Channel catfish soluble FcmuR binds conserved linear epitopes present on Cmu3 and Cmu4

Abstract

A linear epitope on catfish IgM has been identified as the docking site for the catfish soluble FcmuR (IpFcRI). Western blot analyses and latex bead binding assays identified the consensus octapeptide motif FxCxVxHE located at the second cysteine that forms the intrachain disulfide bond of the catfish Cmu3 and Cmu4 immunolglobulin (Ig) domains as the IpFcRI binding sites. Furthermore, molecular modeling of catfish Cmu3 and Cmu4 confirmed that the octapeptide in both of these domains is accessible for IpFcRI interactions. In addition, since this octapeptide motif is also found in other vertebrate Ig domains, IpFcRI binding to Ig heavy (H) and light (L) chains from rainbow trout, chicken, mouse, rabbit, and goat were examined by Western blot analyses and latex bead binding assays. IpFcRI readily bound reduced rainbow trout (Igmu), chicken (Ignu), mouse (Igmu, Iggamma1, Iggamma2a, Iggamma2b, and Igalpha), rabbit (Igmu and Iggamma) and goat (Iggamma) IgH chains, and mouse Igkappa and Iglambda, and chicken Iglambda IgL chains. IpFcRI also bound mouse IgM, IgA and IgG subclasses when examined under native conditions.

Figure 1

IpFcRI binds catfish IgM. A. Latex beads coated with rIpFcRI (black) and beads coated with BSA (gray) were incubated in catfish serum, stained with anti-catfish IgM 9E1 mAb, and analyzed by flow cytometry. rIpFcRI coated beads not incubated in catfish serum (dotted), but stained with anti-catfish IgM 9E1 mAb served as background control. B. Latex beads coated with catfish IgM (black line) and beads coated with BSA (gray) were incubated with rIpFcRI, stained with anti-FLAG M2 mAb, and analyzed by flow cytometry. IgM coated beads not incubated with rIpFcRI (dotted), but stained with anti-FLAG mAb served as background control.

Figure 2

IpFcRI binds reduced catfish IgM. A. Catfish serum and affinity purified catfish IgM were electrophoresed under reducing conditions by 10% SDS-PAGE and visualized by Western blot using rIpFcRI as the primary binding reagent followed by anti-FLAG M2-HRP (left panel) or anti-catfish IgM 1H12 mAb as the primary antibody followed by goat anti-mouse IgG-HRP (right panel). The observed ~120 kD molecular weight protein bands detected by IpFcRI and anti-catfish IgM are partially reduced catfish Igμ chains as verified by MS/MS sequencing. B. Catfish serum and affinity purified catfish IgM were analyzed as above using anti-FLAG M2-HRP. Recombinant IpFcRI-FLAG served as a positive control. Molecular weight size markers are at left.

Figure 3

IpFcRI binds linear epitopes found on catfish Cμ3 and Cμ4 domains. A. Catfish Xpress-tagged-Cμ proteins were electrophoresed under reducing conditions by 10% SDS-PAGE and examined by Western blot. Recombinant IpFcRI was used as the primary binding reagent followed by anti-FLAG M2-HRP. Affinity purified catfish IgM served as positive control. B. Catfish rCμ proteins were analyzed as above using anti-Xpress mAb followed by goat anti-mouse Ig (H + L)-HRP. C. Schematic illustrates where Cμ3 and Cμ4 domains were split into segments, which were cloned and expressed as truncated proteins. D. Catfish rCμ proteins were analyzed using rIpFcRI as the primary binding reagent followed by anti-FLAG M2-HRP. Catfish rCμ3-4 served as the positive control for IpFcRI binding. Molecular weight size markers are at left.

Figure 4

IpFcRI binds to catfish Cμ3 and Cμ4 linear epitopes. A. Synthetic peptides derived from amino acid sequences flanking the second cysteine of catfish Cμ domains are shown with their reactivity for rIpFcRI listed at right. An amino acid-linker consisting of SGSG or SGSGK was added at the N-terminus to improve peptide solubility, amino acid location numbers are shown for catfish IgM (GenBank accession number: P23735), “unk” indicates unknown reactivity due to peptide insolubility. B. Each peptide was spotted onto nitrocellulose and incubated with rIpFcRI as the primary binding reagent. The rIpFcRI reactive spots were developed using anti-FLAG M2-HRP. 2 μg of unreduced and reduced and alkylated affinity purified catfish IgM (IgM and IgM R&A, respectively) served as positive controls, BSA served as negative control. C. Biotinylated peptides were incubated with IpFcRI beads stained with streptavidin-PE and analyzed by flow cytometry. The dashed line indicates cutoff value for a positive reaction. Peptide names are shown on Y-axis and median fluorescence intensity on X-axis.

Figure 5

IpFcRI binds Ig from different species. A. Potential core IpFcRI-binding motifs and their flanking sequences of different vertebrate Ig domains were compared to the catfish core motifs. Consensus amino acids are in upper case and the different core motifs are shown in bold. B. Serum from catfish (CFS), trout, mouse and rabbit; affinity purified catfish IgM, rabbit IgG, chicken IgY and goat IgG were electrophoresed under reducing conditions by 10% SDS-PAGE and examined by Western blot using rIpFcRI as described in Fig. 2. Labeled arrows mark the IgH and IgL chains, and non-specific protein bands that react with the anti-FLAG M2 mAb are marked (*; see sFig. 1). Molecular weight size markers are at left.

Figure 6

IpFcRI binds to different murine Ig isotypes. A. Mouse IgG1, IgG2a, IgG2b, IgG3, and IgA were electrophoresed under reducing conditions by 10% SDS-PAGE and examined by Western blot using rIpFcRI as described in Fig. 2. Molecular weight size markers are at left, IgL chain isotypes are listed below each lane. That the anti-FLAG mAb directly reacts with mouse IgG3 is marked (*) and shown in the bottom panel. B. Latex beads coated with the different mouse Ig proteins (black) and beads coated with BSA (grey) were incubated with rIpFcRI, stained with anti-FLAG M2 mAb and analyzed by flow cytometry. Coated latex beads not incubated with rIpFcRI (dotted), but stained with anti-FLAG M2 mAb served as background staining controls.

Figure 7

Predicted 3-D structure of catfish Cμ3 and Cμ4 domains based on comparative homology modeling. The amino acid sequence of catfish Cμ3 and Cμ4 (aa 204A-434Y, Entrez Protein ID# P23735) was used to query the PDB database using the PHYRE server (Kelley and Sternberg, 2009). The human IgG1 C region PDB accession code 1L6X was used to build the 3D model. The ribbon diagram and space fill model were made in Pymol. The side chain of the amino acids in the FxCxVxHE motif are shown with the domain core residues in grey and putative IpFcRI interacting residues in black. The modeled core motifs in Cμ3 and Cμ4 are shown for comparison. For the web version of this article the side chain of the amino acids in the FxCxVxHE motif are shown with the domain core residues in blue, interchain disulfide bonds in yellow and putative IpFcRI interacting residues in red.