Masakazu Kikuchi

Protein disulfide isomerase mutant lacking its isomerase activity accelerates protein folding in the cell

We investigated the effect of protein disulfide isomerase (PDI) on in vivo protein folding of human lysozyme (h‐LZM) in a specially constructed yeast coexpression system. Coexpression with PDI increased the amounts of intracellular h‐LZM with the native conformation, leading to an increase in h‐LZM secretion. The results indicated that PDI is a real catalyst of protein folding in the cell. The secretion of h‐LZM increased even when both active sites of PDI were disrupted, suggesting that the effect of PDI resulted from a function other than the formation of disulfide bonds. This is the first finding that PDI without isomerase activity accelerates protein folding in vivo.

MOLECULAR CLONING OF THE CDNA ENCODING A NOVEL PROTEIN DISULFIDE ISOMERASE-RELATED PROTEIN (PDIR)

We isolated the cDNA of a novel protein disulfide isomerase (PDI)‐related protein, designated PDIR, from a human placental cDNA library. Deduced from its nucleotide sequence, PDIR has the three CXXC‐like motifs (Cys‐Ser‐Met‐Cys, Cys‐Gly‐His‐Cys and Cys‐Pro‐His‐Cys), which are found in proteins within the PDI superfamily and are responsible for oxidoreductase activity. PDIR has a hydrophobic stretch at its amino terminus, which may serve as a signal sequence, and the putative endoplasmic reticulum (ER) retention signal ‘Lys‐Glu‐Glu‐Leu’ at its carboxy terminus, indicating that PDIR is an ER resident protein. Northern blots showed that PDIR is preferentially expressed in cells actively secreting proteins and that the expression of PDIR is stress‐inducible. These results suggested that PDIR has oxidoreductase activity of disulfide bonds against polypeptides and that it acts as a catalyst of protein folding in the lumen of the ER.

CLONING AND SEQUENCING OF THE CDNA ENCODING HUMAN P5

The cDNA encoding human P5 was cloned and sequenced. The predicted 440-amino-acid (aa) sequence of human P5 contains two thioredoxin-like domains, which are also found in members of the protein disulfide isomerase superfamily. The human and hamster P5 genes reveal 87 and 93% similarity in their nucleotide and deduced aa sequences, respectively.

PDI and glutathione-mediated reduction of the glutathionylated variant of human lysozyme

A mutant human lysozyme, designated as C77A‐a, in which glutathione is bound to Cys95, has been shown to mimic an intermediate in the formation of a disulfide bond during folding of human (h)‐lysozyme. Protein disulfide isomerase (PDI), which is believed to catalyze disulfide bond formation and associated protein folding in the endoplasmic reticulum, attacked the glutathionylated h‐lysozyme C77A‐a to dissociate the glutathione molecule. Structural analyses showed that the protein is folded and that the structure around the disulfide bond, buried in a hydrophobic core, between the protein and the bound glutathione is fairly rigid. Thioredoxin, which has higher reducing activity of protein disulfides than PDI, catalyzed the reduction with lower efficiency. These results strongly suggest that PDI can catalyze the disulfide formation in intermediates with compact structure like the native states in the later step of in vivo protein folding.

Introduction of a non-native disulfide bridge to human lysozyme by cysteine scanning mutagenesis

To examine whether the disulfide bridge between residues 65 and 81 can be replaced by a non-native disulfide bridge in the mutant h-lysozyme C77/95A and whether the formation of such a new disulfide bridge affects the folding of the protein, cysteine scanning mutagenesis has been performed within two discontinuous segments (residues 61-67 for the mutant C65/77/95A, and 74-84 for the mutant C77/81/95A). The position of the Cys residue at 65 or 81 was continuously shifted by site-directed mutagenesis. Of the mutants, only substitution of Cys for Trp64 allowed the secretion of mutant h-lysozyme(W64C) into the medium in a sufficient amount for analysis. After the purification, the mutant enzyme was obtained as two components (W64C-A and W64C-B). The only difference between A and B was that A had a peptide bond cleaved between Ala77 and His78. A non-native disulfide bridge between residues 64-81 was found in both components. Little difference was observed in CD spectra among wild-type and mutant enzymes. It is likely that the tertiary structure of the W64C mutant might be distorted at the location, because the directions of amino acid side chains at positions of 64 and 81 are shown to be opposite to each other in wild-type h-lysozyme by X-ray crystallographic analysis.

Conformation and length of the signal sequence affect processing of secretory protein

Processing of human lysozyme with artificially designed signal sequences was examined in an in vitro translation—translocation system and compared with their secretory capabilities in yeast. It has been shown that the conformation of the C‐terminal region of the signal sequence and the length of the hydrophobic segment are important factors for efficient cleavage of the signal sequence.