Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1997 Nov 17;16(22):6702–6712. doi: 10.1093/emboj/16.22.6702

Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase.

A Vuorela 1, J Myllyharju 1, R Nissi 1, T Pihlajaniemi 1, K I Kivirikko 1
PMCID: PMC1170275  PMID: 9362485

Abstract

Prolyl 4-hydroxylase, the key enzyme of collagen synthesis, is an alpha2beta2 tetramer, the beta subunit of which is protein disulfide isomerase (PDI). Coexpression of the human alpha subunit and PDI in Pichia produced trace amounts of an active tetramer. A much higher, although still low, assembly level was obtained using a Saccharomyces pre-pro sequence in PDI. Coexpression with human type III procollagen unexpectedly increased the assembly level 10-fold, with no increase in the total amounts of the subunits. The recombinant enzyme was active not only in Pichia extracts but also inside the yeast cell, indicating that Pichia must have a system for transporting all the cosubstrates needed by the enzyme into the lumen of the endoplasmic reticulum. The 4-hydroxyproline-containing procollagen polypeptide chains were of full length and formed molecules with stable triple helices even though Pichia probably has no Hsp47-like protein. The data indicate that collagen synthesis in Pichia, and probably also in other cells, involves a highly unusual control mechanism, in that production of a stable prolyl 4-hydroxylase requires collagen expression while assembly of a stable collagen requires enzyme expression. This Pichia system seems ideal for the high-level production of various recombinant collagens for numerous scientific and medical purposes.

Full Text

The Full Text of this article is available as a PDF (322.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  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. Berg R. A., Kao W. W., Kedersha N. L. The assembly of tetrameric prolyl hydroxylase in tendon fibroblasts from newly synthesized alpha-subunits and from preformed cross-reacting protein. Biochem J. 1980 Sep 1;189(3):491–499. doi: 10.1042/bj1890491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bruckner P., Prockop D. J. Proteolytic enzymes as probes for the triple-helical conformation of procollagen. Anal Biochem. 1981 Jan 15;110(2):360–368. doi: 10.1016/0003-2697(81)90204-9. [DOI] [PubMed] [Google Scholar]
  4. Buckholz R. G., Gleeson M. A. Yeast systems for the commercial production of heterologous proteins. Biotechnology (N Y) 1991 Nov;9(11):1067–1072. doi: 10.1038/nbt1191-1067. [DOI] [PubMed] [Google Scholar]
  5. Bulleid N. J., Wilson R., Lees J. F. Type-III procollagen assembly in semi-intact cells: chain association, nucleation and triple-helix folding do not require formation of inter-chain disulphide bonds but triple-helix nucleation does require hydroxylation. Biochem J. 1996 Jul 1;317(Pt 1):195–202. doi: 10.1042/bj3170195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chichester C. O., 3rd, Fuller G. C., Cardinale G. J. In vivo labeling and turnover of prolyl hydroxylase and a related immunoreactive protein. Biochem Biophys Res Commun. 1976 Dec 20;73(4):1056–1062. doi: 10.1016/0006-291x(76)90230-8. [DOI] [PubMed] [Google Scholar]
  7. Chung E., Miller E. J. Collagen polymorphism: characterization of molecules with the chain composition (alpha 1 (3)03 in human tissues. Science. 1974 Mar;183(130):1200–1201. doi: 10.1126/science.183.4130.1200. [DOI] [PubMed] [Google Scholar]
  8. Cregg J. M., Vedvick T. S., Raschke W. C. Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology (N Y) 1993 Aug;11(8):905–910. doi: 10.1038/nbt0893-905. [DOI] [PubMed] [Google Scholar]
  9. De Jong L., Kemp A. Stoicheiometry and kinetics of the prolyl 4-hydroxylase partial reaction. Biochim Biophys Acta. 1984 May 31;787(1):105–111. doi: 10.1016/0167-4838(84)90113-4. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Goffeau A., Barrell B. G., Bussey H., Davis R. W., Dujon B., Feldmann H., Galibert F., Hoheisel J. D., Jacq C., Johnston M. Life with 6000 genes. Science. 1996 Oct 25;274(5287):546, 563-7. doi: 10.1126/science.274.5287.546. [DOI] [PubMed] [Google Scholar]
  12. Hebda P. A., Ebert J., Chou K. L., Shields M., Kao W. W. The association between prolyl hydroxylase metabolism and cell growth in cultured L-929 fibroblasts. Biochim Biophys Acta. 1983 Jul 29;758(2):128–134. doi: 10.1016/0304-4165(83)90293-3. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Helaakoski T., Vuori K., Myllylä R., Kivirikko K. I., Pihlajaniemi T. Molecular cloning of the alpha-subunit of human prolyl 4-hydroxylase: the complete cDNA-derived amino acid sequence and evidence for alternative splicing of RNA transcripts. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4392–4396. doi: 10.1073/pnas.86.12.4392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. John D. C., Bulleid N. J. Intracellular dissociation and reassembly of prolyl 4-hydroxylase:the alpha-subunits associated with the immunoglobulin-heavy-chain binding protein (BiP) allowing reassembly with the beta-subunit. Biochem J. 1996 Aug 1;317(Pt 3):659–665. doi: 10.1042/bj3170659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. Kivirikko K. I., Myllylä R. Recent developments in posttranslational modification: intracellular processing. Methods Enzymol. 1987;144:96–114. doi: 10.1016/0076-6879(87)44175-x. [DOI] [PubMed] [Google Scholar]
  20. Koivu J., Myllylä R., Helaakoski T., Pihlajaniemi T., Tasanen K., Kivirikko K. I. A single polypeptide acts both as the beta subunit of prolyl 4-hydroxylase and as a protein disulfide-isomerase. J Biol Chem. 1987 May 15;262(14):6447–6449. [PubMed] [Google Scholar]
  21. Lamberg A., Helaakoski T., Myllyharju J., Peltonen S., Notbohm H., Pihlajaniemi T., Kivirikko K. I. Characterization of human type III collagen expressed in a baculovirus system. Production of a protein with a stable triple helix requires coexpression with the two types of recombinant prolyl 4-hydroxylase subunit. J Biol Chem. 1996 May 17;271(20):11988–11995. doi: 10.1074/jbc.271.20.11988. [DOI] [PubMed] [Google Scholar]
  22. Majamaa K., Kuutti-Savolainen E. R., Tuderman L., Kivirikko K. I. Turnover of prolyl hydroxylase tetramers and the monomer-size protein in chick-embryo cartilaginous bone and lung in vivo. Biochem J. 1979 Feb 15;178(2):313–322. doi: 10.1042/bj1780313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mayne R., Brewton R. G. New members of the collagen superfamily. Curr Opin Cell Biol. 1993 Oct;5(5):883–890. doi: 10.1016/0955-0674(93)90039-s. [DOI] [PubMed] [Google Scholar]
  24. Myllylä R., Majamaa K., Günzler V., Hanauske-Abel H. M., Kivirikko K. I. Ascorbate is consumed stoichiometrically in the uncoupled reactions catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase. J Biol Chem. 1984 May 10;259(9):5403–5405. [PubMed] [Google Scholar]
  25. Myllylä R., Tuderman L., Kivirikko K. I. Mechanism of the prolyl hydroxylase reaction. 2. Kinetic analysis of the reaction sequence. Eur J Biochem. 1977 Nov 1;80(2):349–357. doi: 10.1111/j.1432-1033.1977.tb11889.x. [DOI] [PubMed] [Google Scholar]
  26. Nagata K. Hsp47: a collagen-specific molecular chaperone. Trends Biochem Sci. 1996 Jan;21(1):22–26. doi: 10.1016/0968-0004(96)80881-4. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Pihlajaniemi T., Rehn M. Two new collagen subgroups: membrane-associated collagens and types XV and XVII. Prog Nucleic Acid Res Mol Biol. 1995;50:225–262. doi: 10.1016/s0079-6603(08)60816-8. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Romanos M. A., Scorer C. A., Clare J. J. Foreign gene expression in yeast: a review. Yeast. 1992 Jun;8(6):423–488. doi: 10.1002/yea.320080602. [DOI] [PubMed] [Google Scholar]
  32. Satoh M., Hirayoshi K., Yokota S., Hosokawa N., Nagata K. Intracellular interaction of collagen-specific stress protein HSP47 with newly synthesized procollagen. J Cell Biol. 1996 Apr;133(2):469–483. doi: 10.1083/jcb.133.2.469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schmitt M. E., Brown T. A., Trumpower B. L. A rapid and simple method for preparation of RNA from Saccharomyces cerevisiae. Nucleic Acids Res. 1990 May 25;18(10):3091–3092. doi: 10.1093/nar/18.10.3091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Scorer C. A., Clare J. J., McCombie W. R., Romanos M. A., Sreekrishna K. Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression. Biotechnology (N Y) 1994 Feb;12(2):181–184. doi: 10.1038/nbt0294-181. [DOI] [PubMed] [Google Scholar]
  35. Tromp G., Kuivaniemi H., Shikata H., Prockop D. J. A single base mutation that substitutes serine for glycine 790 of the alpha 1 (III) chain of type III procollagen exposes an arginine and causes Ehlers-Danlos syndrome IV. J Biol Chem. 1989 Jan 25;264(3):1349–1352. [PubMed] [Google Scholar]
  36. Tuderman L., Myllylä R., Kivirikko K. I. Mechanism of the prolyl hydroxylase reaction. 1. Role of co-substrates. Eur J Biochem. 1977 Nov 1;80(2):341–348. doi: 10.1111/j.1432-1033.1977.tb11888.x. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. 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]
  39. Veijola J., Pihlajaniemi T., Kivirikko K. I. Co-expression of the alpha subunit of human prolyl 4-hydroxylase with BiP polypeptide in insect cells leads to the formation of soluble and insoluble complexes. Soluble alpha-subunit-BiP complexes have no prolyl 4-hydroxylase activity. Biochem J. 1996 Apr 15;315(Pt 2):613–618. doi: 10.1042/bj3150613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. 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]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES