scholarly journals ERp60 does not substitute for protein disulphide isomerase as the β-subunit of prolyl 4-hydroxylase

1996 ◽  
Vol 316 (2) ◽  
pp. 599-605 ◽  
Author(s):  
Peppi KOIVUNEN ◽  
Tarja HELAAKOSKI ◽  
Pia ANNUNEN ◽  
Johanna VEIJOLA ◽  
Seija RÄISÄNEN ◽  
...  
Keyword(s):  
Amino Acid Sequence Identity ◽  
Insect Cells ◽  
Terminal Region ◽  
Cloning And Expression ◽  
Β Subunit ◽  
Hydroxylase Activity ◽  
Α Subunit ◽  
Protein Disulphide Isomerase ◽  
Tetramer Formation ◽  
Almost All

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyses the formation of 4-hydroxyproline in collagens. The vertebrate enzymes are α2β2 tetramers while the Caenorhabditis elegans enzyme is an αβ dimer. The β-subunit is identical to protein disulphide isomerase (PDI), a multifunctional endoplasmic reticulum luminal polypeptide. ERp60 is a PDI isoform that was initially misidentified as a phosphatidylinositol-specific phospholipase C. We report here on the cloning and expression of the human and Drosophila ERp60 polypeptides. The overall amino acid sequence identity and similarity between the processed human ERp60 and PDI polypeptides are 29% and 56% respectively, and those between the Drosophila ERp60 and human PDI polypeptides 29% and 55%. The two ERp60 polypeptides were found to be similar to human PDI within almost all their domains, the only exception being the extreme C-terminal region. Nevertheless, when the human or Drosophila ERp60 was expressed in insect cells together with an α-subunit of human prolyl 4-hydroxylase, no tetramer was formed and no prolyl 4-hydroxylase activity was generated in the cells. Additional experiments with hybrid polypeptides in which the C-terminal regions had been exchanged between the human ERp60 and PDI polypeptides demonstrated that the differences in the C-terminal region are not the only reason for the lack of prolyl 4-hydroxylase tetramer formation by ERp60.

scholarly journals Baculovirus expression of two protein disulphide isomerase isoforms from Caenorhabditis elegans and characterization of prolyl 4-hydroxylases containing one of these polypeptides as their β subunit

1996 ◽  
Vol 317 (3) ◽  
pp. 721-729 ◽  
Author(s):  
Johanna VEIJOLA ◽  
Pia ANNUNEN ◽  
Peppi KOIVUNEN ◽  
Antony P. PAGE ◽  
Taina PIHLAJANIEMI ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Cloning And Expression ◽  
Β Subunit ◽  
Α Subunit ◽  
Protein Disulphide Isomerase ◽  
C Elegans ◽  
Baculovirus Expression ◽  
Expression Studies ◽  
Α Subunits

Protein disulphide isomerase (PDI; EC 5.3.4.1) is a multifunctional polypeptide that is identical to the β subunit of prolyl 4-hydroxylases. We report here on the cloning and expression of the Caenorhabditis elegans PDI/β polypeptide and its isoform. The overall amino acid sequence identity and similarity between the processed human and C. elegans PDI/β polypeptides are 61% and 85% respectively, and those between the C. elegans PDI/β polypeptide and the PDI isoform 46% and 73%. The isoform differs from the PDI/β and ERp60 polypeptides in that its N-terminal thioredoxin-like domain has an unusual catalytic site sequence -CVHC-. Expression studies in insect cells demonstrated that the C. elegans PDI/β polypeptide forms an active prolyl 4-hydroxylase α2β2 tetramer with the human α subunit and an αβ dimer with the C. elegans α subunit, whereas the C. elegans PDI isoform formed no prolyl 4-hydroxylase with either α subunit. Removal of the 32-residue C-terminal extension from the C. elegans α subunit totally eliminated αβ dimer formation. The C. elegans PDI/β polypeptide formed less prolyl 4-hydroxylase with both the human and C. elegans α subunits than did the human PDI/β polypeptide, being particularly ineffective with the C. elegans α subunit. Experiments with hybrid polypeptides in which the C-terminal regions had been exchanged between the human and C. elegans PDI/β polypeptides indicated that differences in the C-terminal region are one reason, but not the only one, for the differences in prolyl 4-hydroxylase formation between the human and C. elegans PDI/β polypeptides. The catalytic properties of the C. elegans prolyl 4-hydroxylase αβ dimer were very similar to those of the vertebrate type II prolyl 4-hydroxylase tetramer, including the Km for the hydroxylation of long polypeptide substrates.

scholarly journals Co-expression of the α subunit of human prolyl 4-hydroxylase with BiP polypeptide in insect cells leads to the formation of soluble and insoluble complexes. Soluble α-subunit-BiP complexes have no prolyl 4-hydroxylase activity

1996 ◽  
Vol 315 (2) ◽  
pp. 613-618 ◽  
Author(s):  
J Veijola ◽  
T Pihlajaniemi ◽  
K Kivirikko
Keyword(s):  
Insect Cells ◽  
Soluble Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Insoluble Fraction ◽  
Type I ◽  
Β Subunit ◽  
Hydroxylase Activity ◽  
Α Subunit ◽  
Catalytically Active ◽  
Α Subunits

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyses the post-translational formation of 4-hydroxyproline in collagens. The vertebrate enzymes are α2β2 tetramers, their β subunit being identical to protein disulphide isomerase (PDI). The function of the PDI-β subunit in prolyl 4-hydroxylases is not fully understood, but it seems to be that of keeping the highly insoluble α subunits in solution. We report here that expression of the α subunit of human type I prolyl 4-hydroxylase in insect cells together with BiP polypeptide leads to the formation of both soluble and insoluble α-subunit–BiP complexes. Formation of the soluble complexes was evident from (1) a marked increase in the amount of the α subunit in the soluble fraction of the cell homogenates when expressed together with BiP, (2) immunoprecipitation experiments and (3) demonstration of the presence of some of the complexes by polyacrylamide gel electrophoresis under non-denaturing conditions. Formation of the insoluble complexes was suggested by an increase in the amount of BiP in the insoluble fraction when expressed together with the α subunit. Nevertheless the soluble α-subunit–BiP complexes had no prolyl 4-hydroxylase activity. This indicates that the function of the PDI-β subunit in the prolyl 4-hydroxylase tetramer is not only that of keeping the α subunits in solution but appears to be more specific, probably that of keeping them in a catalytically active, non-aggregated conformation.

scholarly journals Enzyme Specificity of 2-Nitrotoluene 2,3-Dioxygenase from Pseudomonas sp. Strain JS42 Is Determined by the C-Terminal Region of the α Subunit of the Oxygenase Component

1998 ◽  
Vol 180 (5) ◽  
pp. 1194-1199 ◽  
Author(s):  
Juanito V. Parales ◽  
Rebecca E. Parales ◽  
Sol M. Resnick ◽  
David T. Gibson
Keyword(s):  
Amino Acid Sequence Identity ◽  
Large Subunit ◽  
Small Subunit ◽  
Terminal Region ◽  
Oxidation Products ◽  
Enzyme Specificity ◽  
Α Subunit ◽  
Sequence Identity ◽  
Wide Range ◽  
Genes Encoding

ABSTRACT Biotransformations with recombinant Escherichia coliexpressing the genes encoding 2-nitrotoluene 2,3-dioxygenase (2NTDO) from Pseudomonas sp. strain JS42 demonstrated that 2NTDO catalyzes the dihydroxylation and/or monohydroxylation of a wide range of aromatic compounds. Extremely high nucleotide and deduced amino acid sequence identity exists between the components from 2NTDO and the corresponding components from 2,4-dinitrotoluene dioxygenase (2,4-DNTDO) from Burkholderia sp. strain DNT (formerlyPseudomonas sp. strain DNT). However, comparisons of the substrates oxidized by these dioxygenases show that they differ in substrate specificity, regiospecificity, and the enantiomeric composition of their oxidation products. Hybrid dioxygenases were constructed with the genes encoding 2NTDO and 2,4-DNTDO. Biotransformation experiments with these hybrid dioxygenases showed that the C-terminal region of the large subunit of the oxygenase component (ISPα) was responsible for the enzyme specificity differences observed between 2NTDO and 2,4-DNTDO. The small subunit of the terminal oxygenase component (ISPβ) was shown to play no role in determining the specificities of these dioxygenases.

Heterologous expression of Na+-K+-ATPase in insect cells: intracellular distribution of pump subunits

2001 ◽  
Vol 281 (3) ◽  
pp. C982-C992 ◽  
Author(s):  
Craig Gatto ◽  
Scott M. McLoud ◽  
Jack H. Kaplan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Atpase Activity ◽  
Insect Cells ◽  
Expression System ◽  
Β Subunit ◽  
Α Subunit ◽  
Enzyme Preparations ◽  
High Five Cells

The Na+-K+-ATPase is a heterodimeric plasma membrane protein responsible for cellular ionic homeostasis in nearly all animal cells. It has been shown that some insect cells (e.g., High Five cells) have no (or extremely low) Na+-K+-ATPase activity. We expressed sheep kidney Na+-K+-ATPase α- and β-subunits individually and together in High Five cells via the baculovirus expression system. We used quantitative slot-blot analyses to determine that the expressed Na+-K+-ATPase comprises between 0.5% and 2% of the total membrane protein in these cells. Using a five-step sucrose gradient (0.8–2.0 M) to separate the endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions, we observed functional Na+ pump molecules in each membrane pool and characterized their properties. Nearly all of the expressed protein functions normally, similar to that found in purified dog kidney enzyme preparations. Consequently, the measurements described here were not complicated by an abundance of nonfunctional heterologously expressed enzyme. Specifically, ouabain-sensitive ATPase activity, [3H]ouabain binding, and cation dependencies were measured for each fraction. The functional properties of the Na+-K+-ATPase were essentially unaltered after assembly in the endoplasmic reticulum. In addition, we measured ouabain-sensitive 86Rb+ uptake in whole cells as a means to specifically evaluate Na+-K+-ATPase molecules that were properly folded and delivered to the plasma membrane. We could not measure any ouabain-sensitive activities when either the α-subunit or β-subunit were expressed individually. Immunostaining of the separate membrane fractions indicates that the α-subunit, when expressed alone, is degraded early in the protein maturation pathway (i.e., the endoplasmic reticulum) but that the β-subunit is processed normally and delivered to the plasma membrane. Thus it appears that only the α-subunit has an oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the α-β heterodimer within the endoplasmic reticulum apparently does not require a Na+pump-specific chaperone.

scholarly journals The role of protein disulphide isomerase in the microsomal triacylglycerol transfer protein does not reside in its isomerase activity

1996 ◽  
Vol 315 (2) ◽  
pp. 533-536 ◽  
Author(s):  
Arja LAMBERG ◽  
Matti JAUHIAINEN ◽  
Jari METSO ◽  
Christian EHNHOLM ◽  
Carol SHOULDERS ◽  
...  
Keyword(s):  
Β Subunit ◽  
Lipid Transfer ◽  
Α Subunit ◽  
Protein Disulphide Isomerase ◽  
Isomerase Activity ◽  
Transfer Protein ◽  
Transfer Activity ◽  
Disulphide Bond Formation ◽  
Catalytically Active

The microsomal triacylglycerol transfer protein (MTP), an αβ dimer, is obligatory for the assembly of apoB-containing lipoproteins in liver and intestinal cells. The β subunit is identical with protein disulphide isomerase, a 58 kDa endoplasmic reticulum luminal protein involved in ensuring correct disulphide bond formation of newly synthesized proteins. We report here the expression of the human MTP subunits in Spodoptera frugiperda cells. When the α subunit was expressed alone, the polypeptide formed insoluble aggregates that were devoid of triacylglycerol transfer activity. In contrast, when the α and β subunits were co-expressed, soluble αβ dimers were formed with significant triacylglycerol transfer activity. Expression of the α subunit with a mutant protein disulphide isomerase polypeptide in which both -CGHC- catalytic sites had been inactivated also yielded αβ dimers that had comparable levels of lipid transfer activity relative to wild-type dimers. The results indicate that the role of the β subunit in MTP seems to be to keep the α subunit in a catalytically active, non-aggregated conformation and that disulphide isomerase activity of the β subunit is not required for this function.

scholarly journals H,K-ATPase α subunit C-terminal membrane topology: epitope tags in the insect cell expression system

1999 ◽  
Vol 340 (3) ◽  
pp. 601-611
Author(s):  
Adam J. SMOLKA ◽  
Kellie A. LARSEN ◽  
Clifford W. SCHWEINFEST ◽  
Charles E. HAMMOND
Keyword(s):  
Expression System ◽  
Recombinant Baculovirus ◽  
Transmembrane Domains ◽  
Terminal Region ◽  
Structural Basis ◽  
Sf9 Cells ◽  
Β Subunit ◽  
Α Subunit ◽  
Baculovirus Infection ◽  
P Type

The H,K-ATPase responsible for gastric acidification is a heterodimeric (α and β subunit) P-type ATPase, an integral protein of parietal cell apical membranes, which promotes the electroneutral exchange of K+ for protons, is stimulated by K+ and is inhibited by 2-methyl-8-(phenylmethoxy)imidazo[1,2-α]pyridine-3-acetonitrile (SCH 28080). Hydropathy analysis of the catalytic α subunit has been interpreted in terms of four N-terminal transmembrane domains, a cytoplasmically oriented segment containing ATP binding and phosphorylation sites, and a C-terminal region with four or six putative transmembrane domains. Several lines of evidence implicate the C-terminal region of P-type ATPases in cation-binding and occlusion, conformational changes, and interactions with the β subunit (HKβ), making the definition of topology a prerequisite for understanding the structural basis of these functions. Influenza haemagglutinin epitopes (YPYDVPDYA; flu tag) were inserted in predicted hydrophilic segments of the α subunit (HKα) to establish the membrane orientation of two amino acids with different predicted topologies in the C-terminal four- and six-transmembrane models. Wild-type and mutated HKα and HKβ cDNA species were expressed in insect cells (Sf9) via recombinant baculovirus infection, and expression of H,K-ATPase was verified by immunoblotting with HKα- and HKβ-specific and flu-tag-specific antibodies. Functional assays showed K+-stimulated, SCH 28080-sensitive ATPase activity, confirming neo-native topology in H,K-ATPase heterodimers expressed in Sf9 cells. The topology of flu tags was determined by microsomal protease protection assays in Sf9 cells and immunolabelling of HKα and HKβ in intact and permeabilized Sf9 cells. In addition, MS of native H,K-ATPase tryptic peptides identified cytoplasmically oriented HKα residues. The results indicated cytoplasmic exposure of Leu844 and Phe996, and luminal exposure of Pro898, leading to a revised secondary structure model of the C-terminal third of HKα.

Insect cells infected with a recombinant baculovirus express both O- and N-glycosylated forms of the rat glycoprotein hormone α-subunit

1996 ◽  
Vol 16 (2) ◽  
pp. 141-149 ◽  
Author(s):  
R Delahaye ◽  
P Berreur ◽  
R Salesse ◽  
R Counis
Keyword(s):  
Post Infection ◽  
Insect Cells ◽  
Recombinant Baculovirus ◽  
Chorionic Gonadotrophin ◽  
Biologically Active ◽  
Glycoprotein Hormones ◽  
Β Subunit ◽  
Glycoprotein Hormone ◽  
Α Subunit ◽  
Infected Cells

ABSTRACT Glycoprotein hormones LH, FSH, TSH and chorionic gonadotrophin are heterodimers composed of two non-covalently associated subunits, a common α- and a specific β-subunit. A recombinant baculo-virus containing a cDNA encoding the α-subunit of rat glycoprotein hormones was constructed. Viral-infected cells expressed, 48 h post infection, 7–10mg immunoreactive α-glycopolypeptide/6×108 cells, of which 65·6% was able to associate with native LHβ and formed a biologically active heterodimeric hormone that bound to testicular receptors. The treatment with specific glycanases showed that the recombinant α-subunit was produced as two differently glycosylated forms; an Mr 23 000 form which contained exclusively N-linked carbohydrate units and another of Mr 25 000 which appeared to contain additional O-linked carbohydrate. Data demonstrated that the α-subunit was expressed by insect cells in a manner similar to that by mammalian pituitary gonadotropes producing both the N- and O-glycosylated forms although only the N-glycosylated α-subunit is known to be capable of associating with the β-subunit.

scholarly journals Roles of HIF and 2-Oxoglutarate-Dependent Dioxygenases in Controlling Gene Expression in Hypoxia

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 350
Author(s):  
Julianty Frost ◽  
Mark Frost ◽  
Michael Batie ◽  
Hao Jiang ◽  
Sonia Rocha
Keyword(s):  
Gene Expression ◽  
Hypoxia Inducible Factor ◽  
Transcriptional Regulator ◽  
Hydroxylase Activity ◽  
Control Of Gene Expression ◽  
Prolyl Hydroxylases ◽  
Chromatin Organisation ◽  
Sense And Respond ◽  
Almost All ◽  
And Function

Hypoxia—reduction in oxygen availability—plays key roles in both physiological and pathological processes. Given the importance of oxygen for cell and organism viability, mechanisms to sense and respond to hypoxia are in place. A variety of enzymes utilise molecular oxygen, but of particular importance to oxygen sensing are the 2-oxoglutarate (2-OG) dependent dioxygenases (2-OGDs). Of these, Prolyl-hydroxylases have long been recognised to control the levels and function of Hypoxia Inducible Factor (HIF), a master transcriptional regulator in hypoxia, via their hydroxylase activity. However, recent studies are revealing that dioxygenases are involved in almost all aspects of gene regulation, including chromatin organisation, transcription and translation. We highlight the relevance of HIF and 2-OGDs in the control of gene expression in response to hypoxia and their relevance to human biology and health.

scholarly journals Translational Fusion of Two β-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

Endocrinology ◽  
2007 ◽  
Vol 148 (8) ◽  
pp. 3977-3986 ◽  
Author(s):  
Satarupa Roy ◽  
Sunita Setlur ◽  
Rupali A. Gadkari ◽  
H. N. Krishnamurthy ◽  
Rajan R. Dighe
Keyword(s):  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Expression System ◽  
Biologically Active ◽  
Chain Molecule ◽  
Dependent Manner ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
Lh Receptor

The strategy of translationally fusing the α- and β-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGαβ expressed using Pichia expression system. Using the same expression system, another analog, in which the α-subunit was replaced with the second β-subunit, was expressed (hCGββ) and purified. hCGββ could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGαβ and hCGββ using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGββ interacts with two LH receptor molecules. These studies demonstrate that the presence of the second β-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of α-subunit, the molecule is unable to elicit response. The strategy of fusing two β-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.

scholarly journals Thyroid hormone regulation of mRNAs encoding thyrotropin β-subunit, glycoprotein α-subunit, and thyroid hormone receptors α and β in brain, pituitary gland, liver, and gonads of an adult teleost, Pimephales promelas

2009 ◽  
Vol 202 (1) ◽  
pp. 43-54 ◽  
Author(s):  
Sean C Lema ◽  
Jon T Dickey ◽  
Irvin R Schultz ◽  
Penny Swanson
Keyword(s):  
Thyroid Hormone ◽  
Pituitary Gland ◽  
Teleost Fish ◽  
Hormone Receptors ◽  
Pimephales Promelas ◽  
Early Gene ◽  
Thyroid Hormone Receptors ◽  
Β Subunit ◽  
Glycoprotein Hormone ◽  
Α Subunit

Thyroid hormones (THs) regulate growth, morphological development, and migratory behaviors in teleost fish, yet little is known about the transcriptional dynamics of gene targets for THs in these taxa. Here, we characterized TH regulation of mRNAs encoding thyrotropin subunits and thyroid hormone receptors (TRs) in an adult teleost fish model, the fathead minnow (Pimephales promelas). Breeding pairs of adult minnows were fed diets containing 3,5,3′-triiodo-l-thyronine (T3) or the goitrogen methimazole for 10 days. In males and females, dietary intake of exogenous T3 elevated circulating total T3, while methimazole depressed plasma levels of total thyroxine (T4). In both sexes, this methimazole-induced reduction in T4 led to elevated mRNA abundance for thyrotropin β-subunit (tshβ) in the pituitary gland. Fish treated with T3 had elevated transcript levels for TR isoforms α and β (trα and trβ) in the liver and brain, but reduced levels of brain mRNA for the immediate-early gene basic transcription factor-binding protein (bteb). In the ovary and testis, exogenous T3 elevated gene transcripts for tshβ, glycoprotein hormone α-subunit (gphα), and trβ, while not affecting trα levels. Taken together, these results demonstrate negative feedback of T4 on pituitary tshβ, identify trα and trβ as T3-autoinduced genes in the brain and liver, and provide new evidence that tshβ, gphα, and trβ are THs regulated in the gonad of teleosts. Adult teleost models are increasingly used to evaluate the endocrine-disrupting effects of chemical contaminants, and our results provide a systemic assessment of TH-responsive genes during that life stage.

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