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 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 Prolyl 4-Hydroxylase Is an Essential Procollagen-Modifying Enzyme Required for Exoskeleton Formation and the Maintenance of Body Shape in the Nematode Caenorhabditis elegans

2000 ◽  
Vol 20 (11) ◽  
pp. 4084-4093 ◽  
Author(s):  
Alan D. Winter ◽  
Antony P. Page
Keyword(s):  
Extracellular Matrix ◽  
Caenorhabditis Elegans ◽  
Collagen Synthesis ◽  
Null Alleles ◽  
Β Subunit ◽  
Α Subunit ◽  
C Elegans ◽  
Link Type ◽  
Genes Encoding

ABSTRACT The multienzyme complex prolyl 4-hydroxylase catalyzes the hydroxylation of proline residues and acts as a chaperone during collagen synthesis in multicellular organisms. The β subunit of this complex is identical to protein disulfide isomerase (PDI). The free-living nematode Caenorhabditis elegans is encased in a collagenous exoskeleton and represents an excellent model for the study of collagen biosynthesis and extracellular matrix formation. In this study, we examined prolyl 4-hydroxylase α-subunit (PHY; EC1.14.11.2 )- and β-subunit (PDI; EC 5.3.4.1 )-encoding genes with respect to their role in collagen modification and formation of theC. elegans exoskeleton. We identified genes encoding two PHYs and a single associated PDI and showed that all three are expressed in collagen-synthesizing ectodermal cells at times of maximal collagen synthesis. Disruption of the pdi gene via RNA interference resulted in embryonic lethality. Similarly, the combinedphy genes are required for embryonic development. Interference with phy-1 resulted in a morphologically dumpy phenotype, which we determined to be identical to the uncharacterizeddpy-18 locus. Two dpy-18 mutant strains were shown to have null alleles for phy-1 and to have a reduced hydroxyproline content in their exoskeleton collagens. This study demonstrates in vivo that this enzyme complex plays a central role in extracellular matrix formation and is essential for normal metazoan development.

The glycomes of Caenorhabditis elegans and other model organisms

2002 ◽  
Vol 69 ◽  
pp. 117-134 ◽  
Author(s):  
Stuart M. Haslam ◽  
David Gems ◽  
Howard R. Morris ◽  
Anne Dell
Keyword(s):  
Caenorhabditis Elegans ◽  
Current Knowledge ◽  
Structural Data ◽  
Model Organisms ◽  
Entire Genome ◽  
C Elegans ◽  
The Face ◽  
Area Of Concern ◽  
Nematode Worm

There is no doubt that the immense amount of information that is being generated by the initial sequencing and secondary interrogation of various genomes will change the face of glycobiological research. However, a major area of concern is that detailed structural knowledge of the ultimate products of genes that are identified as being involved in glycoconjugate biosynthesis is still limited. This is illustrated clearly by the nematode worm Caenorhabditis elegans, which was the first multicellular organism to have its entire genome sequenced. To date, only limited structural data on the glycosylated molecules of this organism have been reported. Our laboratory is addressing this problem by performing detailed MS structural characterization of the N-linked glycans of C. elegans; high-mannose structures dominate, with only minor amounts of complex-type structures. Novel, highly fucosylated truncated structures are also present which are difucosylated on the proximal N-acetylglucosamine of the chitobiose core as well as containing unusual Fucα1–2Gal1–2Man as peripheral structures. The implications of these results in terms of the identification of ligands for genomically predicted lectins and potential glycosyltransferases are discussed in this chapter. Current knowledge on the glycomes of other model organisms such as Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster is also discussed briefly.

scholarly journals Characterization of Caenorhabditis elegans Homologs of the Down Syndrome Candidate Gene DYRK1A

Genetics ◽  
2003 ◽  
Vol 163 (2) ◽  
pp. 571-580 ◽  
Author(s):  
William B Raich ◽  
Celine Moorman ◽  
Clay O Lacefield ◽  
Jonah Lehrer ◽  
Dusan Bartsch ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Caenorhabditis Elegans ◽  
Trisomy 21 ◽  
Gene Dosage ◽  
Inducible Promoters ◽  
C Elegans ◽  
Dual Specificity ◽  
Specificity Protein

Abstract The pathology of trisomy 21/Down syndrome includes cognitive and memory deficits. Increased expression of the dual-specificity protein kinase DYRK1A kinase (DYRK1A) appears to play a significant role in the neuropathology of Down syndrome. To shed light on the cellular role of DYRK1A and related genes we identified three DYRK/minibrain-like genes in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. We found these genes to be widely expressed and to localize to distinct subcellular compartments. We isolated deletion alleles in all three genes and show that loss of mbk-1, the gene most closely related to DYRK1A, causes no obvious defects, while another gene, mbk-2, is essential for viability. The overexpression of DYRK1A in Down syndrome led us to examine the effects of overexpression of its C. elegans ortholog mbk-1. We found that animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and that the extent of these defects correlates with mbk-1 gene dosage. Using tissue-specific and inducible promoters, we show that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. Our results suggest that increased gene dosage of human DYRK1A in trisomy 21 may disrupt the function of fully differentiated neurons and that this disruption is reversible.

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 Goα Regulates Volatile Anesthetic Action in Caenorhabditis elegans

Genetics ◽  
2001 ◽  
Vol 158 (2) ◽  
pp. 643-655 ◽  
Author(s):  
Bruno van Swinderen ◽  
Laura B Metz ◽  
Laynie D Shebester ◽  
Jane E Mendel ◽  
Paul W Sternberg ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Novel Mutation ◽  
Volatile Anesthetic ◽  
Loss Of Function ◽  
Wild Type ◽  
Α Subunit ◽  
C Elegans ◽  
Missense Mutant ◽  
Anesthetic Action ◽  
Mutant Phenotypes

Abstract To identify genes controlling volatile anesthetic (VA) action, we have screened through existing Caenorhabditis elegans mutants and found that strains with a reduction in Go signaling are VA resistant. Loss-of-function mutants of the gene goa-1, which codes for the α-subunit of Go, have EC50s for the VA isoflurane of 1.7- to 2.4-fold that of wild type. Strains overexpressing egl-10, which codes for an RGS protein negatively regulating goa-1, are also isoflurane resistant. However, sensitivity to halothane, a structurally distinct VA, is differentially affected by Go pathway mutants. The RGS overexpressing strains, a goa-1 missense mutant found to carry a novel mutation near the GTP-binding domain, and eat-16(rf) mutants, which suppress goa-1(gf) mutations, are all halothane resistant; goa-1(null) mutants have wild-type sensitivities. Double mutant strains carrying mutations in both goa-1 and unc-64, which codes for a neuronal syntaxin previously found to regulate VA sensitivity, show that the syntaxin mutant phenotypes depend in part on goa-1 expression. Pharmacological assays using the cholinesterase inhibitor aldicarb suggest that VAs and GOA-1 similarly downregulate cholinergic neurotransmitter release in C. elegans. Thus, the mechanism of action of VAs in C. elegans is regulated by Goα, and presynaptic Goα-effectors are candidate VA molecular targets.

The genetic and RFLP characterization of the left end of linkage group III in Caenorhabditis elegans

Genome ◽  
1993 ◽  
Vol 36 (4) ◽  
pp. 712-724 ◽  
Author(s):  
Dave Pilgrim
Keyword(s):  
Caenorhabditis Elegans ◽  
Linkage Group ◽  
Genetic Map ◽  
Physical Map ◽  
Group Iii ◽  
C Elegans ◽  
Genomic Regions ◽  
Caenorhabditis Elegans Genome ◽  
Selection Of

A genetic approach was taken to identify new transposable element Tc1 -dependent polymorphisms on the left end of linkage group III in the nematode Caenorhabditis elegans. The cloning of the genomic DNA surrounding the Tc1 allowed the selection of overlapping clones (from the collection being used to assemble the physical map of the C. elegans genome). A contig of approximately 600–800 kbp in the region has been identified, the genetic map of the region has been refined, and 10 new RFLPs as well as at least four previously characterized genetic loci have been positioned onto the physical map, to the resolution of a few cosmids. This analysis demonstrated the ability to combine physical and genetic mapping for the rapid analysis of large genomic regions (0.5–1 Mbp) in genetically amenable eukaryotes.Key words: Caenorhabditis elegans, genome analysis, RFLP, physical map, genetic map.

scholarly journals DYC-1, a Protein Functionally Linked to Dystrophin in Caenorhabditis elegans Is Associated with the Dense Body, Where It Interacts with the Muscle LIM Domain Protein ZYX-1

2008 ◽  
Vol 19 (3) ◽  
pp. 785-796 ◽  
Author(s):  
Claire Lecroisey ◽  
Edwige Martin ◽  
Marie-Christine Mariol ◽  
Laure Granger ◽  
Yannick Schwab ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Dense Body ◽  
Striated Muscles ◽  
Lim Domain ◽  
Functional Link ◽  
C Elegans ◽  
Lim Domain Protein ◽  
Muscle Necrosis ◽  
Domain Protein

In Caenorhabditis elegans, mutations of the dystrophin homologue, dys-1, produce a peculiar behavioral phenotype (hyperactivity and a tendency to hypercontract). In a sensitized genetic background, dys-1 mutations also lead to muscle necrosis. The dyc-1 gene was previously identified in a genetic screen because its mutation leads to the same phenotype as dys-1, suggesting that the two genes are functionally linked. Here, we report the detailed characterization of the dyc-1 gene. dyc-1 encodes two isoforms, which are expressed in neurons and muscles. Isoform-specific RNAi experiments show that the absence of the muscle isoform, and not that of the neuronal isoform, is responsible for the dyc-1 mutant phenotype. In the sarcomere, the DYC-1 protein is localized at the edges of the dense body, the nematode muscle adhesion structure where actin filaments are anchored and linked to the sarcolemma. In yeast two-hybrid assays, DYC-1 interacts with ZYX-1, the homologue of the vertebrate focal adhesion LIM domain protein zyxin. ZYX-1 localizes at dense bodies and M-lines as well as in the nucleus of C. elegans striated muscles. The DYC-1 protein possesses a highly conserved 19 amino acid sequence, which is involved in the interaction with ZYX-1 and which is sufficient for addressing DYC-1 to the dense body. Altogether our findings indicate that DYC-1 may be involved in dense body function and stability. This, taken together with the functional link between the C. elegans DYC-1 and DYS-1 proteins, furthermore suggests a requirement of dystrophin function at this structure. As the dense body shares functional similarity with both the vertebrate Z-disk and the costamere, we therefore postulate that disruption of muscle cell adhesion structures might be the primary event of muscle degeneration occurring in the absence of dystrophin, in C. elegans as well as vertebrates.

scholarly journals Characterization of Hybrid Toluate and Benzoate Dioxygenases

2003 ◽  
Vol 185 (18) ◽  
pp. 5333-5341 ◽  
Author(s):  
Yong Ge ◽  
Lindsay D. Eltis
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Acinetobacter Calcoaceticus ◽  
The Other ◽  
Β Subunit ◽  
Structural Determinants ◽  
Α Subunit ◽  
Benzoate Dioxygenase

ABSTRACT Toluate dioxygenase of Pseudomonas putida mt-2 (TADOmt2) and benzoate dioxygenase of Acinetobacter calcoaceticus ADP1 (BADOADP1) catalyze the 1,2-dihydroxylation of different ranges of benzoates. The catalytic component of these enzymes is an oxygenase consisting of two subunits. To investigate the structural determinants of substrate specificity in these ring-hydroxylating dioxygenases, hybrid oxygenases consisting of the α subunit of one enzyme and the β subunit of the other were prepared, and their respective specificities were compared to those of the parent enzymes. Reconstituted BADOADP1 utilized four of the seven tested benzoates in the following order of apparent specificity: benzoate > 3-methylbenzoate > 3-chlorobenzoate > 2-methylbenzoate. This is a significantly narrower apparent specificity than for TADOmt2 (3-methylbenzoate > benzoate ∼ 3-chlorobenzoate > 4-methylbenzoate ∼ 4-chlorobenzoate ≫ 2-methylbenzoate ∼ 2-chlorobenzoate [Y. Ge, F. H. Vaillancourt, N. Y. Agar, and L. D. Eltis, J. Bacteriol. 184:4096-4103, 2002]). The apparent substrate specificity of the αBβT hybrid oxygenase for these benzoates corresponded to that of BADOADP1, the parent from which the α subunit originated. In contrast, the apparent substrate specificity of the αTβB hybrid oxygenase differed slightly from that of TADOmt2 (3-chlorobenzoate > 3-methylbenzoate > benzoate ∼ 4-methylbenzoate > 4-chlorobenzoate > 2-methylbenzoate > 2-chlorobenzoate). Moreover, the αTβB hybrid catalyzed the 1,6-dihydroxylation of 2-methylbenzoate, not the 1,2-dihydroxylation catalyzed by the TADOmt2 parent. Finally, the turnover of this ortho-substituted benzoate was much better coupled to O2 utilization in the hybrid than in the parent. Overall, these results support the notion that the α subunit harbors the principal determinants of specificity in ring-hydroxylating dioxygenases. However, they also demonstrate that the β subunit contributes significantly to the enzyme's function.

scholarly journals Calcineurin, a Calcium/Calmodulin-dependent Protein Phosphatase, Is Involved in Movement, Fertility, Egg Laying, and Growth inCaenorhabditis elegans

2002 ◽  
Vol 13 (9) ◽  
pp. 3281-3293 ◽  
Author(s):  
Jaya Bandyopadhyay ◽  
Jiyeon Lee ◽  
Jungsoo Lee ◽  
Jin Il Lee ◽  
Jae-Ran Yu ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Protein Phosphatase ◽  
Body Wall ◽  
Egg Laying ◽  
Α Subunit ◽  
C Elegans ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein ◽  
Identification And Characterization

Calcineurin is a Ca2+-calmodulin–dependent serine/threonine protein phosphatase that has been implicated in various signaling pathways. Here we report the identification and characterization of calcineurin genes in Caenorhabditis elegans (cna-1 and cnb-1), which share high homology with Drosophila and mammalian calcineurin genes. C. elegans calcineurin binds calcium and functions as a heterodimeric protein phosphatase establishing its biochemical conservation in the nematode. Calcineurin is expressed in hypodermal seam cells, body-wall muscle, vulva muscle, neuronal cells, and in sperm and the spermatheca. cnb-1 mutants showed pleiotropic defects including lethargic movement and delayed egg-laying. Interestingly, these characteristic defects resembled phenotypes observed in gain-of-function mutants ofunc-43/Ca2+-calmodulin–dependent protein kinase II (CaMKII) and goa-1/Go-protein α-subunit. Double mutants of cnb-1 andunc-43(gf) displayed an apparent synergistic severity of movement and egg-laying defects, suggesting that calcineurin may have an antagonistic role in CaMKII-regulated phosphorylation signaling pathways in C. elegans.

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