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 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 Assembly of the elongated collagen prolyl 4-hydroxylase α2β2 heterotetramer around a central α2 dimer

2017 ◽  
Vol 474 (5) ◽  
pp. 751-769 ◽  
Author(s):  
M. Kristian Koski ◽  
Jothi Anantharajan ◽  
Petri Kursula ◽  
Prathusha Dhavala ◽  
Abhinandan V. Murthy ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Coiled Coil ◽  
Β Subunit ◽  
Dimerization Domain ◽  
Α Subunit ◽  
Protein Dimer ◽  
Symmetric Molecule ◽  
Asymmetric Shape ◽  
Proline Rich Peptide ◽  
Α Subunits

Collagen prolyl 4-hydroxylase (C-P4H), an α2β2 heterotetramer, is a crucial enzyme for collagen synthesis. The α-subunit consists of an N-terminal dimerization domain, a central peptide substrate-binding (PSB) domain, and a C-terminal catalytic (CAT) domain. The β-subunit [also known as protein disulfide isomerase (PDI)] acts as a chaperone, stabilizing the functional conformation of C-P4H. C-P4H has been studied for decades, but its structure has remained elusive. Here, we present a three-dimensional small-angle X-ray scattering model of the entire human C-P4H-I heterotetramer. C-P4H is an elongated, bilobal, symmetric molecule with a length of 290 Å. The dimerization domains from the two α-subunits form a protein–protein dimer interface, assembled around the central antiparallel coiled-coil interface of their N-terminal α-helices. This region forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/β-subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/β-subunits locate at the far ends of the βααβ complex. The PDI/β-subunit interacts extensively with the CAT domain. The asymmetric shape of two truncated C-P4H-I variants, also characterized in the present study, agrees with this assembly. Furthermore, data from these truncated variants show that dimerization between the α-subunits has an important role in achieving the correct PSB–CAT assembly competent for catalytic activity. Kinetic assays with various proline-rich peptide substrates and inhibitors suggest that, in the competent assembly, the PSB domain binds to the procollagen substrate downstream from the CAT domain.

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.

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.

Studies of human pituitary LH containing internally cleaved β subunit

1991 ◽  
Vol 6 (1) ◽  
pp. 101-109 ◽  
Author(s):  
A. Stockell Hartree ◽  
R. C. Shownkeen
Keyword(s):  
Receptor Binding ◽  
Proteolytic Enzyme ◽  
Enzyme Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Peptide Bond ◽  
Β Subunit ◽  
Human Pituitary ◽  
Α Subunit ◽  
Peptide Bond Cleavage

ABSTRACT We have investigated the origin of the internal peptide bond cleavage found in the β subunit of a proportion of pituitary human LH (hLH) molecules, as well as the effects of this cleavage (or nick) on the interaction between α and β subunits and on binding of hLH to its receptor. The content of cleaved β subunit, assessed by the intensity of staining of an approximately 10 kDa component on sodium dodecyl sulphate-polyacrylamide gel electrophoresis of reduced hLH, was variable in batches of hLH prepared from pooled acetone-preserved human pituitary glands. There was evidence of a similar cleavage in purified hTSH, but not in the purified hFSH or human chorionic gonadotrophin examined. Although intact hLH was relatively resistant to cleavage in solution, urea dissociation of hormone followed by dialysis resulted in an increased content of nicked β subunit, which was largely prevented by incorporation of the proteolytic enzyme inhibitor phenylmethylsulphonyl fluoride (PMSF) and the metal-chelating agent EDTA. Hormone that was virtually free of nicked β subunit was obtained by urea dissociation of hLH subunits in the presence of PMSF and EDTA followed by dialysis to remove urea, reassociation of subunits at 37 °C (pH 7) and purification of reassociated hLH dimer by gel filtration on Sephadex G-100 in the presence of 1-anilinonaphthalene-8-sulphonic acid (ANS). When hLH was incubated at 37 °C at pH 55 or pH 95 in the absence of enzyme inhibitors, some cleaved β subunit was found in the hLH—ANS dimer form of the hormone, but most of the nicked subunit appeared in fractions of lower molecular weight and with low receptor-binding activity. Fractions isolated as hLH—ANS dimer had receptor-binding activities which were negatively correlated with their content of cleaved β subunit, the most active fraction (21 000 i.u./mg) being virtually free of this component. Our studies suggest (1) that the cleavage is caused by proteolytic enzyme activity present in human pituitary tissue and in purified preparations of hLH, (2) that free hLH-β subunit is cleaved far more readily than when it is combined with α subunit in the intact hormone, (3) that the presence of the cleavage in free hLH-β prevents refolding of this subunit to the correct conformation that permits its combination with α subunit to regenerate native hormone, and (4) that the site of cleavage is likely to be at a peptide bond located on the surface of the intact hLH molecule.

The reaction between hemoglobin α-subunit and haptoglobin

1976 ◽  
Vol 54 (11) ◽  
pp. 1011-1015 ◽  
Author(s):  
Frank A. Terpstra ◽  
David B. Smith
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Human Hemoglobin ◽  
Gel Filtration Chromatography ◽  
Separation Techniques ◽  
Α Subunit ◽  
Α Subunits

The interaction between human hemoglobin α-subunit and porcine haptoglobin was investigated by polyacrylamide gel electrophoresis, gel filtration chromatography, sedimentation through excess α-subunit and gel filtration in an α-subunit-containing medium. No interaction was detected by the first two methods indicating dissociation of the complex during the application of these separation techniques. The latter two methods, in which the complex is studied in a medium of excess subunits, showed that haptoglobin became saturated with the binding of two α-subunits.

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 Crystallographic evidence for oxidative Met→Asp conversion in human hemoglobin

2014 ◽  
Vol 70 (a1) ◽  
pp. C310-C310
Author(s):  
Jayashree Soman ◽  
Michael Strader ◽  
Wayne Hicks ◽  
Tigist Kassa ◽  
Eileen Singleton ◽  
...  
Keyword(s):  
Electron Density ◽  
Dose Dependence ◽  
Side Chain ◽  
Β Subunit ◽  
Α Subunit ◽  
In The Wild ◽  
Ferryl Species ◽  
Coordinated Water ◽  
Α Subunits

The mutants HbA Bristol-Alesha (βV(E11)67M) and HbF Toms River (γV(E11)67M) [1,2] are examples of a `silent' posttranslational modification in which the side chain of the substituted amino acid is chemically modified (Met→Asp) resulting in a disparity between the DNA and protein sequences. In both cases the patients' hemolysate contained both V67M and V67D isoforms. But in the analogous α subunit mutant, Hb Evans αV(E11)62M, the conversion to Asp was not identified and DNA sequencing confirmed the Met replacement [3]. Our crystal structures of the three (ferrous) CO-bound recombinant V(E11)M mutants show the MetE11 side chain in similar conformations. But the air-oxidized β mutant crystals clearly showed a `bifurcated' and smaller electron density pattern for the E11 side chain, indicating the appearance of Asp. Also, the ligand electron-density at the iron atom in the oxidized β subunit appears to be an oxoferryl Fe4+=O rather than a Fe3+OH2 ferric complex. In contrast, there was little change in the electron density for αMetE11 in oxidized αV62M crystals. The ligand in the ferric α subunit is clearly a coordinated water molecule. But again, a ferryl Fe4+=O complex appears to occur in the wild-type β subunit. This strongly suggest that β subunits have a greater propensity to form highly reactive ferryl species, and that the ferryl species play a role in the Met→Asp conversion. Our autoxidation and proteomics studies showed that although all three recombinant VE11M mutants had similar, high rates of autooxidation and a strong H2O2 dose dependence on sulfoxide and sulfone formation, no Asp formation was detected in α subunits whereas MetE11 is converted to Asp to levels as high as 15% in vitro in β and γ subunits. We propose that the Met→Asp conversion specifically involves H2O2 mediated oxidation of the ferrous heme to an oxoferryl state, and because the transient ferryl intermediates are much less stable in the α subunits, there is no oxidative conversion.

scholarly journals Intracellular dissociation and reassembly of prolyl 4-hydroxylase:the α-subunits associated with the immunoglobulin-heavy-chain binding protein (BiP) allowing reassembly with the β-subunit

1996 ◽  
Vol 317 (3) ◽  
pp. 659-665 ◽  
Author(s):  
David C. A. JOHN ◽  
Neil J. BULLEID
Keyword(s):  
Heavy Chain ◽  
Binding Protein ◽  
Immunoglobulin Heavy Chain ◽  
Β Subunit ◽  
Intact Cells ◽  
Α Subunit ◽  
Free System ◽  
A Cell ◽  
Α Subunits

Prolyl 4-hydroxylase (P4-H) consists of two distinct polypeptides; the catalytically more important α-subunit and the β-subunit, which is identical to the multifunctional enzyme protein disulphide isomerase. The enzyme appears to be assembled in vivo into an α2β2 tetramer from newly synthesized α-subunits associating with an endogenous pool of β-subunits. Using a cell-free system, we have shown previously that enzyme assembly is redox-dependent and that assembled α-subunits are intramolecularly disulphide-bonded [John and Bulleid (1994) Biochemistry 33, 14018–14025]. Here we have studied this assembly process within intact cells by expressing both subunits in COS-1 cells. Newly synthesized α-subunits were shown to assemble with the β-subunit, to form insoluble aggregates, or to remain soluble but not associate with the β-subunit. Treatment of cells with dithiothreitol (DTT) led to dissociation of P4-H into subunits and on removal of DTT the enzyme reassembled. This reassembly was ATP-dependent, suggesting an interaction with an ATP-dependent chaperone. This was confirmed when immunoglobulin-heavy-chain binding protein (BiP) and α-subunits were co-immunoprecipitated with antibodies against the α-subunit and BiP, respectively. These results indicate that unassembled α-subunits are maintained in an assembly-competent form by interacting with the molecular chaperone BiP.

scholarly journals Variation in pituitary expression of mRNAs encoding the putative inhibin co-receptor (betaglycan) and type-I and type-II activin receptors during the chicken ovulatory cycle

2005 ◽  
Vol 186 (3) ◽  
pp. 447-455 ◽  
Author(s):  
T M Lovell ◽  
P G Knight ◽  
R T Gladwell
Keyword(s):  
Quantitative Pcr ◽  
Anterior Pituitary ◽  
Transforming Growth Factor ◽  
Plasma Concentrations ◽  
Type I ◽  
Mrna Levels ◽  
Β Subunit ◽  
Ovulatory Cycle ◽  
Α Subunit ◽  
Lh Surge

Secretion of LH and FSH from the anterior pituitary is regulated primarily by hypothalamic GnRH and ovarian steroid hormones. More recent evidence indicates regulatory roles for certain members of the transforming growth factor β (TGFβ) superfamily including inhibin and activin. The aim of this study was to identify expression of mRNAs encoding key receptors and ligands of the inhibin/activin system in the hen pituitary gland and to monitor their expression throughout the 24–25-h ovulatory cycle. Hens maintained on long days (16 h light/8 h dark) were killed 20, 12, 6 and 2 h before predicted ovulation of a midsequence egg (n=8 per group). Anterior pituitary glands were removed, RNA extracted and cDNA synthesized. Plasma concentrations of LH, FSH, progesterone and inhibin A were measured. Real-time quantitative PCR was used to quantify pituitary expression of mRNAs encoding betaglycan, activin receptor (ActR) subtypes (type I, IIA), GnRH receptor (GnRH-R), LH β subunit, FSH β subunit and GAPDH. Levels of mRNA for inhibin/activin βA and βB subunits, inhibin α subunit, follistatin and ActRIIB mRNA in pituitary were undetectable by quantitative PCR (<2 amol/reaction). Significant changes in expression (P<0.05) of ActRIIA and betaglycan mRNA were found, both peaking 6 h before ovulation just prior to the preovulatory LH surge and reaching a nadir 2 h before ovulation, just after the LH surge. There were no significant changes in expression of ActRI mRNA throughout the cycle although values were correlated with mRNA levels for both ActRIIA (r=0.77; P<0.001) and beta-glycan (r=0.45; P<0.01). Expression of GnRH-R mRNA was lowest 20 h before ovulation and highest (P<0.05) 6 h before ovulation; values were weakly correlated with betaglycan (r=0.33; P=0.06) and ActRIIA (r=0.34; P=0.06) mRNA levels. Expression of mRNAs encoding LH β and FSH β subunit were both lowest (P<0.05) after the LH surge, 2 h before ovulation. These results are consistent with an endocrine, but not a local intrapituitary, role of inhibin-related proteins in modulating gonadotroph function during the ovulatory cycle of the hen, potentially through interaction with betaglycan and ActRIIA. In contrast to mammals, intrapituitary expression of inhibin/activin subunits and follistatin appears to be extremely low or absent in the domestic fowl.

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