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. 1999 Jan 4;18(1):65–74. doi: 10.1093/emboj/18.1.65

The acidic C-terminal domain of protein disulfide isomerase is not critical for the enzyme subunit function or for the chaperone or disulfide isomerase activities of the polypeptide.

P Koivunen 1, A Pirneskoski 1, P Karvonen 1, J Ljung 1, T Helaakoski 1, H Notbohm 1, K I Kivirikko 1
PMCID: PMC1171103  PMID: 9878051

Abstract

Protein disulfide isomerase (PDI) is a multifunctional polypeptide that acts as a subunit in the animal prolyl 4-hydroxylases and the microsomal triglyceride transfer protein, and as a chaperone that binds various peptides and assists their folding. We report here that deletion of PDI sequences corresponding to the entire C-terminal domain c, previously thought to be critical for chaperone activity, had no inhibitory effect on the assembly of recombinant prolyl 4-hydroxylase in insect cells or on the in vitro chaperone activity or disulfide isomerase activity of purified PDI. However, partially overlapping critical regions for all these functions were identified at the C-terminal end of the preceding thioredoxin-like domain a'. Point mutations introduced into this region identified several residues as critical for prolyl 4-hydroxylase assembly. Circular dichroism spectra of three mutants suggested that two of these mutations may have caused only local alterations, whereas one of them may have led to more extensive structural changes. The critical region identified here corresponds to the C-terminal alpha helix of domain a', but this is not the only critical region for any of these functions.

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Selected References

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  1. Annunen P., Helaakoski T., Myllyharju J., Veijola J., Pihlajaniemi T., Kivirikko K. I. Cloning of the human prolyl 4-hydroxylase alpha subunit isoform alpha(II) and characterization of the type II enzyme tetramer. The alpha(I) and alpha(II) subunits do not form a mixed alpha(I)alpha(II)beta2 tetramer. J Biol Chem. 1997 Jul 11;272(28):17342–17348. doi: 10.1074/jbc.272.28.17342. [DOI] [PubMed] [Google Scholar]
  2. Cai H., Wang C. C., Tsou C. L. Chaperone-like activity of protein disulfide isomerase in the refolding of a protein with no disulfide bonds. J Biol Chem. 1994 Oct 7;269(40):24550–24552. [PubMed] [Google Scholar]
  3. Dai Y., Wang C. A mutant truncated protein disulfide isomerase with no chaperone activity. J Biol Chem. 1997 Oct 31;272(44):27572–27576. doi: 10.1074/jbc.272.44.27572. [DOI] [PubMed] [Google Scholar]
  4. Darby N. J., Creighton T. E. Functional properties of the individual thioredoxin-like domains of protein disulfide isomerase. Biochemistry. 1995 Sep 19;34(37):11725–11735. doi: 10.1021/bi00037a009. [DOI] [PubMed] [Google Scholar]
  5. Darby N. J., Kemmink J., Creighton T. E. Identifying and characterizing a structural domain of protein disulfide isomerase. Biochemistry. 1996 Aug 13;35(32):10517–10528. doi: 10.1021/bi960763s. [DOI] [PubMed] [Google Scholar]
  6. Darby N. J., Penka E., Vincentelli R. The multi-domain structure of protein disulfide isomerase is essential for high catalytic efficiency. J Mol Biol. 1998 Feb 13;276(1):239–247. doi: 10.1006/jmbi.1997.1504. [DOI] [PubMed] [Google Scholar]
  7. Edman J. C., Ellis L., Blacher R. W., Roth R. A., Rutter W. J. Sequence of protein disulphide isomerase and implications of its relationship to thioredoxin. Nature. 1985 Sep 19;317(6034):267–270. doi: 10.1038/317267a0. [DOI] [PubMed] [Google Scholar]
  8. Freedman R. B., Hirst T. R., Tuite M. F. Protein disulphide isomerase: building bridges in protein folding. Trends Biochem Sci. 1994 Aug;19(8):331–336. doi: 10.1016/0968-0004(94)90072-8. [DOI] [PubMed] [Google Scholar]
  9. Hawkins H. C., Freedman R. B. The reactivities and ionization properties of the active-site dithiol groups of mammalian protein disulphide-isomerase. Biochem J. 1991 Apr 15;275(Pt 2):335–339. doi: 10.1042/bj2750335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hayano T., Hirose M., Kikuchi M. Protein disulfide isomerase mutant lacking its isomerase activity accelerates protein folding in the cell. FEBS Lett. 1995 Dec 27;377(3):505–511. doi: 10.1016/0014-5793(95)01410-1. [DOI] [PubMed] [Google Scholar]
  11. Helaakoski T., Annunen P., Vuori K., MacNeil I. A., Pihlajaniemi T., Kivirikko K. I. Cloning, baculovirus expression, and characterization of a second mouse prolyl 4-hydroxylase alpha-subunit isoform: formation of an alpha 2 beta 2 tetramer with the protein disulfide-isomerase/beta subunit. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4427–4431. doi: 10.1073/pnas.92.10.4427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Holmgren A. Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoamide. J Biol Chem. 1979 Oct 10;254(19):9627–9632. [PubMed] [Google Scholar]
  13. John D. C., Grant M. E., Bulleid N. J. Cell-free synthesis and assembly of prolyl 4-hydroxylase: the role of the beta-subunit (PDI) in preventing misfolding and aggregation of the alpha-subunit. EMBO J. 1993 Apr;12(4):1587–1595. doi: 10.1002/j.1460-2075.1993.tb05803.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kemmink J., Darby N. J., Dijkstra K., Nilges M., Creighton T. E. Structure determination of the N-terminal thioredoxin-like domain of protein disulfide isomerase using multidimensional heteronuclear 13C/15N NMR spectroscopy. Biochemistry. 1996 Jun 18;35(24):7684–7691. doi: 10.1021/bi960335m. [DOI] [PubMed] [Google Scholar]
  15. Kemmink J., Darby N. J., Dijkstra K., Nilges M., Creighton T. E. The folding catalyst protein disulfide isomerase is constructed of active and inactive thioredoxin modules. Curr Biol. 1997 Apr 1;7(4):239–245. doi: 10.1016/s0960-9822(06)00119-9. [DOI] [PubMed] [Google Scholar]
  16. Kemmink J., Darby N. J., Dijkstra K., Scheek R. M., Creighton T. E. Nuclear magnetic resonance characterization of the N-terminal thioredoxin-like domain of protein disulfide isomerase. Protein Sci. 1995 Dec;4(12):2587–2593. doi: 10.1002/pro.5560041216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kivirikko K. I., Myllyharju J. Prolyl 4-hydroxylases and their protein disulfide isomerase subunit. Matrix Biol. 1998 Feb;16(7):357–368. doi: 10.1016/s0945-053x(98)90009-9. [DOI] [PubMed] [Google Scholar]
  18. Kivirikko K. I., Myllylä R., Pihlajaniemi T. Protein hydroxylation: prolyl 4-hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit. FASEB J. 1989 Mar;3(5):1609–1617. [PubMed] [Google Scholar]
  19. Kivirikko K. I., Myllylä R. Posttranslational enzymes in the biosynthesis of collagen: intracellular enzymes. Methods Enzymol. 1982;82(Pt A):245–304. doi: 10.1016/0076-6879(82)82067-3. [DOI] [PubMed] [Google Scholar]
  20. Kivirikko K. I., Pihlajaniemi T. Collagen hydroxylases and the protein disulfide isomerase subunit of prolyl 4-hydroxylases. Adv Enzymol Relat Areas Mol Biol. 1998;72:325–398. doi: 10.1002/9780470123188.ch9. [DOI] [PubMed] [Google Scholar]
  21. Klappa P., Ruddock L. W., Darby N. J., Freedman R. B. The b' domain provides the principal peptide-binding site of protein disulfide isomerase but all domains contribute to binding of misfolded proteins. EMBO J. 1998 Feb 16;17(4):927–935. doi: 10.1093/emboj/17.4.927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Koivunen P., Helaakoski T., Annunen P., Veijola J., Räisänen S., Pihlajaniemi T., Kivirikko K. I. ERp60 does not substitute for protein disulphide isomerase as the beta-subunit of prolyl 4-hydroxylase. Biochem J. 1996 Jun 1;316(Pt 2):599–605. doi: 10.1042/bj3160599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. LaMantia M. L., Lennarz W. J. The essential function of yeast protein disulfide isomerase does not reside in its isomerase activity. Cell. 1993 Sep 10;74(5):899–908. doi: 10.1016/0092-8674(93)90469-7. [DOI] [PubMed] [Google Scholar]
  24. Lamberg A., Jauhiainen M., Metso J., Ehnholm C., Shoulders C., Scott J., Pihlajaniemi T., Kivirikko K. I. The role of protein disulphide isomerase in the microsomal triacylglycerol transfer protein does not reside in its isomerase activity. Biochem J. 1996 Apr 15;315(Pt 2):533–536. doi: 10.1042/bj3150533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lyles M. M., Gilbert H. F. Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer. Biochemistry. 1991 Jan 22;30(3):613–619. doi: 10.1021/bi00217a004. [DOI] [PubMed] [Google Scholar]
  26. Lyles M. M., Gilbert H. F. Mutations in the thioredoxin sites of protein disulfide isomerase reveal functional nonequivalence of the N- and C-terminal domains. J Biol Chem. 1994 Dec 9;269(49):30946–30952. [PubMed] [Google Scholar]
  27. Martin J. L., Bardwell J. C., Kuriyan J. Crystal structure of the DsbA protein required for disulphide bond formation in vivo. Nature. 1993 Sep 30;365(6445):464–468. doi: 10.1038/365464a0. [DOI] [PubMed] [Google Scholar]
  28. Martin J. L. Thioredoxin--a fold for all reasons. Structure. 1995 Mar 15;3(3):245–250. doi: 10.1016/s0969-2126(01)00154-x. [DOI] [PubMed] [Google Scholar]
  29. Noiva R., Freedman R. B., Lennarz W. J. Peptide binding to protein disulfide isomerase occurs at a site distinct from the active sites. J Biol Chem. 1993 Sep 15;268(26):19210–19217. [PubMed] [Google Scholar]
  30. Noiva R., Lennarz W. J. Protein disulfide isomerase. A multifunctional protein resident in the lumen of the endoplasmic reticulum. J Biol Chem. 1992 Feb 25;267(6):3553–3556. [PubMed] [Google Scholar]
  31. Otsu M., Omura F., Yoshimori T., Kikuchi M. Protein disulfide isomerase associates with misfolded human lysozyme in vivo. J Biol Chem. 1994 Mar 4;269(9):6874–6877. [PubMed] [Google Scholar]
  32. Peitsch M. C. ProMod and Swiss-Model: Internet-based tools for automated comparative protein modelling. Biochem Soc Trans. 1996 Feb;24(1):274–279. doi: 10.1042/bst0240274. [DOI] [PubMed] [Google Scholar]
  33. Pelham H. R. The retention signal for soluble proteins of the endoplasmic reticulum. Trends Biochem Sci. 1990 Dec;15(12):483–486. doi: 10.1016/0968-0004(90)90303-s. [DOI] [PubMed] [Google Scholar]
  34. Pihlajaniemi T., Helaakoski T., Tasanen K., Myllylä R., Huhtala M. L., Koivu J., Kivirikko K. I. Molecular cloning of the beta-subunit of human prolyl 4-hydroxylase. This subunit and protein disulphide isomerase are products of the same gene. EMBO J. 1987 Mar;6(3):643–649. doi: 10.1002/j.1460-2075.1987.tb04803.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Prockop D. J., Kivirikko K. I. Collagens: molecular biology, diseases, and potentials for therapy. Annu Rev Biochem. 1995;64:403–434. doi: 10.1146/annurev.bi.64.070195.002155. [DOI] [PubMed] [Google Scholar]
  36. Puig A., Gilbert H. F. Protein disulfide isomerase exhibits chaperone and anti-chaperone activity in the oxidative refolding of lysozyme. J Biol Chem. 1994 Mar 11;269(10):7764–7771. [PubMed] [Google Scholar]
  37. Puig A., Lyles M. M., Noiva R., Gilbert H. F. The role of the thiol/disulfide centers and peptide binding site in the chaperone and anti-chaperone activities of protein disulfide isomerase. J Biol Chem. 1994 Jul 22;269(29):19128–19135. [PubMed] [Google Scholar]
  38. Quan H., Fan G., Wang C. C. Independence of the chaperone activity of protein disulfide isomerase from its thioredoxin-like active site. J Biol Chem. 1995 Jul 21;270(29):17078–17080. doi: 10.1074/jbc.270.29.17078. [DOI] [PubMed] [Google Scholar]
  39. Ricci B., Sharp D., O'Rourke E., Kienzle B., Blinderman L., Gordon D., Smith-Monroy C., Robinson G., Gregg R. E., Rader D. J. A 30-amino acid truncation of the microsomal triglyceride transfer protein large subunit disrupts its interaction with protein disulfide-isomerase and causes abetalipoproteinemia. J Biol Chem. 1995 Jun 16;270(24):14281–14285. doi: 10.1074/jbc.270.24.14281. [DOI] [PubMed] [Google Scholar]
  40. Rost B., Sander C. Secondary structure prediction of all-helical proteins in two states. Protein Eng. 1993 Nov;6(8):831–836. doi: 10.1093/protein/6.8.831. [DOI] [PubMed] [Google Scholar]
  41. Rupp K., Birnbach U., Lundström J., Van P. N., Söling H. D. Effects of CaBP2, the rat analog of ERp72, and of CaBP1 on the refolding of denatured reduced proteins. Comparison with protein disulfide isomerase. J Biol Chem. 1994 Jan 28;269(4):2501–2507. [PubMed] [Google Scholar]
  42. Song J. L., Wang C. C. Chaperone-like activity of protein disulfide-isomerase in the refolding of rhodanese. Eur J Biochem. 1995 Jul 15;231(2):312–316. doi: 10.1111/j.1432-1033.1995.tb20702.x. [DOI] [PubMed] [Google Scholar]
  43. Veijola J., Annunen P., Koivunen P., Page A. P., Pihlajaniemi T., Kivirikko K. I. Baculovirus expression of two protein disulphide isomerase isoforms from Caenorhabditis elegans and characterization of prolyl 4-hydroxylases containing one of these polypeptides as their beta subunit. Biochem J. 1996 Aug 1;317(Pt 3):721–729. doi: 10.1042/bj3170721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Veijola J., Koivunen P., Annunen P., Pihlajaniemi T., Kivirikko K. I. Cloning, baculovirus expression, and characterization of the alpha subunit of prolyl 4-hydroxylase from the nematode Caenorhabditis elegans. This alpha subunit forms an active alpha beta dimer with the human protein disulfide isomerase/beta subunit. J Biol Chem. 1994 Oct 28;269(43):26746–26753. [PubMed] [Google Scholar]
  45. Vuori K., Myllylä R., Pihlajaniemi T., Kivirikko K. I. Expression and site-directed mutagenesis of human protein disulfide isomerase in Escherichia coli. This multifunctional polypeptide has two independently acting catalytic sites for the isomerase activity. J Biol Chem. 1992 Apr 15;267(11):7211–7214. [PubMed] [Google Scholar]
  46. Vuori K., Pihlajaniemi T., Marttila M., Kivirikko K. I. Characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide-isomerase subunit synthesized in a baculovirus expression system. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7467–7470. doi: 10.1073/pnas.89.16.7467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Vuori K., Pihlajaniemi T., Myllylä R., Kivirikko K. I. Site-directed mutagenesis of human protein disulphide isomerase: effect on the assembly, activity and endoplasmic reticulum retention of human prolyl 4-hydroxylase in Spodoptera frugiperda insect cells. EMBO J. 1992 Nov;11(11):4213–4217. doi: 10.1002/j.1460-2075.1992.tb05515.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Walker K. W., Lyles M. M., Gilbert H. F. Catalysis of oxidative protein folding by mutants of protein disulfide isomerase with a single active-site cysteine. Biochemistry. 1996 Feb 13;35(6):1972–1980. doi: 10.1021/bi952157n. [DOI] [PubMed] [Google Scholar]
  49. Wetterau J. R., Combs K. A., McLean L. R., Spinner S. N., Aggerbeck L. P. Protein disulfide isomerase appears necessary to maintain the catalytically active structure of the microsomal triglyceride transfer protein. Biochemistry. 1991 Oct 8;30(40):9728–9735. doi: 10.1021/bi00104a023. [DOI] [PubMed] [Google Scholar]
  50. Wetterau J. R., Combs K. A., Spinner S. N., Joiner B. J. Protein disulfide isomerase is a component of the microsomal triglyceride transfer protein complex. J Biol Chem. 1990 Jun 15;265(17):9800–9807. [PubMed] [Google Scholar]
  51. Yao Y., Zhou Y., Wang C. Both the isomerase and chaperone activities of protein disulfide isomerase are required for the reactivation of reduced and denatured acidic phospholipase A2. EMBO J. 1997 Feb 3;16(3):651–658. doi: 10.1093/emboj/16.3.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zheng W. D., Quan H., Song J. L., Yang S. L., Wang C. C. Does DsbA have chaperone-like activity? Arch Biochem Biophys. 1997 Jan 15;337(2):326–331. doi: 10.1006/abbi.1996.9783. [DOI] [PubMed] [Google Scholar]

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