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Cloning of the Human Prolyl 4-Hydroxylase α Subunit Isoform α(II) and Characterization of the Type II Enzyme Tetramer

THE α(I) AND α(II) SUBUNITS DO NOT FORM A MIXED α(I)α(II)β2 TETRAMER*
  • Pia Annunen
    Affiliations
    Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, FIN-90220 Oulu, Finland
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  • Tarja Helaakoski
    Affiliations
    Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, FIN-90220 Oulu, Finland
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  • Johanna Myllyharju
    Affiliations
    Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, FIN-90220 Oulu, Finland
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  • Johanna Veijola
    Affiliations
    Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, FIN-90220 Oulu, Finland
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  • Taina Pihlajaniemi
    Affiliations
    Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, FIN-90220 Oulu, Finland
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  • Kari I. Kivirikko
    Correspondence
    To whom correspondence should be addressed: Dept. of Medical Biochemistry, University of Oulu, Kajaanintie 52A, FIN-90220 Oulu, Finland. Tel.: 358-8-5375801; Fax: 358-8-5375810
    Affiliations
    Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, FIN-90220 Oulu, Finland
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  • Author Footnotes
    * This work was supported by grants from the Research Council for Health within the Academy of Finland and from FibroGen Inc., South San Francisco, CA.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:July 11, 1997DOI:https://doi.org/10.1074/jbc.272.28.17342
      Prolyl 4-hydroxylase (proline hydroxylase, EC1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an α2β2tetramer, the β subunit of which is identical to protein disulfide-isomerase (PDI, EC 5.3.4.1). We report here on cloning of the recently discovered α(II) subunit from human sources. The mRNA for the α(II) subunit was found to be expressed in a variety of human tissues, and the presence of the corresponding polypeptide and the (α(II))2β2 tetramer was demonstrated in cultured human WI-38 and HT-1080 cells. The type II tetramer was found to represent about 30% of the total prolyl 4-hydroxylase in these cells and about 5–15% in various chick embryo tissues. The results of coexpression in insect cells argued strongly against the formation of a mixed α(I)α(II)β2 tetramer. PDI/β polypeptide containing a histidine tag in its N terminus was found to form prolyl 4-hydroxylase tetramers as readily as the wild-type PDI/β polypeptide, and histidine-tagged forms of prolyl 4-hydroxylase appear to offer an excellent source for a simple large scale purification of the recombinant enzyme. The properties of the purified human type II enzyme were very similar to those of the type I enzyme, but theK i of the former for poly(l-proline) was about 200–1000 times that of the latter. In agreement with this, a minor difference, about 3–6-fold, was found between the two enzymes in the K m values for three peptide substrates. The existence of two forms of prolyl 4-hydroxylase in human cells raises the possibility that mutations in one enzyme form may not be lethal despite the central role of this enzyme in the synthesis of all collagens.
      Prolyl 4-hydroxylase (proline hydroxylase, EC 1.14.11.2) catalyzes the hydroxylation of proline in -Xaa-Pro-Gly- triplets in collagens and other proteins with collagen-like sequences. The enzyme plays a central role in the synthesis of all collagens, as the 4-hydroxyproline residues formed in the reaction are essential for the folding of the newly synthesized collagen polypeptide chains into triple helical molecules. The vertebrate enzyme is an α2β2tetramer in which the α subunits contribute to most parts of the two catalytic sites (for reviews, see Refs.
      • Kivirikko K.I.
      • Myllylä R.
      • Pihlajaniemi T.
      ,
      • Kivirikko K.I.
      • Myllylä R.
      • Pihlajaniemi T.
      ,
      • Prockop D.J.
      • Kivirikko K.I.
      ). The β subunit is identical to the enzyme protein disulfide-isomerase (PDI, EC5.3.4.1)
      The abbreviations used are: PDI, protein disulfide-isomerase; His-PDI, PDI containing a histidine affinity tag in its N terminus; PAGE, polyacrylamide gel electrophoresis.
      1The abbreviations used are: PDI, protein disulfide-isomerase; His-PDI, PDI containing a histidine affinity tag in its N terminus; PAGE, polyacrylamide gel electrophoresis.
      and has PDI activity even when present in the prolyl 4-hydroxylase tetramer (
      • Pihlajaniemi T.
      • Helaakoski T.
      • Tasanen K.
      • Myllylä R.
      • Huhtala M.-L.
      • Koivu J.
      • Kivirikko K.I.
      ,
      • Koivu J.
      • Myllylä R.
      • Helaakoski T.
      • Pihlajaniemi T.
      • Tasanen K.
      • Kivirikko K.I.
      ,
      • Parkkonen T.
      • Kivirikko K.I.
      • Pihlajaniemi T.
      ). The PDI polypeptide also has several other functions (
      • Kivirikko K.I.
      • Myllylä R.
      • Pihlajaniemi T.
      ,
      • Kivirikko K.I.
      • Myllylä R.
      • Pihlajaniemi T.
      ,
      • Prockop D.J.
      • Kivirikko K.I.
      ,
      • Noiva R.
      • Lennarz W.J.
      ,
      • Freedman R.B.
      • Hirst T.R.
      • Tuite M.F.
      ).
      Prolyl 4-hydroxylase had long been assumed to be of one type only, with no isoenzymes (
      • Kivirikko K.I.
      • Myllylä R.
      • Pihlajaniemi T.
      ,
      • Kivirikko K.I.
      • Myllylä R.
      • Pihlajaniemi T.
      ,
      • Prockop D.J.
      • Kivirikko K.I.
      ), but recently an isoform of the α subunit, termed the α(II) subunit, was cloned from mouse tissues (
      • Helaakoski T.
      • Annunen P.
      • MacNeil I.A.
      • Vuori K.
      • Pihlajaniemi T.
      • Kivirikko K.I.
      ). Correspondingly, the previously known α subunit is now called the α(I) subunit. The α(II) subunit was found to form an (α(II))2β2 tetramer with the PDI/β subunit when the two polypeptides were coexpressed in insect cells. The properties of the new type II enzyme were found to be very similar to those of the type I tetramer, with the distinct difference that it was inhibited by poly(l-proline) only at very high concentrations (
      • Helaakoski T.
      • Annunen P.
      • MacNeil I.A.
      • Vuori K.
      • Pihlajaniemi T.
      • Kivirikko K.I.
      ).
      The α subunit of prolyl 4-hydroxylase cloned from the nematodeCaenorhabditis elegans (
      • Veijola J.
      • Koivunen P.
      • Annunen P.
      • Pihlajaniemi T.
      • Kivirikko K.I.
      ) has been found to have features of both types of mouse α subunit, suggesting that C. elegans may have only one type of α subunit (
      • Helaakoski T.
      • Annunen P.
      • MacNeil I.A.
      • Vuori K.
      • Pihlajaniemi T.
      • Kivirikko K.I.
      ). This forms active prolyl 4-hydroxylase in insect cell coexpression experiments with either the C. elegans or the human PDI/β polypeptide, but surprisingly, the enzymes containing the C. elegans α subunit are αβ dimers (
      • Veijola J.
      • Koivunen P.
      • Annunen P.
      • Pihlajaniemi T.
      • Kivirikko K.I.
      ,
      • Veijola J.
      • Annunen P.
      • Koivunen P.
      • Page A.P.
      • Pihlajaniemi T.
      • Kivirikko K.I.
      ).
      We report here that the existence of α(II) subunit mRNA is not limited to the mouse, as a corresponding mRNA is expressed in a variety of human tissues. All the data so far available on the existence of the type II prolyl 4-hydroxylase tetramer are based on insect cell coexpression experiments, but we now demonstrate that this enzyme is indeed present in cultured human fibroblasts and represents about 30% of their total prolyl 4-hydroxylase activity. We also studied whether the α(I) and α(II) subunits can form a mixed α(I)α(II)β2 tetramer, and whether any differences are found between the type I and II enzymes in their K m values for various peptide substrates, as the two mouse enzymes differ so markedly from each other with respect to inhibition by poly(l-proline). A new affinity purification procedure was developed that is based on the use of a histidine tag in the N terminus of the PDI/β polypeptide, and this makes it possible to obtain large amounts of any form of the recombinant enzyme by very simple steps.

      DISCUSSION

      The data reported here indicate that the existence of an mRNA for the α(II) subunit of prolyl 4-hydroxylase is not limited to the mouse (
      • Helaakoski T.
      • Annunen P.
      • MacNeil I.A.
      • Vuori K.
      • Pihlajaniemi T.
      • Kivirikko K.I.
      ), as an mRNA coding for a highly similar α(II) subunit was also found in human tissues. Furthermore, the present data indicate that the α(II) subunit is translated into the corresponding polypeptide in human cells. The α(II) subunit mRNA was found to be expressed in a variety of tissues, but distinct differences were found in the expression patterns of the α(II) and α(I) subunit mRNAs between tissues.
      Quantification of the proportions of the two types of prolyl 4-hydroxylase tetramer by Western blotting in cultured human WI-38 lung fibroblasts and HT-1080 fibrosarcoma cells indicated that the type II tetramer represents about 30% of the total enzyme protein in these two cell types. Correspondingly, about 30% of the total prolyl 4-hydroxylase activity in extracts from these two cell types was found in the flow-through fractions of poly(l-proline) affinity columns, suggesting that the (α(II))2β2tetramer represents about 30% of the total prolyl 4-hydroxylase activity. The type II prolyl 4-hydroxylase is also likely to be present in chick embryo tissues, as a fraction of the total enzyme activity was found to pass through the poly(l-proline) affinity column in the case of all the chick embryo tissues studied. Nevertheless, the proportion of type II enzyme activity may be lower in chick embryo tissues than in human fibroblasts, about 5–15% of total prolyl 4-hydroxylase activity. This percentage agrees with early reports indicating that up to at least 80% of the total prolyl 4-hydroxylase activity present in crude extracts from whole chick embryos is bound to a poly(l-proline) affinity column (
      • Tuderman L.
      • Kuutti E-R.
      • Kivirikko K.I.
      ), and that at least 80% of the total prolyl 4-hydroxylase activity present in extracts from whole chick embryo homogenates is inhibited by poly(l-proline) (
      • Prockop D.J.
      • Kivirikko K.I.
      ).
      The present insect cell expression data argue strongly against the presence of a protein containing the α(I) and α(II) subunits in a single molecule. No information is currently available on sequences in the α subunits that are involved in the α2β2 tetramer assembly, but the C-terminal domains of the α subunits, which show the highest degrees of amino acid sequence identity between the α(I) and α(II) subunits and theC. elegans α subunit (
      • Helaakoski T.
      • Annunen P.
      • MacNeil I.A.
      • Vuori K.
      • Pihlajaniemi T.
      • Kivirikko K.I.
      ,
      • Veijola J.
      • Koivunen P.
      • Annunen P.
      • Pihlajaniemi T.
      • Kivirikko K.I.
      ), are known to contain residues involved in the binding of all the cosubstrates to a catalytic site (
      • Lamberg A.
      • Pihlajaniemi T.
      • Kivirikko K.I.
      ,
      • Myllyharju J.
      • Kivirikko K.I.
      ). It seems probable that the regions involved in tetramer assembly contain some sequences that prevent incorporation of the α(I) and α(II) subunits into the same molecule.
      Although no information is available on the sequences in the α subunits that are critical for tetramer assembly, several observations suggest that in the case of the PDI/β subunit some such sequences are located close to the C-terminal domain of the polypeptide (
      • Veijola J.
      • Annunen P.
      • Koivunen P.
      • Page A.P.
      • Pihlajaniemi T.
      • Kivirikko K.I.
      ,
      • Koivunen P.
      • Helaakoski T.
      • Annunen P.
      • Veijola J.
      • Räisänen S.
      • Pihlajaniemi T.
      • Kivirikko K.I.
      ). The present data demonstrate that the N terminus of the PDI/β polypeptide is not critical for tetramer assembly, as the His-PDI polypeptide was found to form an active prolyl 4-hydroxylase as readily as the wild-type PDI/β polypeptide. Our additional experiments have demonstrated that the His-PDI polypeptide also efficiently forms an αβ dimer with the C. elegans prolyl 4-hydroxylase α subunit.
      J. Myllyharju and K. I. Kivirikko, unpublished observations.
      The histidine-tagged forms of prolyl 4-hydroxylase appear to offer an excellent source of the enzyme for simple large scale purification in experiments such as attempts at crystallization.
      Poly(l-proline) has been regarded as a highly effective competitive inhibitor with respect to the polypeptide substrate of prolyl 4-hydroxylases from all the vertebrate sources studied, and an efficient polypeptide substrate for all plant prolyl 4-hydroxylases (
      • Kivirikko K.I.
      • Myllylä R.
      • Pihlajaniemi T.
      ,
      • Kivirikko K.I.
      • Myllylä R.
      • Pihlajaniemi T.
      ,
      • Prockop D.J.
      • Kivirikko K.I.
      ). It is therefore highly surprising that the human and mouse (
      • Helaakoski T.
      • Annunen P.
      • MacNeil I.A.
      • Vuori K.
      • Pihlajaniemi T.
      • Kivirikko K.I.
      ) type II prolyl 4-hydroxylases are inhibited by poly(l-proline) only at very high concentrations. This property of the type II enzyme agrees with that reported for crude preparations of prolyl 4-hydroxylase from the nematode Ascaris lumbricoides (
      • Fujimoto D.
      • Prockop D.J.
      ) and for the recombinant C. elegansprolyl 4-hydroxylase αβ dimer (
      • Veijola J.
      • Koivunen P.
      • Annunen P.
      • Pihlajaniemi T.
      • Kivirikko K.I.
      ,
      • Veijola J.
      • Annunen P.
      • Koivunen P.
      • Page A.P.
      • Pihlajaniemi T.
      • Kivirikko K.I.
      ). As these findings suggest that distinct differences are likely to exist in the structures of the peptide binding sites of various prolyl 4-hydroxylases, a comparison was made here between the K m values of the human type I and type II enzymes for three peptide substrates: the polypeptide (Pro-Pro-Gly)10, which is the most commonly used synthetic peptide substrate for prolyl 4-hydroxylase, the pentapeptide Gly-Val-Pro-Gly-Val, which has been introduced as a model peptide for elastin (
      • Atreya P.L.
      • Ananthanarayanan V.S.
      ), a protein that also contains 4-hydroxyproline (
      • Kivirikko K.I.
      • Myllylä R.
      • Pihlajaniemi T.
      ,
      • Kivirikko K.I.
      • Myllylä R.
      • Pihlajaniemi T.
      ,
      • Prockop D.J.
      • Kivirikko K.I.
      ), and protocollagen, a biologically prepared collagenous substrate for the enzyme. Small but significant differences were found between the type I and type II enzymes in these experiments, in that the K m values for all three peptide substrates with the type II enzyme were about 3–6 times those with the type I enzyme. Nevertheless, these differences are very small when compared with the at least 200–1000-fold differences between their K i values for poly(l-proline).
      Mutations have been characterized in the genes for many types of collagen and for lysyl hydroxylase, a collagen hydroxylase closely related to prolyl 4-hydroxylase in its catalytic properties (
      • Prockop D.J.
      • Kivirikko K.I.
      ,
      • Kivirikko K.I.
      ,
      • Hyland J.
      • Ala-Kokko L.
      • Royce P.
      • Steinmann B.
      • Kivirikko K.I.
      • Myllylä R.
      ,
      • Hautala T.
      • Heikkinen J.
      • Kivirikko K.I.
      • Myllylä R.
      ,
      • Ha V.T.
      • Marshall M.K.
      • Elsas L.J.
      • Pinnell S.R.
      • Yeowell H.N.
      ). No mutations have been identified in the gene coding for the α(I) subunit of prolyl 4-hydroxylase, and due to the central role of this enzyme in the synthesis of all collagens, such mutations have generally been assumed to be lethal. The present data indicating the presence of two isoforms of prolyl 4-hydroxylase α subunit in human tissues raises the possibility, however, that mutations in the gene coding for one type may not be lethal, especially if cells are capable of up-regulating the expression of the other type in cases when one type is inactive.

      Acknowledgments

      We thank Riitta Polojärvi, Hanna-Mari Jauho, and Anne Kokko for their expert technical assistance.

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