title: Identification and characterization of structural domains of human ERp57: association with calreticulin requires several domains.
abstract: The amino acid sequence of ERp57, which functions in the endoplasmic reticulum together with the lectins calreticulin and calnexin to achieve folding of newly synthesized glycoproteins, is highly similar to that of protein disulfide isomerase (PDI), but they have their own distinct roles in protein folding. We have characterized the domain structure of ERp57 by limited proteolysis and N-terminal sequencing and have found it to be similar but not identical to that of PDI. ERp57 had three major protease-sensitive regions, the first of which was located between residues 120 and 150, the second between 201 and 215, and the third between 313 and 341, the data thus being consistent with a four-domain structure abb'a'. Recombinant expression in Escherichia coli was used to verify the domain boundaries. Each single domain and a b'a' double domain could be produced in the form of soluble, folded polypeptides, as verified by circular dichroism spectra and urea gradient gel electrophoresis. When the ability of ERp57 and its a and a' domains to fold denatured RNase A was studied by electrospray mass analyses, ERp57 markedly enhanced the folding rate at early time points, although less effectively than PDI, but was an ineffective catalyst of the overall process. The a and a' domains produced only minor, if any, increases in the folding rate at the early stages and no increase at the late stages. Interaction of the soluble ERp57 domains with the P domain of calreticulin was studied by chemical cross-linking in vitro. None of the single ERp57 domains nor the b'a' double domain could be cross-linked to the P domain, whereas cross-linking was obtained with a hybrid ERpabb'PDIa'c polypeptide but not with ERpabPDIb'a'c, indicating that multiple domains are involved in this protein-protein interaction and that the b' domain of ERp57 cannot be replaced by that of PDI.
authors.affiliation: Department of Medical Biochemistry and Molecular Biology, University of Oulu, PO Box 5000, FIN-90014 Oulu, Finland.
authors.forename: Laura
authors.initials: L
authors.lastname: Silvennoinen
authors.forename: Johanna
authors.initials: J
authors.lastname: Myllyharju
authors.forename: Margherita
authors.initials: M
authors.lastname: Ruoppolo
authors.forename: Stefania
authors.initials: S
authors.lastname: Orrù
authors.forename: Marianna
authors.initials: M
authors.lastname: Caterino
authors.forename: Kari I
authors.initials: KI
authors.lastname: Kivirikko
authors.forename: Peppi
authors.initials: P
authors.lastname: Koivunen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D037282:Calreticulin / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007313:Insecta; D007535:Isomerases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D012995:Solubility; D014508:Urea
chemical_list: D037282:Calreticulin; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D014508:Urea; D012259:Ribonuclease, Pancreatic; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human