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.

A Novel Plant Protein-disulfide Isomerase Involved in the Oxidative Folding of Cystine Knot Defense Proteins

“…The PDI-like protein hP5, which contains a Gln instead of the Lys in domain a 0 , has greatly compromised isomerase activity. Replacing Gln with Lys in the active site of hP5 increases the isomerase activity, and conversely a Lys to Gln exchange in hPDI results in decreased isomerase activity (29). OaPDI exhibits lower isomerase activity (70%) than hPDI, which is probably due to the Lys to Gln exchange in both active site motifs, reinforcing the importance of the Lys for isomerase activity.…”

“…OaPDI exhibits lower isomerase activity (70%) than hPDI, which is probably due to the Lys to Gln exchange in both active site motifs, reinforcing the importance of the Lys for isomerase activity. However, the loss in activity in OaPDI is not as dramatic as observed with human mutant proteins (29), indicating that the isomerase activity of PDI proteins may be further influenced by the microenvironment of the active site.…”

“…Human PDI, human P5 (hP5) (Fig. 2A), and mutants of these proteins have been studied extensively (19,20,29). Human PDI and hP5 were both purified to homogeneity (Fig.…”

“…A distinguishing feature of OaPDI is that it contains a Gln residue following both active site motifs, in contrast to hPDI, which contains a Lys at the corresponding position in both domains. Gln occurs only very rarely at this position among known PDIs, and the Lys reportedly is important for isomerase activity of hPDI (29). The PDI-like protein hP5, which contains a Gln instead of the Lys in domain a 0 , has greatly compromised isomerase activity.…”

Identification and enzymatic activities of four protein disulfide isomerase (PDI) isoforms of Leishmania amazonensis

“…PDI proteins are essential enzymes that catalyze thiol-disulfide interchange, ensuring the proper folding and conformation of proteins, acting as coreceptors of cell reorganization, and preventing cell toxicity associated with ER stress and protein misfolding (Kimura et al 2005;Kimura et al 2004;Tian et al 2004). Most members of the PDI family can function both as oxidoreductases/isomerases and as molecular chaperones in the ER of eukaryotic cells (Ferrari and Soling 1999).…”