Novel Protein-disulfide Isomerases from the Early-diverging Protist Giardia lamblia

Leigh A Knodler,Robert Noiva,Kapil Mehta,J Michael Mccaffery,Stephen B Aley,Staffan G Svärd,Todd G Nystul,David S Reiner,Jeffrey D Silberman,Frances D Gillin

doi:10.1074/jbc.274.42.29805

Abstract

Protein-disulfide isomerase is essential for formation and reshuffling of disulfide bonds during nascent protein folding in the endoplasmic reticulum. The two thioredoxin-like active sites catalyze a variety of thiol-disulfide exchange reactions. We have characterized three novel protein-disulfide isomerases from the primitive eukaryote Giardia lamblia. Unlike other protein-disulfide isomerases, the giardial enzymes have only one active site. The active-site sequence motif in the giardial proteins (CGHC) is characteristic of eukaryotic protein-disulfide isomerases, and not other members of the thioredoxin superfamily that have one active site, such as thioredoxin and Dsb proteins from Gram-negative bacteria. The three giardial proteins have very different amino acid sequences and molecular masses (26, 50, and 13 kDa). All three enzymes were capable of rearranging disulfide bonds, and giardial protein-disulfide isomerase-2 also displayed oxidant and reductant activities. Surprisingly, the three giardial proteins also had Ca(2+)-dependent transglutaminase activity. This is the first report of protein-disulfide isomerases with a single active site that have diverse roles in protein cross-linking. This study may provide clues to the evolution of key functions of the endoplasmic reticulum in eukaryotic cells, protein disulfide formation, and isomerization.

Highlights

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) U64730, U65017, and AF164117
We found three very different genes that encode proteins of ϳ26, 50, and 13 kDa with only one CGHC active site and so are unlike most other eukaryotic proteindisulfide isomerase (PDI)
With a predicted molecular mass of 25,846 Da, giardial PDIs (gPDIs)-1 is slightly smaller than the yeast Mpd1 and Mpd2 proteins (36.4 and 32.4 kDa) [33, 34] and similar to the bacterial Dsb proteins (DsbA is 21.5 kDa), which have one active site [15, 27]. gPDI-3, with a predicted molecular mass of 12,648 Da, is the smallest eukaryotic protein containing the PDI active site reported to date

Summary

Introduction

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) U64730 (gPDI-1), U65017 (gPDI-2), and AF164117 (gPDI-3). Like Dsb proteins, PDI is a member of the thioredoxin superfamily, but has two thioredoxin-like active sites (CGHC) that are involved in disulfide bond formation and rearrangement reactions [3]. To gain insight into the evolution of thiol-disulfide exchange mechanisms in eukaryotes and because of their likely importance in giardial biology, we characterized genes encoding giardial PDIs (gPDIs).

Results

Conclusion