Identification of the Linear Fc-Binding Site on the Bovine IgG1 Fc Receptor (boFcγRIII) Using Synthetic Peptides

Abstract

The bovine IgG1 Fc receptor (boFcγRIII) is a homologue to human FcγRIII (CD16) that binds bovine IgGI with medium-low affinity. In order to identify the Fc-binding site on the bovine IgG1 Fc receptor (boFcγRIII), peptides derived from the second extracellular domain (EC2) of boFcγRIII were synthesized and conjugated with the carrier protein. With a Dot-blot assay, the ability of the peptides to bind bovine IgG1 was determined, and the IgG1-binding peptide was also identified via truncation and mutation. The minimal peptide AQRVVN corresponding to the sequence 98-103 of boFcγRIII bound bovine IgG1 in Dot-blot, suggesting that it represents a linear ligand-binding site located in the putative A-B loop of the boFcγRIII EC2 domain. Mutation analysis of the peptide showed that the residues of Ala⁹⁸, Gln⁹⁹, Val¹⁰¹, Val¹⁰² and Asn¹⁰³ within the Fc-binding site are critical for IgG1 binding on boFcγRIII. The functional peptide identified in this paper is of great value to the IgG-Fc interaction study and FcR-targeting drug development.

Keywords: BoFcγRIII; Fc-binding site; bovine IgG1; synthetic peptides.

Figure 1

Alignment of the protein sequences of the EC2 domains of boFcγRIIIA (X99695), moFcγRIII (NP_034318), huFcγRIIIA (NP_000560) and huFcγRIIIB (NP_000561). The black highlights show that three or four sequences are the same, and the dotted lines represent deletions of amino acids. β-Sheets of the huFcγRIIIA EC2 domain (named from A to G) are shown by blank arrows below the sequence according to previous report [9].

Figure 2

Bovine IgG1 binding to the boFcγRIII peptides. The boFcγRIII peptides (1 µg/dot) spotted on the nitrocellulose membranes were tested with the HRP-IgG1 at 37 °C for 1 h, followed by AEC color development using the recombinant boFcγRII (rboRII) and BSA as the positive control and negative control, respectively (A). To determine the specificity of Fc-binding, the peptide membrane was incubated with the Fab and Fc fragments (10 µg/mL), purified bovine IgG1 (10 µg/mL) and PBS, respectively, at 37 °C for 1 h, followed by HRP-IgG1 detection (B). The colored membranes were scanned under a TSR-3000 Reader, and relative optical density (ROD) values were analyzed using AIS software.

Figure 3

IgG1 binding of the N-truncated boRIII1 peptides. The N-truncated peptides derived from boRIII1 spotted on the nitrocellulose membranes were tested with the HRP-IgG1 as described above. The colored membranes were then scanned under the TSR-3000 Strip Reader, and the ROD values were obtained.

Figure 4

IgG1 binding of the C-truncated boRIII1 peptides. The N-truncated peptides derived from boRIII1 spotted on the nitrocellulose membranes were tested with the HRP-IgG1 as described above. The colored membranes were then scanned under the TSR-3000 Strip Reader, and the ROD values were obtained.

Figure 5

IgG1 binding of the Ala-substituted 1N1-C8 peptides. The Ala-substituted peptides derived from 1N1-C8 dotted on the nitrocellulose membranes were tested with the HRP-IgG1 as described above. The colored membranes were then scanned under the TSR-3000 Strip Reader, and the ROD values were obtained.

Figure 6

Structure model of the extracellular domains of boFcγRIII. The structure model of boFcγRIII in monomer was built based on the human IgG1 Fc fragment–FcγRIII complex crystal structure (PDB DOI: 1E4K) with ProMod3 on the SWISS-MODEL Server, in which the Fc-binding site located on the first loop of the EC2 domain was shown in box (A), and the residues with side chains were labeled (B).