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

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.

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Structural Characterization of Act c 10.0101 and Pun g 1.0101—Allergens from the Non-Specific Lipid Transfer Protein Family

Molecules ◽

10.3390/molecules26020256 ◽

2021 ◽

Vol 26 (2) ◽

pp. 256

Author(s):

Andrea O’Malley ◽

Swanandi Pote ◽

Ivana Giangrieco ◽

Lisa Tuppo ◽

Anna Gawlicka-Chruszcz ◽

...

Keyword(s):

Immunoglobulin E ◽

Lipid Transfer Protein ◽

Mass Spectrometry Analysis ◽

Lipid Transfer ◽

Helical Structures ◽

Spectrometry Analysis ◽

X Ray Crystallography ◽

Transfer Protein ◽

Specific Lipid

(1) Background: Non-specific lipid transfer proteins (nsLTPs), which belong to the prolamin superfamily, are potent allergens. While the biological role of LTPs is still not well understood, it is known that these proteins bind lipids. Allergen nsLTPs are characterized by significant stability and resistance to digestion. (2) Methods: nsLTPs from gold kiwifruit (Act c 10.0101) and pomegranate (Pun g 1.0101) were isolated from their natural sources and structurally characterized using X-ray crystallography (3) Results: Both proteins crystallized and their crystal structures were determined. The proteins have a very similar overall fold with characteristic compact, mainly α-helical structures. The C-terminal sequence of Act c 10.0101 was updated based on our structural and mass spectrometry analysis. Information on proteins’ sequences and structures was used to estimate the risk of cross-reactive reactions between Act c 10.0101 or Pun g 1.0101 and other allergens from this family of proteins. (4) Conclusions: Structural studies indicate a conformational flexibility of allergens from the nsLTP family and suggest that immunoglobulin E binding to some surface regions of these allergens may depend on ligand binding. Both Act c 10.0101 and Pun g 1.0101 are likely to be involved in cross-reactive reactions involving other proteins from the nsLTP family.

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Binding of plasma-derived lipid transfer protein to lipoprotein substrates. The role of binding in the lipid transfer process.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)38912-3 ◽

1985 ◽

Vol 260 (23) ◽

pp. 12593-12599 ◽

Cited By ~ 27

Author(s):

R E Morton

Keyword(s):

Transfer Process ◽

Lipid Transfer Protein ◽

Lipid Transfer ◽

Transfer Protein

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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

Biochemical Journal ◽

10.1042/bj3170721 ◽

1996 ◽

Vol 317 (3) ◽

pp. 721-729 ◽

Cited By ~ 26

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.

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Sphingomyelin Deacylase, the Enzyme Involved in the Pathogenesis of Atopic Dermatitis, Is Identical to the β-Subunit of Acid Ceramidase

International Journal of Molecular Sciences ◽

10.3390/ijms21228789 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8789

Author(s):

Yasuhiro Teranishi ◽

Hiroshi Kuwahara ◽

Masaru Ueda ◽

Tadashi Takemura ◽

Masanori Kusumoto ◽

...

Keyword(s):

Atopic Dermatitis ◽

Essential Role ◽

Etiologic Factor ◽

Gel Chromatography ◽

Β Subunit ◽

Acid Ceramidase ◽

Rat Skin ◽

Α Subunit ◽

Sds Page

A ceramide deficiency in the stratum corneum (SC) is an essential etiologic factor for the dry and barrier-disrupted skin of patients with atopic dermatitis (AD). Previously, we reported that sphingomyelin (SM) deacylase, which hydrolyzes SM and glucosylceramide at the acyl site to yield their lysoforms sphingosylphosphorylcholine (SPC) and glucosylsphingosine, respectively, instead of ceramide and/or acylceramide, is over-expressed in AD skin and results in a ceramide deficiency. Although the enzymatic properties of SM deacylase have been clarified, the enzyme itself remains unidentified. In this study, we purified and characterized SM deacylase from rat skin. The activities of SM deacylase and acid ceramidase (aCDase) were measured using SM and ceramide as substrates by tandem mass spectrometry by monitoring the production of SPC and sphingosine, respectively. Levels of SM deacylase activity from various rat organs were higher in the order of skin > lung > heart. By successive chromatography using Phenyl-5PW, Rotofor, SP-Sepharose, Superdex 200 and Shodex RP18-415, SM deacylase was purified to homogeneity with a single band of an apparent molecular mass of 43 kDa with an enrichment of > 14,000-fold. Analysis by MALDI-TOF MS/MS using a protein spot with SM deacylase activity separated by 2D-SDS-PAGE allowed its amino acid sequence to be determined and identified as the β-subunit of aCDase, which consists of α- and β-subunits linked by amino bonds and a single S-S bond. Western blotting of samples treated with 2-mercaptoethanol revealed that, whereas recombinant human aCDase was recognized by antibodies to the α-subunit at ~56 kDa and ~13 kDa and the β-subunit at ~43 kDa, the purified SM deacylase was detectable only by the antibody to the β-subunit at ~43 kDa. Breaking the S-S bond of recombinant human aCDase with dithiothreitol elicited the activity of SM deacylase with ~40 kDa upon gel chromatography. These results provide new insights into the essential role of SM deacylase expressed as an aCDase-degrading β-subunit that evokes the ceramide deficiency in AD skin.

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Cryoprotectin: a plant lipid–transfer protein homologue that stabilizes membranes during freezing

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2002.1079 ◽

2002 ◽

Vol 357 (1423) ◽

pp. 909-916 ◽

Cited By ~ 28

Author(s):

Dirk K. Hincha

Keyword(s):

Lipid Transfer Protein ◽

Thylakoid Membranes ◽

Cold Climates ◽

Lipid Transfer ◽

Plant Lipid ◽

Freeze Thaw ◽

Transfer Protein ◽

Thaw Cycle ◽

Freeze Thaw Cycle

Plants from temperate and cold climates are able to increase their freezing tolerance during exposure to low non–freezing temperatures. It has been shown that several genes are induced in a coordinated manner during this process of cold acclimation. The functional role of most of the corresponding cold–regulated proteins is not yet known. We summarize our knowledge of those cold–regulated proteins that are able to stabilize membranes during a freeze–thaw cycle. Special emphasis is placed on cryoprotectin, a lipid–transfer protein homologue that was isolated from cold–acclimated cabbage leaves and that protects isolated chloroplast thylakoid membranes from freeze–thaw damage.

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Pituitary transcription factor Prop-1 stimulates porcine pituitary glycoprotein hormone α subunit gene expression

Journal of Molecular Endocrinology ◽

10.1677/jme.1.02010 ◽

2006 ◽

Vol 37 (2) ◽

pp. 341-352 ◽

Cited By ~ 14

Author(s):

Takanobu Sato ◽

Kousuke Kitahara ◽

Takao Susa ◽

Takako Kato ◽

Yukio Kato

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Binding Sites ◽

Gh3 Cells ◽

Pituitary Hormone ◽

Transcriptional Level ◽

Β Subunit ◽

Glycoprotein Hormone ◽

Α Subunit

Recently, we have reported that a Prophet of Pit-1 homeodomain factor, Prop-1, is a novel transcription factor for the porcine follicle-stimulating hormone β subunit (FSHβ) gene. This study subsequently aimed to examine the role of Prop-1 in the gene expression of two other porcine gonadotropin subunits, pituitary glycoprotein hormone α subunit (αGSU), and luteinizing hormone β subunit (LHβ). A series of deletion mutants of the porcine αGSU (up to −1059 bp) and LHβ (up to −1277 bp) promoters were constructed in the reporter vector, fused with the secreted alkaline phosphatase gene (pSEAP2-Basic). Transient transfection studies using GH3 cells were carried out to estimate the activation of the porcine αGSU and LHβ promoters by Prop-1, which was found to activate the αGSU promoter of −1059/+12 bp up to 11.7-fold but not the LHβ promoter. Electrophoretic mobility shift assay and DNase I footprinting analysis revealed that Prop-1 binds to six positions, −1038/−1026, −942/−928, −495/−479, −338/−326, −153/−146, and −131/−124 bp, that comprise the A/T cluster. Oligonucleotides of six Prop-1 binding sites were directly connected to the minimum promoter of αGSU, fused in the pSEAP2-Basic vector, followed by transfecting GH3 cells to determine the cis-acting activity. Finally, we concluded that at least five Prop-1 binding sites are the cis-acting elements for αGSU gene expression. The present results revealed a notable feature of the proximal region, where three Prop-1-binding sites are close to and/or overlap the pituitary glycoprotein hormone basal element, GATA-binding element, and junctional regulatory element. To our knowledge, this is the first demonstration of the role of Prop-1 in the regulation of αGSU gene expression. These results, taken together with our previous finding that Prop-1 is a transcription factor for FSHβ gene, confirm that Prop-1 modulates the synthesis of FSH at the transcriptional level. On the other hand, the defects of Prop-1 are known to cause dwarfism and combined pituitary hormone deficiency accompanying hypogonadism. Accordingly, the present observations provide a novel view to understand the hypogonadism caused by Prop-1 defects at the molecular level through the regulatory mechanism of αGSU and FSHβ gene expressions.

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Detection of protein disulphide-isomerase in sheep pancreas fractions

Bioscience Reports ◽

10.1007/bf01115120 ◽

1982 ◽

Vol 2 (5) ◽

pp. 343-349 ◽

Cited By ~ 1

Author(s):

David A. Hillson ◽

Jacqueline Anderson

Keyword(s):

Specific Activity ◽

Protein Biosynthesis ◽

Major Site ◽

General Role ◽

Protein Disulphide Isomerase ◽

Isomerase Activity ◽

Liver Homogenates ◽

Specific Activities

Conclusions The use of diethylpyrocarbonate to inhibit endogenous ribonuclease in sheep pancreas allows the detection of protein-disulphide-isomerase activity in homogenates, at specific activities of up to 4 units/g. This is higher than the specific activity in sheep liver homogenates (about 2 units/g) or in homogenates of other sheep tissues (16). It is thus evident that high levels of protein-disulphide-isomerase activity are present in sheep pancreas. This is consistent both with the postulated general role of protein disulphide-isomerase in protein biosynthesis (10,11) and with the in vitro action of the enzyme on its conventional substrate scrambled ribonuclease, since pancreas is the major site of ribonuclease synthesis.

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