Functional analysis of the CXXC motif using phage antibodies that cross-react with protein disulphide-isomerase family proteins

Abstract

Polyclonal antibodies that had been raised against particular PDI (protein disulphide-isomerase) family proteins did not cross-react with other PDI family proteins. To evade immune tolerance to the important self-motif Cys-Xaa-Xaa-Cys, which is present in PDI family proteins, we used the phage display library [established by Griffiths, Williams, Hartley, Tomlinson, Waterhouse, Crosby, Kontermann, Jones, Low, Allison et al. (1994) EMBO J. 13, 3245-3260] to isolate successfully the phage antibodies that can cross-react with human and bovine PDIs, human P5, human PDI-related protein and yeast PDI. By measuring the binding of scFv (single-chain antibody fragment of variable region) to synthetic peptides and to mutants of PDI family proteins in a surface plasmon resonance apparatus, we identified clones that recognized sequences containing the CGHC motif or the CGHCK sequence. By using the isolated phage antibodies, we demonstrated for the first time that a lysine residue following the CXXC motif significantly increases the isomerase activities of PDI family proteins. Moreover, we demonstrated that the affinity of isolated scFvs for mutant PDI family proteins is proportional to the isomerase activities of their active sites.

Figure 1. Western-blot analysis of PDI family proteins using polyclonal antibodies

The ability of an anti-bPDI antibody (A) and an anti-human P5 antibody (B) to detect PDI family proteins was tested by Western-blot analysis. Lane 1, marker proteins; lane 2, hPDI; lane 3, hP5; lane 4, hPDIR; lane 5, bPDI; lane 6, yPDI. Approx. 5 μg of each protein was used.

Figure 2. Detection of PDI family proteins by the 5E phage antibody

The ability of the 5E phage antibody to detect PDI family proteins was tested by Western-blot analysis. Lane 1, marker proteins; lane 2, hPDR; lane 3, hPDI; lane 4, bPDI; lane 5, hP5; lane 6, yPDI. The 5E phage antibody (10¹¹ pfu/ml) and each protein (approx. 5 μg) were used. The asterisk indicates a breakdown product of hPDIR.

Figure 3. Comparison of the CXXC motif and its neighbouring sequences in PDI family proteins

The most conserved region (APWCGHCK) is indicated by dark grey boxes.

Figure 4. Dissociation constants (K_D) of scFvs for 5E and 18F1 and ELISA of PDI family proteins with 5E

The data analysis was performed using the BIA evaluation version 3.1 software.

Figure 5. Sensorgrams for the binding of the 5E scFv to immobilized hPDIR (A) or hPDI (B)

All analytes [1 (7.5 μM), 2 (3.75 μM) and 3 (1.675 μM)] of the 5E scFv, which are shown in the inset, were injected over immobilized hPDIR (A) or hPDI (B) on the sensor chip. Progress in the binding of the 5E scFv to immobilized hPDI and hPDIR was assessed by following the increase in the signal [expressed in terms of RU (resonance unit)].

Figure 6. The domain structures of hPDI and yPDI, the sequences of their mutants and recognition of the mutants by the 5E phage antibody by Western blotting

(A) Domains a and a′ (striped boxes), redox-active Trx domains; domains b and b′ (grey boxes), redox-inactive Trx domains; and domain c (white box), a putative calcium-binding domain. The C-terminal KDEL and HDEL sequences are the possible ER-retention signals. (B) Detection of hPDI and yPDI mutants by the 5E phage antibody by Western blotting. Lane 1, marker proteins; lane 2, hPDI-m1; lane 3, hPDI-m2; lane 4, hPDI-m12; lane 5, yPDI-m1; lane 6, yPDI-m2; lane 7, yPDI-m12. hPDI and yPDI are shown in Figure 2. The 5E phage antibody (10¹¹ pfu/ml) was used with each protein (approx. 5 μg).

Figure 7. The domain structure of hPDIR, the sequences of its mutants and recognition of the mutants by the 5E phage antibody by Western blotting

(A) Domains a, a⁰ and a′ (striped boxes), redox-active Trx domains; domain b (grey box), redoxinactive Trx domain; and domain c (white box), a putative calcium-binding domain. The C-terminal KEEL sequence is a possible ER-retention signal. (B) Detection of the mutants by the 5E phage antibody by Western blotting. Lane 1, marker proteins; lane 2, hPDIR-m1; lane 3, hPDIR-m3; lane 4, hPDIR-m2; lane 5, hPDIR-m12; lane 6, hPDIR-m13; lane 7, hPDIR-m23; lane 8, hPDIR-m123; lane 9, hPDIR-CGHS; lane 10, hPDIR-SGHC; lane 11, hPDIR-m12-GH; lane 12, hPDIR-m23-GH. hPDIR is shown in Figure 2. The 5E phage antibody (10¹¹ pfu/ml) was used with each protein (approx. 5 μg).

Figure 8. The domain structure of hP5, the sequences of its mutants and recognition of the mutants by the 5E phage antibody by Western blotting

(A) Domains a⁰ and a (striped boxes), redox-active Trx domains; domain b (grey box), redox-inactive Trx domain; and domain c (white box), a putative calcium-binding domain. The C-terminal KDEL sequence is a possible ER-retention signal. (B) Detection of the mutants by the 5E phage antibody by Western blotting. Lane 1, marker proteins; lane 2, hP5-m1; lane 3, hP5-m2; lane 4, hP5-m12; lane 5, hP5-Q40K; lane 6, hP5-m2Q40K. hP5 is shown in Figure 2. The 5E phage antibody (10¹¹ pfu/ml) was used with each protein (approx. 5 μg).