Kristina Vuori

Research Article1 November 1992free access 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. K. Vuori K. Vuori Collagen Research Unit, University of Oulu, Finland. Search for more papers by this author T. Pihlajaniemi T. Pihlajaniemi Collagen Research Unit, University of Oulu, Finland. Search for more papers by this author R. Myllylä R. Myllylä Collagen Research Unit, University of Oulu, Finland. Search for more papers by this author K.I. Kivirikko K.I. Kivirikko Collagen Research Unit, University of Oulu, Finland. Search for more papers by this author K. Vuori K. Vuori Collagen Research Unit, University of Oulu, Finland. Search for more papers by this author T. Pihlajaniemi T. Pihlajaniemi Collagen Research Unit, University of Oulu, Finland. Search for more papers by this author R. Myllylä R. Myllylä Collagen Research Unit, University of Oulu, Finland. Search for more papers by this author K.I. Kivirikko K.I. Kivirikko Collagen Research Unit, University of Oulu, Finland. Search for more papers by this author Author Information K. Vuori1, T. Pihlajaniemi1, R. Myllylä1 and K.I. Kivirikko1 1Collagen Research Unit, University of Oulu, Finland. The EMBO Journal (1992)11:4213-4217https://doi.org/10.1002/j.1460-2075.1992.tb05515.x PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Protein disulphide isomerase (PDI) is a highly unusual multifunctional polypeptide, identical to the beta-subunit of prolyl 4-hydroxylase. It has two -Cys-Gly-His-Cys- sequences which represent two independently acting catalytic sites of PDI activity. We report here on the expression in baculovirus vectors of various mutant PDI/beta-subunits together with a wild-type alpha-subunit of the human prolyl 4-hydroxylase alpha 2 beta 2 tetramer in Spodoptera frugiperda insect cells. When either one or both of the -Cys-Gly-His-Cys- sequences was converted to -Ser-Gly-His-Cys-, a tetramer was formed as with wild-type PDI/beta-subunit. This tetramer was fully active prolyl 4-hydroxylase. The data demonstrate that PDI activity of the PDI/beta-subunit is not required for tetramer assembly or for the prolyl 4-hydroxylase activity of the tetramer, and thus other sequences of the PDI/beta-subunit may be critical for keeping the alpha-subunits in a catalytically active, non-aggregated conformation. Measurements of the PDI activities of tetramers containing the various mutant PDI/beta-subunits demonstrated that the activity of the wild-type tetramer is almost exclusively due to the C-terminal PDI catalytic sites, which explains the finding that the PDI activity of the PDI/beta-subunit present in the tetramer is about half that in the free polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS) Previous ArticleNext Article Volume 11Issue 111 November 1992In this issue RelatedDetailsLoading ...

Protein disulfide isomerase (PDI, EC 5.3.4.1) is a highly unusual multifunctional polypeptide, being identical to the beta subunit of prolyl 4-hydroxylase, a cellular thyroid hormone binding protein and a component of the microsomal triglyceride transfer protein complex, and highly similar to a polypeptide acting in vitro as a glycosylation site binding protein. It has two -Cys-Gly-His-Cys- sequences which, it has been proposed, act as catalytic sites for the isomerase activity, but few data have been available to indicate whether one or both of them do indeed act as catalytic sites and whether the two presumed catalytic sites act independently or cooperatively. We report here on the expression of human PDI in Escherichia coli with three different signal sequences. All three polypeptide variants were secreted into the periplasmic space as fully active enzymes. Oligonucleotide-directed mutagenesis was used to convert either one or both of the -Cys-Gly-His-Cys- sequences to -Ser-Gly-His-Cys-. The PDI activity of both polypeptides containing a single modified sequence was about 50% of that of the wild-type polypeptide, whereas the polypeptide with two modified sequences had no isomerase activity. It is thus concluded that both -Cys-Gly-His-Cys- sequences act as catalytic sites for the isomerase activity, and the two catalytic sites appear to operate independently of one another.

Prolyl 4-hydroxylase [procollagen-proline, 2-oxyglutarate 4-dioxygenase; procollagen-L-proline, 2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2], an alpha 2 beta 2 tetramer, catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here on the isolation of cDNA clones encoding the alpha-subunit of the enzyme from human tumor HT-1080, placenta, and fibroblast cDNA libraries. Eight overlapping clones covering almost all of the corresponding 3000-nucleotide mRNA, including all the coding sequences, were characterized. These clones encode a polypeptide of 517 amino acid residues and a signal peptide of 17 amino acids. Previous characterization of cDNA clones for the beta-subunit of prolyl 4-hydroxylase has indicated that its C terminus has the amino acid sequence Lys-Asp-Glu-Leu, which, it has been suggested, is necessary for the retention of a polypeptide within the lumen of the endoplasmic reticulum. The alpha-subunit does not have this C-terminal sequence, and thus one function of the beta-subunit in the prolyl 4-hydroxylase tetramer appears to be to retain the enzyme within this cell organelle. Interestingly, three of the cDNA clones for the alpha-subunit contained a 64-nucleotide sequence homologous but not identical to the corresponding 64-nucleotide sequence found in four other cDNA clones. Nuclease S1 mapping experiments demonstrated that this difference was due to the existence of two types of mRNA present in approximately equal amounts. Southern blot analyses of human genomic DNA with a cDNA probe for the alpha-subunit suggested the presence of only one gene encoding the two types of mRNA, which appear to result from mutually exclusive alternative splicing of primary transcripts of one gene.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer, the beta subunit of which is a highly unusual multifunctional polypeptide, being identical to protein disulfide-isomerase (EC 5.3.4.1). We report here the cloning of a second mouse alpha subunit isoform, termed the alpha (II) subunit. This polypeptide consists of 518 aa and a signal peptide of 19 aa. The processed polypeptide is one residue longer than the mouse alpha (I) subunit (the previously known type), the cloning of which is also reported here. The overall amino acid sequence identity between the mouse alpha (II) and alpha (I) subunits is 63%. The mRNA for the alpha (II) subunit was found to be expressed in a variety of mouse tissues. When the alpha (II) subunit was expressed together with the human protein disulfide-isomerase/beta subunit in insect cells by baculovirus vectors, an active prolyl 4-hydroxylase was formed, and this protein appeared to be an alpha (II) 2 beta 2 tetramer. The activity of this enzyme was very similar to that of the human alpha (I) 2 beta 2 tetramer, and most of its catalytic properties were also highly similar, but it differed distinctly from the latter in that it was inhibited by poly(L-proline) only at very high concentrations. This property may explain why the type II enzyme was not recognized earlier, as an early step in the standard purification procedure for prolyl 4-hydroxylase is affinity chromatography on a poly(L-proline) column.

Prolyl4-hydroxylase, an a .# , tetramer, plays a central role in collagen synthesis as it catalyzes the formation of 4-hydroxyproline residues by the hydroxylation of proline in X-Pro-Gly sequences.We report here that the human gene for the catalytically important (Y subunit is more than 69 kilobase pairs and consists of 16 exons.The exons that encode solely protein sequences vary from 54 to 240 base pairs (bp), and the introns vary from 750 to more than 16,000 bp.The 133 bp of 5"untranslated sequences of the mRNA are coded by two exons, and these sequences contain inverted repeats with a potential for stem-loop formation, which may be involved in translational control of the expression of this gene.The 5'- flanking region contains a TATA motif at -29 relative to the major transcription site but no CCAAT motif.The 5'-flanking region and the downstream sequences contain several motifs that may act as binding sites for various transcription factors.Evidence has previously been reported for a mutually exclusive alternative splicing of RNA transcripts of this gene.The present data indicate that the mutually exclusive sequences found in the mRNAs are coded by two consecutive, homologous 71-bp exons 9 and 10.These exons are identical in their first 5 bp and the overall identity between them is 61% at the nucleotide level and 58% at the level of the coded amino acids.Both types of mRNA were found to be expressed in all of the tissues studied, but in some tissues the type coding for exon 9 or 10 sequences was more abundant than the other type.Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens and other proteins with collagen-like amino acid sequences by the hydroxylation of proline residues in repeating X-Pro-Gly triplets.This enzyme plays a central role in collagen synthesis, since the 4-hydroxyproline cils for Medicine and Natural Sciences within the Academy of Finland.

Comment on: Finlay D, et al. Cancer Res 2007; 67:11704-11.

Abstract Platelet-derived growth factor receptor (PDGFR)α ranks third among the top 11 amplified genes in high-grade glioblastoma multiforme (GBM) and is frequently overexpressed in low-grade gliomas. However, the molecular mechanisms by which PDGFRα promotes glioma growth and invasion are largely unknown. Here we report that PDGFRα induces glioma cell invasion through tyrosine phosphorylation of Dock180, a Rac1 guanine nucleotide exchange factor (GEF). By immunohistochemical analysis of primary glioma tissue specimens, we found that PDGFRα, PDGF-A and Dock180 are co-expressed in invasive areas but not the central regions of the clinical biopsies. Ectopic expression of PDGF-A by human glioma LN444 cells with endogenous PDGFRα significantly promotes glioma growth and invasion in the brain of mice. Cellular depletion of Dock180 by RNAi in LN444 and LN443 cells inhibits PDGF-A-promoted cell migration in vitro and suppressed PDGF-A-expressing LN444 glioma infiltration and growth in the brain. In vitro, PDGF-A stimulates cell migration, invasion and induces tyrosine phosphorylation of Dock180, whereas AG1296, an inhibitor of PDGFRα inhibits the PDGF-A-induced phosphorylation of Dock180 and glioma cell migration. By co-expression of Dock180, its various deletion mutants and an oncogenic mutant PDGFRαΔ8,9, we identified tyrosine residue (Y) 1811 of Dock180 as a major phosphorylation (p-Y) site of Dock180. Y1811 is located within the CrkII-binding domain at the C-terminus of Dock180 and a potential tyrosine phosphorylation site for Src kinase. Y1811 is also highly conserved in Dock180 protein from various species. Mutation of the Y to a phenylalanine (F) at Y1811 of Dock180 significantly attenuates PDGFRα-induced phosphorylation and subsequently decreases Rac1 GTPase activity. Co-expression of Dock180 and PDGFRα and CrkII promotes PDGF-A-induced association of Dock180 with CrkII and p130Cas whereas the Y1811F mutant markedly disrupts their association. Inhibition of Src by its inhibitors and a dominant negative Src mutant attenuates PDGF-A-stimulated p-Y of wild-type Dock180, Rac1 activity and cell migration while a constitutively active Src mutant induces p-Y of wild-type Dock180 but not Dock180 Y1811F mutant. Finally, re-introduction of wild-type Dock180 into LN444/PDGF-A/shRNA-Dock180 cells that endogenous Dock180 was depleted rescued PDGFRα-promoted CrkII association, Rac1 activity and glioma cell motility in vitro, and tumor growth and invasion the brain whereas re-expression of Dock180 Y1811F mutant in these cells failed to restore PDGFRα-stimulated cellular behaviors. Taken together, this data reveals a molecular mechanism by which PDGFRα stimulates glioma cell invasion through tyrosine phosphorylation at Y1811 of a Rac1 GEF, Dock180, leading to increases in Rac1 activity and glioma cell invasion in the brain. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5208.

In this study, genetic parameters of test-day (TD) somatic cell score (SCS) and lactation average (LA)clinical mastitis (CM) were estimated using a random regression model (RRM) that combine two differentdata models. A multitrait RRM (mt-RRM) was then developed for the genetic evaluation of mastitis.Estimates of breeding values (EBVs) from the mt-RRM were compared to corresponding multitrait LAmodel (biv-LAM) and univariate LA models (univ-LAM). A total of 147500 and about 5.6 million recordsfrom 27500 and 1.4 million Finnish Ayrshire cows were used for estimation of genetic parameters andprediction of breeding values, respectively. Heritabilities of CM1 and CM2 traits: (CM1, -7 to 30 andCM2, 31 to 300 DIM) were 0.026 and 0.016, respectively, while for TD SCS they ranged from 0.06 to0.11. During first lactation, the genetic correlations between TD SCS and CM1 and between TD SCS andCM2 varied from 0.40 to 0.77 and from 0.34 to 0.71, respectively. In genetic evaluation of mastitis, modelcomparisons have showed that mt-RRM has high model predictive ability and high standard deviation ofbreeding values. Moreover, it has added advantages of making efficient use of available TD SCSinformation and offers proofs for bulls and cows. Therefore, mt-RRM can be used as best practical modelin the future evaluation of animals for mastitis resistance.