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ABSTRACT: We previously reported that anti-amyloid-beta (Aβ) single-chain antibody (scFv59) brain delivery via recombinant adeno-associated virus (rAAV) was effective in reducing cerebral Aβ load in an Alzheimer's disease (AD) mouse model without inducing inflammation. Here, we investigated the prophylactic effects and mechanism of a muscle-directed gene therapy modality in an AD mouse model. We injected rAAV serotype 1 encoding scFv59 into the right thigh muscles of 3-month-old mice. Nine months later, high levels of scFv59 expression were confirmed in the thigh muscles by both immunoblotting and immunohistochemistry. As controls, model mice were similarly injected with rAAV1 encoding antihuman immunodeficiency virus Gag antibody (scFvGag). AAV1-mediated scFv59 gene delivery was effective in decreasing Aβ deposits in the brain. Compared with the scFvGag group, levels of Aβ in cerebrospinal fluid (CSF) decreased significantly while Aβ in serum tended to increase in the scFv59 group. AAV1-mediated scFv59 gene delivery may alter the equilibrium of Aβ between the blood and brain, resulting in an increased efflux of Aβ from the brain owing to antibody-mediated sequestration/clearance of peripheral Aβ. Our results suggest that muscle-directed scFv59 delivery via rAAV1 may be a prophylactic option for AD and that levels of CSF Aβ may be used to evaluate the efficacy of anti-Aβ immunotherapy.
Journal of Molecular Neuroscience 09/2012; · 2.50 Impact Factor
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ABSTRACT: Accumulation of amyloid-β protein (Aβ) in the brain is thought to be a causal event in Alzheimer's disease (AD). Immunotherapy targeting Aβ holds great promise for reducing Aβ in the brain. Here, we evaluated the efficacy and safety of anti-Aβ single-chain antibody (scFv59) delivery via recombinant adeno-associated virus (rAAV) on reducing Aβ deposits in an AD mouse model (TgAβPPswe/PS1dE9). First, delivery of scFv59 to the brain was optimized by injecting rAAV serotypes 1, 2, and 5 into the right lateral ventricle. Symmetrical high expression of scFv59 was found throughout the hippocampus and partly in the neocortex in both hemispheres via rAAV1 or rAAV5, while scFv59 expression via rAAV2 was mostly limited to one hemisphere. rAAV1, however, induced apoptosis and microglial activation but rAAV5 did not. Therefore, rAAV5 was selected for therapeutic scFv59 delivery in TgAβPPswe/PS1dE9 mice. rAAV5 was similarly injected into the ventricle of 10-month-old TgAβPPswe/PS1dE9 mice and 5 months later its efficacy and safety were evaluated. Immunoreactive Aβ deposits reduced in the hippocampus. Aβ42 levels in cerebrospinal fluid (CSF) tended to increase and the Aβ40 : 42 ratio decreased in CSF, suggesting that Aβ42 was relocated from the parenchyma to CSF. Hemorrhages associated with a focal increase in blood vessel amyloid were found in the brain. While immunotherapy has great potential for clearing cerebral Aβ, caution for cerebrovascular effects should be exercised when rAAV-mediated anti-Aβ immunotherapy is applied.
Journal of Alzheimer's disease: JAD 06/2011; 27(1):23-38. · 3.74 Impact Factor
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Yasuhiro Nishiyama,
Stephanie Planque,
Yukie Mitsuda,
Giovanni Nitti, Hiroaki Taguchi,
Lei Jin,
Jindrich Symersky,
Stephane Boivin,
Marcin Sienczyk,
Maria Salas,
Carl V. Hanson,
Sudhir Paul
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ABSTRACT: 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 (VH) domain framework (FR) residues. Substitution of the FR cavity VH Lys-19 residue by an Ala residue resulted in attenuated binding of the 421–433 region peptide probe. The CDRs and VH 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 VH1 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.
Journal of Biological Chemistry 10/2009; 284(44):30627-30642. · 4.77 Impact Factor
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Yasuhiro Nishiyama,
Stephanie Planque,
Yukie Mitsuda,
Giovanni Nitti, Hiroaki Taguchi,
Lei Jin,
Jindrich Symersky,
Stephane Boivin,
Marcin Sienczyk,
Maria Salas,
Carl V Hanson,
Sudhir Paul
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ABSTRACT: 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.
Journal of Biological Chemistry 10/2009; 284(44):30627-42. · 4.77 Impact Factor
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ABSTRACT: Nucleophilic sites in the paired variable domains of the light and heavy chains (VL and VH domains) of Ig can catalyze peptide bond hydrolysis. Amyloid beta (Abeta)-binding Igs are under consideration for immunotherapy of Alzheimer disease. We searched for Abeta-hydrolyzing human IgV domains (IgVs) in a library containing a majority of single chain Fv clones mimicking physiological VL-VH-combining sites and minority IgV populations with nonphysiological structures generated by cloning errors. Random screening and covalent selection of phage-displayed IgVs with an electrophilic Abeta analog identified rare IgVs that hydrolyzed Abeta mainly at His14-Gln15. Inhibition of IgV catalysis and irreversible binding by an electrophilic hapten suggested a nucleophilic catalytic mechanism. Structural analysis indicated that the catalytic IgVs are nonphysiological structures, a two domain heterodimeric VL (IgVL2-t) and single domain VL clones with aberrant polypeptide tags (IgVL-t'). The IgVs hydrolyzed Abeta at rates superior to naturally occurring Igs by 3-4 orders of magnitude. Forced pairing of the single domain VL with VH or VL domains resulted in reduced Abeta hydrolysis, suggesting catalysis by the unpaired VL domain.Angstrom level amino acid displacements evident in molecular models of the two domain and unpaired VL domain clones explain alterations of catalytic activity. In view of their superior catalytic activity, the VL domain IgVs may help attain clearance of medically important antigens more efficiently than natural Igs.
Journal of Biological Chemistry 11/2008; 283(52):36724-33. · 4.77 Impact Factor
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ABSTRACT: Immunoglobulins (Igs) in uninfected humans recognize residues 421-433 located in the B cell superantigenic site (SAg) of the HIV envelope protein gp120 and catalyze its hydrolysis by a serine protease-like mechanism. The catalytic activity is encoded by germline Ig variable (V) region genes, and is expressed at robust levels by IgMs and IgAs but poorly by IgGs. Mucosal IgAs are highly catalytic and neutralize HIV, suggesting that they constitute a first line of defense against HIV. Lupus patients produce the Igs at enhanced levels. Homology of the 421-433 region with an endogenous retroviral sequence and a bacterial protein may provide clues about the antigen driving anti-SAg synthesis in lupus patients and uninfected subjects. The potency and breadth of HIV neutralization revives hopes of clinical application of catalytic anti-421-433 Igs as immunotherapeutic and topical microbicide reagents. Adaptive improvement of anti-SAg catalytic Igs in HIV infected subjects is not customary. Further study of the properties of the naturally occurring anti-SAg catalytic Igs should provide valuable guidance in designing a prophylactic vaccine that amplifies protective catalytic immunity to HIV.
Autoimmunity Reviews 07/2008; 7(6):473-9. · 6.62 Impact Factor
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ABSTRACT: Immunoglobulins (Igs) that bind amyloid beta peptide (Abeta) are under clinical trials for immunotherapy of Alzheimer disease (AD). We have identified IgMs and recombinant Ig fragments that hydrolyze Abeta. Hydrolysis of peripheral Abeta by the IgMs may induce increased Abeta release from the brain. The catalytic IgMs are increased in AD patients, presumably reflecting a protective autoimmune response. Reduced Abeta aggregation and neurotoxicity attributable to the catalytic function were evident. These findings provide a foundation for development of catalytic Igs for AD immunotherapy.
Autoimmunity Reviews 06/2008; 7(5):391-7. · 6.62 Impact Factor
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ABSTRACT: The antigen-binding sites of antibodies (Abs) can express enzyme-like nucleophiles that react covalently with electrophilic compounds. We examined the irreversible and specific inactivation of antibodies (Abs) to Factor VIII (FVIII) responsible for failure of FVIII replacement therapy in hemophilia A (HA) patients. Electrophilic analogs of FVIII (E-FVIII) and its C2 domain (E-C2) were prepared by placing the strongly electrophilic phosphonate groups at surface-exposed Lys side chains of diverse antigenic epitopes. IgG Abs to FVIII from HA patients formed stable immune complexes with E-FVIII and E-C2 that were refractory to dissociation by SDS treatment and boiling, procedures that dissociate noncovalent Ab-antigen complexes. The rate-limiting step in the reaction was formation of the initial noncovalent complexes. Conversion of the initial complexes to the irreversible state occurred rapidly. The antigenic epitopes of E-FVIII were largely intact, and most of the Abs were consumed covalently. E-FVIII expressed poor FVIII cofactor activity in clotting factor assays. Nonspecific interference by E-FVIII in clotting factor function was not evident. Treatment with E-FVIII, and to a lesser extent E-C2, irreversibly relieved the FVIII inhibitory effect of HA IgG in clotting factor assays. Small FVIII peptides did not display useful reactivity, highlighting the diverse epitope specificities of the Abs and the conformational character of FVIII epitopes. E-FVIII is a prototype reagent able to attain irreversible and specific inactivation of pathogenic Abs.
Journal of Biological Chemistry 06/2008; 283(18):11876-86. · 4.77 Impact Factor
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Hiroaki Taguchi,
Stephanie Planque,
Yasuhiro Nishiyama,
Jindrich Symersky,
Stephane Boivin,
Paul Szabo,
Robert P Friedland,
Paul A Ramsland,
Allen B Edmundson,
Marc E Weksler,
Sudhir Paul
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ABSTRACT: We describe IgM class human autoantibodies that hydrolyze amyloid beta peptide 1-40 (Abeta40). A monoclonal IgM from a patient with Waldenström's macroglobulinemia hydrolyzed Abeta40 at the Lys-28-Gly-29 bond and Lys-16-Ala-17 bonds. The catalytic activity was inhibited stoichiometrically by an electrophilic serine protease inhibitor. Treatment with the catalytic IgM blocked the aggregation and toxicity of Abeta40 in neuronal cell cultures. IgMs purified from the sera of patients with Alzheimer disease (AD) hydrolyzed Abeta40 at rates superior to IgMs from age-matched humans without dementia. IgMs from non-elderly humans expressed the least catalytic activity. The reaction rate was sufficient to afford appreciable degradation at physiological Abeta and IgM concentrations found in peripheral circulation. Increased Abeta concentrations in the AD brain are thought to induce neurodegenerative effects. Peripheral administration of Abeta binding antibodies has been suggested as a potential treatment of AD. Our results suggest that catalytic IgM autoantibodies can help clear Abeta, and they open the possibility of using catalytic Abs for AD immunotherapy.
Journal of Biological Chemistry 03/2008; 283(8):4714-22. · 4.77 Impact Factor
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Stephanie Planque,
Yukie Mitsuda, Hiroaki Taguchi,
Maria Salas,
Mary-Kate Morris,
Yasuhiro Nishiyama,
Robert Kyle,
Pablo Okhuysen,
Miguel Escobar,
Robert Hunter,
Haynes W Sheppard,
Carl Hanson,
Sudhir Paul
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ABSTRACT: Antibody hydrolysis of the superantigenic gp120 site and HIV-1 neutralization was studied as a potential anti-HIV mechanism in uninfected humans. gp120 hydrolysis by purified serum and salivary antibodies was determined by electrophoresis and peptide sequencing, the proteolytic mechanism was analyzed using electrophilic peptide analogs, and viral neutralization was studied using peripheral blood mononuclear cells as hosts. Polyclonal and monoclonal IgA but not IgG preparations selectively catalyzed the cleavage of HIV gp120 at rates sufficient to predict biologically relevant protection against the virus. The IgA hydrolytic reaction proceeded by noncovalent recognition of gp120 residues 421-433, a component of the superantigenic site of gp120, coordinated with peptide bond cleavage via a serine protease-like mechanism. The Lys-432-Ala-433 bond was one of the cleavage sites. Infection of peripheral blood mononuclear cells by a primary isolate of HIV was neutralized by the IgA but not IgG fractions. The neutralizing activity was specifically inhibited by an electrophilic inhibitor of the catalytic activity. The existence of catalytic IgAs to gp120 in uninfected humans suggests their role in resistance to HIV.
AIDS Research and Human Retroviruses 01/2008; 23(12):1541-54. · 2.25 Impact Factor
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ABSTRACT: Rare monoclonal antibodies (Abs) can form irreversible complexes with antigens by enzyme-like covalent nucleophile-electrophile pairing. To determine the feasibility of applying irreversible antigen inactivation by Abs as the basis of vaccination against microbes, we studied the polyclonal nucleophilic Ab response induced by the electrophilic analog of a synthetic peptide corresponding to the principal neutralizing determinant (PND) of human immunodeficiency virus type-1 (HIV) gp120 located in the V3 domain. Abs from mice immunized with the PND analog containing electrophilic phosphonates (E-PND) neutralized a homologous HIV strain (MN) approximately 50-fold more potently than control Abs from mice immunized with PND. The IgG fractions displayed binding to intact HIV particles. HIV complexes formed by anti-E-PND IgG dissociated noticeably more slowly than the complexes formed by anti-PND IgG. The slower dissociation kinetics are predicted to maintain long-lasting blockade of host cell receptor recognition by gp120. Pretreatment of the anti-PND IgG with a haptenic electrophilic phosphonate compound resulted in more rapid dissociation of the HIV-IgG complexes, consistent with the hypothesis that enhanced Ab nucleophilic reactivity induced by electrophilic immunization imparts irreversible character to the complexes. These results suggest that electrophilic immunization induces a sufficiently robust nucleophilic Ab response to enhance the anti-microbial efficacy of candidate polypeptide vaccines.
Journal of Biological Chemistry 11/2007; 282(43):31250-6. · 4.77 Impact Factor
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ABSTRACT: Rare monoclonal antibodies (Abs) can form irreversible complexes with antigens by enzyme-like covalent nucleophile-electrophile
pairing. To determine the feasibility of applying irreversible antigen inactivation by Abs as the basis of vaccination against
microbes, we studied the polyclonal nucleophilic Ab response induced by the electrophilic analog of a synthetic peptide corresponding
to the principal neutralizing determinant (PND) of human immunodeficiency virus type-1 (HIV) gp120 located in the V3 domain.
Abs from mice immunized with the PND analog containing electrophilic phosphonates (E-PND) neutralized a homologous HIV strain
(MN) ∼50-fold more potently than control Abs from mice immunized with PND. The IgG fractions displayed binding to intact HIV
particles. HIV complexes formed by anti-E-PND IgG dissociated noticeably more slowly than the complexes formed by anti-PND
IgG. The slower dissociation kinetics are predicted to maintain long-lasting blockade of host cell receptor recognition by
gp120. Pretreatment of the anti-PND IgG with a haptenic electrophilic phosphonate compound resulted in more rapid dissociation
of the HIV-IgG complexes, consistent with the hypothesis that enhanced Ab nucleophilic reactivity induced by electrophilic
immunization imparts irreversible character to the complexes. These results suggest that electrophilic immunization induces
a sufficiently robust nucleophilic Ab response to enhance the anti-microbial efficacy of candidate polypeptide vaccines.
Journal of Biological Chemistry 10/2007; 282(43):31250-31256. · 4.77 Impact Factor
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ABSTRACT: IgG class antibodies express catalytic activities rarely and at very low levels. Here, we studied polyclonal IgA and IgG preparations from healthy human sera and saliva for the ability to hydrolyze model peptidyl-aminomethylcoumarin (peptide-AMC) substrates. These substrates permit objective evaluation of the catalytic potential of the antibody classes with minimal effects of noncovalent interactions occurring at sites remote from the reaction center. The IgA preparations hydrolyzed Glu-Ala-Arg-AMC at rates 3-orders of magnitude greater than IgG preparations from the same individuals. The cleavage occurred preferentially on the C terminal side of a basic residue. The activity was confirmed using monoclonal IgAs isolated from patients with multiple myeloma. Active site-directed inhibitors of serine proteases inhibited the catalytic activity and were bound irreversibly by the IgA, suggesting the involvement of a serine protease-like mechanism similar to that utilized by previously described IgM antibodies. These observations suggest that mechanisms underlying B cell clonal selection favor the retention and improvement of catalytic activity in the IgA, but not the IgG compartment of the immune response.
Molecular Biotechnology 07/2007; 36(2):113-22. · 2.17 Impact Factor
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ABSTRACT: Antibodies (Abs) to the superantigenic determinant of HIV gp120 (gp120(SAg)) are potential protective agents against HIV infection. We report that the light chain subunits of Abs cloned from lupus patients using phage library methods bind and hydrolyze gp120(SAg) independent of the heavy chain. Unlike frequent gp120(SAg) recognition by intact Abs attributable to V(H) domain structural elements, the isolated light chains expressed this activity rarely. Four light chains capable of gp120(SAg) recognition were identified by fractionating phage displayed light chains using peptide probes containing gp120 residues 421-433, a gp120(SAg) component. Three light chains expressed non-covalent gp120(SAg) binding and one expressed gp120(SAg) hydrolyzing activity. The hydrolytic light chain was isolated by covalent phage fractionation using an electrophilic analog of residues 421-433. This light chain hydrolyzed a reporter gp120(SAg) substrate and full-length gp120. Other peptide substrates and proteins were hydrolyzed at lower rates or not at all. Consistent with the expected nucleophilic mechanism of hydrolysis, the light chain reacted selectively and covalently with the electrophilic gp120(SAg) peptide analog. The hydrolytic reaction entailed a fast initial step followed by a slower rate limiting step, suggesting rapid substrate acylation and slow deacylation. All four gp120(SAg)-recognizing light chains contained sequence diversifications relative to their germline gene counterparts. These observations indicate that in rare instances, the light chain subunit can bind and hydrolyze gp120(SAg) without the participation of the heavy chain. The pairing of such light chains with heavy chains capable of gp120(SAg) recognition represents a potential mechanism for generating protective Abs with enhanced HIV binding strength and anti-viral proteolytic activity.
Molecular Immunology 05/2007; 44(10):2707-18. · 2.90 Impact Factor
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ABSTRACT: Antibodies (Abs) with proteolytic and other catalytic activities have been characterized in the blood and mucosal secretions of humans and experimental animals. The catalytic activity can be traced to nucleophilic sites of innate origin located in Ab germline variable regions. Discoveries of the natural chemical reactivity of Abs were initially met with bewilderment, as the notion had taken hold that catalytic activities can be introduced into Abs by artificial means, but somatically operative selection pressures are designed only to adapt non-covalent Ab binding to antigen ground states. Unsurprisingly, initial efforts to engineer Abs with catalytic activity were oriented towards improving the non-covalent binding at the atoms immediately within the transition state reaction center. Slowly, however, dogmatic approaches to Ab catalysis have given way to the realization that efficient and specific catalytic Abs can be prepared by improving the natural nucleophilic reactivity combined with non-covalent recognition of epitope regions remote from the reaction center. The field remains beset, however, with controversy. This article attempts to provide a rational basis for natural Ab catalysis, in the hope that understanding this phenomenon will stimulate medical and basic science advances in the field.
Immunology Letters 03/2006; 103(1):8-16. · 2.53 Impact Factor
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ABSTRACT: Antibodies (Abs) and enzymes are structural and functional relatives. Abs with promiscuous peptidase activity are ubiquitous in healthy humans, evidently derived from germline variable domain immunoglobulin genes encoding the serine protease-like nucleophilic function. Exogenous and endogenous electrophilic antigens can bind the nucleophilic sites covalently, and recent evidence suggests that immunization with such antigens can induce proteolytic antibodies. Previously, Ab catalytic activities have been linked to pathogenic autoimmune reactions, but recent studies indicate that proteolytic Abs may also serve beneficial functions. An example is the rapid and selective cleavage of the HIV-1 coat protein gp120 by IgMs found in uninfected humans. The selectivity of this reaction appears to derive from recognition of gp120 as a superantigen. A second example is the cleavage of amyloid beta-peptide by IgM and IgG from aged humans, a phenomenon that may represent a specific proteolytic response to a neurotoxic endogenous peptide implicated in the pathogenesis of Alzheimer's disease.
Springer Seminars in Immunopathology 04/2005; 26(4):485-503. · 4.17 Impact Factor
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ABSTRACT: A panel of human monoclonal and recombinant antibody light chains was screened for cleavage of the synthetic peptide corresponding to a neutralizing epitope of hepatitis C virus (residues 192-205 of envelope glycoprotein E1). One of the 39 light chains studied hydrolyzed the Val197-Ser198 bond of the peptide with Km and kcat values of 223 +/- 7 microM and 0.087 +/- 0.001 min(-1).
Bioorganic & Medicinal Chemistry Letters 10/2004; 14(17):4529-32. · 2.55 Impact Factor
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ABSTRACT: We report the selective catalytic cleavage of the HIV coat protein gp120, a B cell superantigen, by IgM antibodies (Abs) from uninfected humans and mice that had not been previously exposed to gp120. The rate of IgM-catalyzed gp120 cleavage was greater than of other polypeptide substrates, including the bacterial superantigen protein A. The kinetic parameters of gp120 cleavage varied over a broad range depending on the source of the IgMs, and turnover numbers as great as 2.1/min were observed, suggesting that different Abs possess distinct gp120 recognition properties. IgG Abs failed to cleave gp120 detectably. The Fab fragment of a monoclonal IgM cleaved gp120, suggesting that the catalytic activity belongs to the antibody combining site. The electrophoretic profile of gp120 incubated with a monoclonal human IgM suggested hydrolysis at several sites. One of the cleavage sites was identified as the Lys(432)-Ala(433) peptide bond, located within the region thought to be the Ab-recognizable superantigenic determinant. A covalently reactive peptide analog (CRA) corresponding to gp120 residues 421-431 with a C-terminal amidino phosphonate diester mimetic of the Lys(432)-Ala(433) bond was employed to probe IgM nucleophilic reactivity. The peptidyl CRA inhibited the IgM-catalyzed cleavage of gp120 and formed covalent IgM adducts at levels exceeding a control hapten CRA devoid of the peptide sequence. These observations suggest that IgMs can selectively cleave gp120 by a nucleophilic mechanism and raise the possibility of their role as defense enzymes.
Journal of Biological Chemistry 10/2004; 279(38):39611-9. · 4.77 Impact Factor
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ABSTRACT: We report the selective catalytic cleavage of the HIV coat protein gp120, a B cell superantigen, by IgM antibodies (Abs) from
uninfected humans and mice that had not been previously exposed to gp120. The rate of IgM-catalyzed gp120 cleavage was greater
than of other polypeptide substrates, including the bacterial superantigen protein A. The kinetic parameters of gp120 cleavage
varied over a broad range depending on the source of the IgMs, and turnover numbers as great as 2.1/min were observed, suggesting
that different Abs possess distinct gp120 recognition properties. IgG Abs failed to cleave gp120 detectably. The Fab fragment
of a monoclonal IgM cleaved gp120, suggesting that the catalytic activity belongs to the antibody combining site. The electrophoretic
profile of gp120 incubated with a monoclonal human IgM suggested hydrolysis at several sites. One of the cleavage sites was
identified as the Lys432-Ala433 peptide bond, located within the region thought to be the Ab-recognizable superantigenic determinant. A covalently reactive
peptide analog (CRA) corresponding to gp120 residues 421–431 with a C-terminal amidino phosphonate diester mimetic of the
Lys432-Ala433 bond was employed to probe IgM nucleophilic reactivity. The peptidyl CRA inhibited the IgM-catalyzed cleavage of gp120 and
formed covalent IgM adducts at levels exceeding a control hapten CRA devoid of the peptide sequence. These observations suggest
that IgMs can selectively cleave gp120 by a nucleophilic mechanism and raise the possibility of their role as defense enzymes.
Journal of Biological Chemistry 09/2004; 279(38):39611-39619. · 4.77 Impact Factor
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ABSTRACT: We report the chemical activity of immunoglobulin micro and kappa/lambda subunits expressed on the surface of B cells and in secreted IgM antibodies (Abs) found in the preimmune repertoire. Most of the nucleophilic reactivity of B cells measured by formation of covalent adducts of a hapten amidino phosphonate diester was attributed to micro and kappa/lambda subunits of the B cell receptor. Secreted IgM Abs displayed superior nucleophilic reactivity than IgG Abs. IgM Abs catalyzed the cleavage of model peptide substrates at rates up to 344-fold greater than IgG Abs. Catalytic activities were observed in polyclonal IgM Abs from immunologically naïve mice and humans without immunological disease, as well as monoclonal IgM Abs to unrelated antigens. Comparison of several IgM Abs indicated divergent activity levels and substrate preferences, with the common requirement of a basic residue flanking the cleavage site. Fab fragments of a monoclonal IgM Ab expressed catalytic activity, confirming the V domain location of the catalytic site. The catalytic reaction was inhibited by the covalently reactive hapten probe and diisopropylfluorophosphate, suggesting a serine protease-like mechanism. These observations indicate the existence of serine protease-like BCRs and secreted IgM Abs as innate immunity components with potential roles in B cell development and Ab effector functions.
Journal of Biological Chemistry 05/2004; 279(14):14024-32. · 4.77 Impact Factor
