antibodies

Nishiyama

Bhatia²,

Bangale

et al. 2004

Journal of Biological Chemistry

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We report the selective inactivation of proteolytic antibodies (Abs) to an autoantigen, the neuropeptide vasoactive intestinal peptide (VIP), by a covalently reactive analog (CRA) of VIP containing an electrophilic phosphonate diester at the Lys 20 residue. The VIP-CRA was bound irreversibly by a monoclonal Ab that catalyzes the hydrolysis of VIP. The reaction with the VIP-CRA proceeded more rapidly than with a hapten CRA devoid of the VIP sequence. The covalent binding occurred preferentially at the light chain subunit of the Ab. Covalent VIP-CRA binding was inhibited by VIP devoid of the phosphonate diester group. These results indicate the importance of noncovalent VIP recognition in guiding Ab nucleophilic attack on the phosphonate group. Consistent with the covalent binding data, the VIP-CRA inhibited catalysis by the recombinant light chain of this Ab with potency greater than the hapten-CRA. Catalytic hydrolysis of VIP by a polyclonal VIPase autoantibody preparation that cleaves multiple peptide bonds located between residues 7 and 22 essentially was inhibited completely by the VIP-CRA, suggesting that the electrophilic phosphonate at Lys 20 enjoys sufficient conformational freedom to react covalently with Abs that cleave different peptide bonds in VIP. These results suggest a novel route to antigen-specific covalent targeting of pathogenic Abs.Specific antigen recognition by the variable domains underlies the pathogenic effects of certain Abs 1 produced as a result of autoimmune, allergic, and anti-transplant reactions. For instance, Abs found in myasthenia gravis (reviewed in Ref. 1) and hemophilia (reviewed in Ref.2) bind important epitopes of the acetylcholine receptor and Factor VIII, respectively, that interfere with the biological activity of these proteins by a steric hindrance mechanism. Other Abs utilize their constant region to mediate pathogenic effects, but antigen recognition by Ab variable domains is the stimulus initiating these effects, e.g. Ab recognition of erythrocyte antigens stimulates complement activation by the constant region in autoimmune hemolytic anemia and incompatible blood transfusions. Similarly, allergen recognition by IgE bound to receptors for the constant region on the surface of mast cells stimulates their degranulation. In other diseases, the mechanism of Ab pathogenicity is less clear. For example, Abs to nucleic acids in lupus (reviewed in Ref.3) and to thyroglobulin in Hashimoto's thyroiditis (reviewed in Ref. 4) are unambiguously disease-associated but additional immune abnormalities are also evident in these diseases and the precise functional effects of the Abs remain debatable. Recently, a novel variable domain mechanism underlying Ab pathogenicity has emerged, viz. the catalytic cleavage of antigens. Hydrolytic catalysts such as Abs to polypeptides (5-8) and nucleic acids (9) hold the potential of permanent antigen inactivation. Moreover, catalysts are endowed with turnover capability, i.e. a single Ab molecule can hydrolyze multiple antigen molecules, sugges...

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“…Hydrolysis of peptide-MCA substrates (Peptide International, Louisville, KY or Bachem Biosciences, King of Prussia, PA) was determined in 96-well plates by fluorimetric detection of aminomethylcoumarin (Varian Cary Eclipse; ex 360 nm, em 470 nm) with authentic aminomethylcoumarin as standard (6). Cleavage of [Tyr 10 -125 I]VIP by mAb c23.5 was measured as the radioactivity rendered soluble in trichloroacetic acid (17). Kinetic parameters for cleavage of increasing concentrations of peptide-MCA substrates were determined from the Michaelis-Menten equation,…”

Section: Methodsmentioning

confidence: 99%

“…In some blots, reaction products were identified by immunoblotting using peroxidase-conjugated goat anti-gp120 Abs (Fitzgerald, Concord, MA; catalog #60-H14) (16). N-terminal sequencing of protein bands from electrophoresis gels was done as described previously (17). Hydrolysis of peptide-MCA substrates (Peptide International, Louisville, KY or Bachem Biosciences, King of Prussia, PA) was determined in 96-well plates by fluorimetric detection of aminomethylcoumarin (Varian Cary Eclipse; ex 360 nm, em 470 nm) with authentic aminomethylcoumarin as standard (6).…”

Section: Methodsmentioning

confidence: 99%

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Specific HIV gp120-cleaving Antibodies Induced by Covalently Reactive Analog of gp120

Paul

Planque

Zhou

et al. 2003

Journal of Biological Chemistry

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We report the results of efforts to strengthen and direct the natural nucleophilic activity of antibodies (Abs) for the purpose of specific cleavage of the human immunodeficiency virus-1 coat protein gp120. Phosphonate diester groups previously reported to form a covalent bond with the active site nucleophile of serine proteases (Paul, S., Tramontano, A., Gololobov, G., Zhou, Y. X., Taguchi, H., Karle, S., Nishiyama, Y., Planque, S., and George, S. (2001) J. Biol. Chem. 276, 28314 -28320) were placed on Lys side chains of gp120. Seven monoclonal Abs raised by immunization with the covalently reactive analog of gp120 displayed irreversible binding to this compound (binding resistant to dissociation with the denaturant SDS). Catalytic cleavage of biotinylated gp120 by three monoclonal antibodies was observed. No cleavage of albumin and the extracellular domain of the epidermal growth factor receptor was detected. Cleavage of model peptide substrates occurred on the C-terminal side of basic amino acids, and K m for this reaction was ϳ200-fold greater than that for gp120 cleavage, indicating Ab specialization for the gp120 substrate. A hapten phosphonate diester devoid of gp120 inhibited the catalytic activity with exceptional potency, confirming that the reaction proceeds via a serine protease mechanism. Irreversible binding of the hapten phosphonate diester by polyclonal IgG from mice immunized with gp120 covalently reactive analog was increased compared with similar preparations from animals immunized with control gp120, indicating induction of Ab nucleophilicity. These findings suggest the feasibility of raising antigen-specific proteolytic antibodies on demand by covalent immunization.

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Cleavage specificity of a proteolytic antibody light chain and effects of the heavy chain variable domain 1 1Edited by A.R.Fersht

Cited by 68 publications

References 32 publications

Catalytic IgG from Patients with Hemophilia A Inactivate Therapeutic Factor VIII

Catalytic IgG from Patients with Hemophilia A Inactivate Therapeutic Factor VIII

Toward Selective Covalent Inactivation of Pathogenic Antibodies

Specific HIV gp120-cleaving Antibodies Induced by Covalently Reactive Analog of gp120

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