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Mutagenesis of the Arg-Gly-Asp triplet in human complement component C3 does not abolish binding of iC3b to the
A Taniguchi-Sidle1, D E Isenman
1Department of Biochemistry, University of Toronto, Ontario, Canada.
Insights
The leukocyte integrin complement receptor type III (CR3) does not bind iC3b via the Arg-Gly-Asp (RGD) triplet. Mutagenesis studies confirm that RGD and surrounding residues are not essential for CR3-iC3b interaction.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Leukocyte integrin CR3 (CD11b/CD18) mediates cell adhesion by binding complement receptor type III (CR3) to iC3b.
- Integrin-ligand interactions often involve an Arg-Gly-Asp (RGD) motif, but CR3-iC3b binding has shown inconsistencies with this rule.
Purpose of the Study:
- To investigate the specific role of the RGD motif and adjacent sequences in the C3 protein for CR3-mediated binding.
- To resolve conflicting data regarding the RGD-dependence of CR3-iC3b interactions.
Main Methods:
- Site-directed mutagenesis of human C3 cDNA to alter specific residues within the iC3b binding region.
- Expression and purification of recombinant mutant C3 proteins.
- Assay of CR3-mediated binding using iC3b-coated erythrocytes and CR3-bearing leukocytes.
Main Results:
- Replacing the RGD triplet with AAA in iC3b did not abolish CR3-mediated binding.
- Mutations in the positively charged R1391 residue and the conserved 1397-DATMS sequence also failed to disrupt CR3 binding.
- CR3 binding to iC3b is independent of the RGD motif and its immediate surrounding residues.
Conclusions:
- The interaction between CR3 and iC3b is not mediated by the canonical Arg-Gly-Asp (RGD) sequence.
- Alternative binding mechanisms or motifs likely account for CR3-iC3b recognition.
Abstract:
The leukocyte integrin complement receptor type III (CR3, CD11b/CD18) binds the C3 cleavage product iC3b. Many other integrins bind their ligands via an Arg-Gly-Asp (RGD) triplet. Both the RGD-containing C3 peptide 1390TRYRGDQDATMS1401 (pro-C3 numbering) and the RGD-like fibrinogen peptide GGAKQAGDV, which binds to the platelet integrin glycoprotein IIb-IIIa, were shown to inhibit the iC3b-CR3 interaction, suggesting that this binding is also RGD-mediated (Wright, S.D., Weitz, J.I., Huang, A. J., Levin, S.M., Silverstein, S.C., and Loike, J.D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7734-7738). However, unlike other integrin-ligand interactions, that of CR3 and iC3b is unaffected by the hexapeptide GRGDSP, and substitutions in the RGD triplet of C3 from other species appear to be tolerated. It was, therefore, proposed (Grossberger, D., Marcuz, A., du Pasquier, L., and Lambris, J.D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1323-1327) that the highly conserved DATMS portion of the inhibitory C3 peptide may have been responsible for its binding. To address these inconsistencies and directly assess the role of the 1390-1401 segment within the complete iC3b molecule in mediating binding to CR3, a human C3 cDNA was altered by site-directed mutagenesis and the expressed recombinant proteins were examined in a CR3-specific assay. Replacement of RGD by AAA did not abolish rosetting of the corresponding iC3b-coated erythrocytes to human CR3-bearing leukocytes. In addition, mutant iC3b molecules in which the positively charged R1391 (corresponding to K in the fibrinogen peptide) and the highly conserved 1397DATMS sequence were replaced by Q and NAAMA respectively, were still bound by CR3. We conclude that the iC3b-CR3 interaction is not mediated by the RGD triplet or its neighboring residues.