Protein disulfide isomerases (PDIs) constitute a family of structurally related enzymes which catalyze disulfide bonds formation, reduction, or isomerization of newly synthesized proteins in the lumen of the endoplasmic reticulum (ER). They act also as chaperones, and are, therefore, part of a quality-control system for the correct folding of the proteins in the same subcellular compartment. While their functions in the ER have been thoroughly studied, much less is known about their roles in non-ER locations, where, however, they have been shown to be involved in important biological processes. At least three proteins of this family from higher vertebrates have been found in unusual locations (i.e., the cell surface, the extracellular space, the cytosol, and the nucleus), reached through an export mechanism which has not yet been understood. In some cases their function in the non-ER location is clearly related to their redox properties, but in most cases their mechanism of action has still to be disclosed, although their propensity to associate with other proteins or even with DNA might be the main factor responsible for their activities.
To study the role of oligosaccharides on the properties of glycoproteins, five glycoproteins (yeast external invertase, bovine serum fetuin, glucoamylase from Aspergillus niger, and chicken egg white ovotransferrin and avidin) of previously established glycan patterns were purified to homogeneity and deglycosylated with endo- and exo-glycosidases in native conditions. Thermal stability and conformational changes were measured by high-resolution differential scanning microcalorimetry and circular dicroism spectroscopy before and after they were deglycosylated. It was found that deglycosylation decreases protein thermal stability, as judged by the decrease in denaturation temperature and denaturation enthalpy, while it does not affect substantially the conformation as indicated by the CD spectra in the far UV range. The destabilization effect of deglycosylation seems to depend on the carbohydrate content, i.e., the maximum effect was observed for the most heavily glycosylated protein, irrespective of the types (N-linked or O-linked) or patterns (mono- or multi-branched) of the covalently attached carbohydrate chains. In addition, studies of the reversibility to heat denaturation revealed that deglycosylated proteins have a poorer thermal reversibility in calorimetric scans than their native counterparts and tend to aggregate during thermal inactivation at acidic pH. These results suggest that carbohydrate moieties, in addition to the apparent stabilizing effect, may prevent the unfolded or partially folded protein molecules from aggregation. Our results support the hypothesis that the general function of protein glycosylation is to aid in folding of the nascent polypeptide chain and in stabilization of the conformation of the mature glycoprotein.
Abbreviations used: 1α,25-(OH) 2 D 3 -1α,25-dihydroxycholecalciferol and calcitriol; APE/Ref-1 -apurinic (apyrimidinic) endonuclease/redox-factor 1; CRP -C-reactive protein; ER -endoplasmic reticulum; ERAD endoplasmic reticulum associated protein degradation; mTORC -mammalian target of rapamycin complex; PDI -protein disulfide isomerase; PKC -protein kinases C; PLA 2 -phospholipases A 2 ; Plc -peptide loading complex; PLC -phospholipase C; PrP SC -scrapie prion protein (misfolded prions); STAT -signal transducer and activator of transcription; STAT3 -signal transducer and activator of transcription 3; SV40 virus -simian virus 40; VDR -vitamin D receptor Review ERp57/GRP58: A PROTEIN WITH MULTIPLE FUNCTIONSCARLO TURANO*, ELISA GAUCCI, CATERINA GRILLO and SILVIA CHICHIARELLI Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza -Università di Roma, Italy Abstract: The protein ERp57/GRP58 is a stress-responsive protein and a component of the protein disulfide isomerase family. Its functions in the endoplasmic reticulum are well known, concerning mainly the proper folding and quality control of glycoproteins, and participation in the assembly of the major histocompatibility complex class 1. However, ERp57 is present in many other subcellular locations, where it is involved in a variety of functions, primarily suggested by its participation in complexes with other proteins and even with DNA. While in some instances these roles need to be confirmed by further studies, a great number of observations support the participation of ERp57 in signal transduction from the cell surface, in regulatory processes taking place in the nucleus, and in multimeric protein complexes involved in DNA repair.
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