The interaction of 1-anilino-8-naphthalene sulphonate with human Clq

Alcolea, JoséM.; Antón, Luis C.; Marqués, Guillermo; Sánchez-Corral, Pilar; Vivanco, Fernando

doi:10.1016/0161-5890(86)90169-0

Cited by 6 publications

(5 citation statements)

References 22 publications

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“…Human Clq was purified as previously described (Alcolea et al, 1986). Radioiodination was performed by the peroxidase method (Tenner et al, 1981).…”

Section: Isolation Of Complement Componentsmentioning

confidence: 99%

C3 binds covalently to the Cγ3 domain of IgG immune aggregates during complement activation by the alternative pathway

Antón

Alcolea

Sánchez-Corral

et al. 1989

Biochemical Journal

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Ovalbumin-antiovalbumin IgG immune aggregates were incubated with normal human serum in the presence of iodo[1-14C]acetamide, in conditions in which only the alternative pathway of complement was activated. The [14C]C3b-IgG covalent complexes formed were digested with pepsin, and analysed by SDS/polyacrylamide-gel electrophoresis and fluorography. Covalent complexes of [14C]C3-Fd and [14C]C3-pFc' were visualized, demonstrating that, during complement activation by the alternative pathway, C3 is covalently incorporated into the C gamma 3 domain of IgG, as well as into the Fd region. The C gamma 2 domain becomes protected from pepsin action by the bound C3b. All the covalent linkages between C3 and the IgG were sensitive to hydroxylamine. When [14C]C3-pFc' covalent complexes were treated with 1 M-NH2OH and loaded onto a Bio-Gel P-4 column, a radioactive peak of 3 kDa was obtained. The material released from [14C]C3-pFc' and [14C]C3-F(ab')2 complexes after treatment with 1 M-NH2OH was mixed and analysed in the Bio-Gel P-4 column. A similar radioactive peak of 3 kDa was obtained. When this peak, either from [14C]C3-pFc' alone or from the mixture of [14C]C3-F(ab')2 and [14C]C3-pFc', was fractionated by h.p.l.c., virtually the same radioactive peptide profile was obtained, indicating that very similar C3 peptides remained covalently bound to both regions (Fab and C gamma 3) of the antibody molecule. It is suggested that C3 bound to the C gamma 3 domain of IgG may interfere with the Fc-Fc interactions of immune aggregates and thus may be involved in several biological properties displayed by these complement-activating aggregates.

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“…Human Clq was purified as previously described (Alcolea et al, 1986). Radioiodination was performed by the peroxidase method (Tenner et al, 1981).…”

Section: Isolation Of Complement Componentsmentioning

confidence: 99%

C3 binds covalently to the Cγ3 domain of IgG immune aggregates during complement activation by the alternative pathway

Antón

Alcolea

Sánchez-Corral

et al. 1989

Biochemical Journal

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“…Therefore it would appear that the changes in ANS fluorescence with unmodified Clq arose from structural changes in Clq upon binding to aggregated IgG. Alcolea and co-workers [5] have shown that ANS binds to the globular head regions of Clq. Thus, our data imply that the binding of oligomeric IgG to Clq leads to changes in these sections of the molecule.…”

Section: Conformational Changes In Clq Upon Binding To Iggmentioning

confidence: 99%

“…In this context, Alcolea and co-workers [5] have shown that ANS binds to Clq with approx. 20-fotd higher affinity than to IgG.…”

Section: Inhibition Of Clq Binding To Immune Complexes By Ansmentioning

confidence: 99%

“…ANS binds to Clq with an affinity constant of 2 × 106 M -l, and to IgG with an affinity constant of 9 x 10 4 M -1 [5]. It would be expected that addition of monomer IgG to a solution of Clq-bound ANS should have no effect on the fluorescence of the sample, because, firstly, monomer IgG binds to Clq with low affinity [14], and, secondly, a ma-jority of the ANS molecules will remain bound to their high affinity Clq binding sites.…”

Section: Conformational Changes In Clq Upon Binding To Iggmentioning

confidence: 99%

“…In an attempt to detect structural changes within Clq upon binding to aggregated lgG, we have exploited the fact that the fluorescent probe, 1-anilino-8-naphthalenesulfonate (ANS) binds to Clq with 20-fold higher affinity than to IgG [5]. The data presented below show that there is a change in the fluorescence of Clq-bound ANS when the protein binds to oligomers of IgG.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Conformational changes in C1q upon binding to IgG oligomers

Vandenberg¹,

Easterbrook‐Smith²

1986

FEBS Letters

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The interaction between Clq and immune complexes is inhibited by 1-anilino-8-naphthalenesulfonate (ANS) in the concentration range of 2-4 mM. ANS binds to Clq with a 20-fold higher afl]nity than to IgG [(1986) Mol. Immunol. 23, 39-44] and therefore it is possible to label only Clq with ANS in the presence of lgG. Under such conditions no inhibition is observed. Addition of monomer IgG to a solution of C lqbound ANS did not significantly alter the fluorescence of the ANS. However when oligomeric IgG was added there was a 2-fold increase in fluorescence over the same lgG concentration range. When Clq was pretreated with diethylpyrocarbonate there was little change in the fluorescence when IgG oligomers were added to Clq:ANS solutions. These results suggest that Clq undergoes conformational changes upon binding to lgG oligomers.

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Covalent binding of C3b to monoclonal antibodies selectively up‐regulates heavy chain epitope recognition by T cells

et al. 1994

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Protein C3 of the complement system is known for its role in the nonspecific immune response. Covalent binding of C3b to antigen upon complement activation also plays a significant role in specific T cell immune response. C3b-antigen complexes can bind to complement receptors on the antigen-presenting cell, and the C3b antigen link (most often an ester link) remains fairly stable inside the cells. In this study, IgG1,kappa and IgG2a,kappa murine monoclonal antibodies (mAb) were used as antigens; covalent complexes between mAb and C3b were produced and purified in vitro from purified proteins; human B cell lines and T cell clones were raised from tumor patients who received mAb injections for cancer therapy or diagnosis. Recognition of epitopes of these mAb by T cell clones when the mAb were processed alone or bound to C3b was compared. IgG or IgG-C3b complexes presented by B cell lines were able to stimulate proliferation of kappa light chain-specific T cell clones at similar concentrations. In contrast, IgG-C3b complex recognition by heavy chain-specific T cell clones required 100-fold less IgG-C3b than uncomplexed IgG. As C3b was shown to be covalently bound only to the IgG heavy chains in the complexes, C3b chaperoning is restricted to only the IgG heavy chain and selectively influences intracellular steps of IgG heavy chain processing. This differential modulation of C3b suggests an early dissociation of IgG heavy and light chains in antigen-presenting cells.

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The interaction of 1-anilino-8-naphthalene sulphonate with human Clq

Cited by 6 publications

References 22 publications

C3 binds covalently to the Cγ3 domain of IgG immune aggregates during complement activation by the alternative pathway

C3 binds covalently to the Cγ3 domain of IgG immune aggregates during complement activation by the alternative pathway

Conformational changes in C1q upon binding to IgG oligomers

Covalent binding of C3b to monoclonal antibodies selectively up‐regulates heavy chain epitope recognition by T cells

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