Purification and characterization of beta-galactoside-binding proteins from Caenorhabditis elegans.

Two carbohydrate-binding proteins (subunit molecular masses, 32 and 16 kDa, respectively) were isolated for the first time from a nematode, Caenorhabditis elegans. They were specifically extracted with lactose and adsorbed on asialofetuin-Sepharose in the absence of a metal ion. Although these two proteins were co-eluted from a gel filtration column at a position corresponding to an apparent molecular size of 30 kDa under non-denaturing conditions, they could be separated by reversed-phase chromatography. The 32 kDa protein, the main component, was further characterized. Together with its solubility, saccharide specificity and metal independence, some other structural properties, including its amino acid composition, UV spectrum, and partial amino acid sequence, strongly suggested that the 32 kDa protein is a member of a class of soluble beta-galactoside-binding lectins which had previously been only found in vertebrates.     

Functional characterization of five eIF4E isoforms in Caenorhabditis elegans

Recognition of the 5'-cap structure of mRNA by eIF4E is a critical step in the recruitment of most mRNAs to the ribosome. In Caenorhabditis elegans, approximately 70% of mRNAs contain an unusual 2,2,7-trimethylguanosine cap structure as a result of trans-splicing onto the 5' end of the pre-mRNA. The characterization of three eIF4E isoforms in C. elegans (IFE-1, IFE-2, and IFE-3) was reported previously. The present study describes two more eIF4E isoforms expressed in C. elegans, IFE-4 and IFE-5. We analyzed the requirement of each isoform for viability by RNA interference. IFE-3, the most closely related to mammalian eIF4E-1, binds only 7-methylguanosine caps and is essential for viability. In contrast, three closely related isoforms (IFE-1, IFE-2, and IFE-5) bind 2,2, 7-trimethylguanosine caps and are partially redundant, but at least one functional isoform is required for viability. IFE-4, which binds only 7-methylguanosine caps, is most closely related to an unusual eIF4E isoform found in plants (nCBP) and mammals (4E-HP) and is not essential for viability in any combination of IFE knockout. ife-2, ife-3, ife-4, and ife-5 mRNAs are themselves trans-spliced to SL1 spliced leaders. ife-1 mRNA is trans-spliced to an SL2 leader, indicating that its gene resides in a downstream position of an operon.     

Cloning and characterization of antioxidant enzyme methionine sulfoxide-S-reductase from Caenorhabditis elegans

Methionine (Met) residues in proteins are susceptible to oxidation. The resulting methionine sulfoxide can be reduced back to methionine by methionine sulfoxide-S-reductase (MsrA). The MsrA gene, isolated from Caenorhabditis elegans, was cloned and expressed in Escherichia coli. The resultant enzyme was able to revert both free Met and Met in proteins in the presence of either NADPH or dithiothreitol (DTT). However, approximately seven times higher enzyme activity was observed in the presence of DTT than of NADPH. The enzyme had an absolute specificity for the reduction of l-methionine-S-sulfoxide but no specificity for the R isomer. K(m) and k(cat) values for the enzyme were approximately 1.18 mM and 3.64 min(-1), respectively. Other kinetics properties of the enzyme were also evaluated.     

Molecular and functional evolution of human DHRS2 and DHRS4 duplicated genes

Human DHRS2 and DHRS4 genes code for similar NADP-dependent short-chain carbonyl-reductase enzymes having different substrate specificity. Human DHRS2 and DHRS4 enzymes share several common sequence motives including residues responsible for coenzyme binding as well as for the intimate catalytic oxido-reductase mechanism, while their substrate-binding sequences have very low similarity. We found that DHRS2 and DHRS4 genes are syntenic outparalogues originated from a duplication of the DHRS4 gene that took place before the formation of the mammalian clade. DHRS2 gene evolved more rapidly and underwent positive selection on more sites than the DHRS4 gene. DHRS2 sites under positive selection were mainly located on the enzyme active site thus showing that substrate specificity drove the divergence from the DHRS4 enzyme. Rapid divergent evolution brought the human DHRS2 enzyme to have subcellular localization, synthesis regulation and specialized cellular functions very different from those of the human DHRS4 enzyme.     

Purification and characterization of recombinant Caenorhabditis elegans metallothionein

The roundworm Caenorhabditis elegans adapted for survival at high concentrations of Cd(II) expresses two isoforms of metallothionein, CeMT-I and CeMT-II. To characterize one of these proteins CeMT-II was prepared as its Cd containing form by expressing its cDNA heterologously in Escherichia coli. The purified 63-amino-acid protein was identified as the desired product by ion-spray mass spectrometry and was found to resemble in most of its chemical and spectroscopic features the metallothioneins of other animal phyla. The recombinant protein contains a total of 18 cysteine residues and, as documented by electrophoresis and mass spectrometry, binds firmly six Cd ions through the cysteine's side chains. The (113)Cd NMR spectrum features six (113)Cd resonances. Their chemical shift positions between 615 and 675 ppm denote the existence of clusters of tetrahedrally coordinated cadmium thiolate complexes. The metal thiolate coordination dominates also the electronic far-UV absorption spectrum. It is characterized by a massive absorption profile with Cd thiolate shoulders at 255 and 235 nm. Upon replacement of Cd by Zn the profile was blue-shifted by 30 nm.     

Cloning and characterization of a Caenorhabditis elegans D2-like dopamine receptor

The neurotransmitter dopamine plays an important role in the regulation of behavior in both vertebrates and invertebrates. In mammals, dopamine binds and activates two classes of dopamine receptors, D1-like and D2-like receptors. However, D2-like dopamine receptors in Caenorhabditis elegans have not yet been characterized. We have cloned a cDNA encoding a putative C. elegans D2-like dopamine receptor. The deduced amino acid sequence of the cloned cDNA shows higher sequence similarities to vertebrate D2-like dopamine receptors than to D1-like receptors. Two splice variants that differ in the length of their predicted third intracellular loops were identified. The receptor heterologously expressed in cultured cells showed high affinity binding to [125I]iodo-lysergic acid diethylamide. Dopamine showed the highest affinity for this receptor among several amine neurotransmitters tested. Activation of the heterologously expressed receptor led to the inhibition of cyclic AMP production, confirming that this receptor has the functional property of a D2-like receptor. We have also analyzed the expression pattern of this receptor and found that the receptor is expressed in several neurons including all the dopaminergic neurons in C. elegans.     

Functional characterization of Caenorhabditis elegans DNA topoisomerase IIIalpha

To investigate the function of a DNA topoisomerase III enzyme in Caenorhabditis elegans, the full-length cDNA of C.elegans DNA topoisomerase IIIα was cloned. The deduced amino acid sequence exhibited identities of 48 and 39% with those of human DNA topoisomerase IIIα and Saccharomyces cerevisiae DNA topoisomerase III, respectively. The overexpressed polypeptide showed an optimal activity for removing negative DNA supercoils at a relatively high temperature of 52–57°C, which is similar to the optimum temperatures of other eukaryotic DNA topoisomerase III enzymes. When topoisomerase IIIα expression was interfered with by a cognate double-stranded RNA injection, pleiotropic phenotypes with abnormalities in germ cell proliferation, oogenesis and embryogenesis appeared. These phenotypes were well correlated with mRNA expression localized in the meiotic cells of gonad and early embryonic cells.     

Purification and characterization of wild-type and mutant TK1 type kinases from Caenorhabditis elegans

Caenorhabditis elegans has a single deoxynucleoside kinase-like gene. The sequence is similar to that of human TK1, but besides accepting thymidine as a substrate, the C. elegans TK1 (CeTK1) also phosphorylates deoxyguanosine. In contrast to human TK1, the CeTK1 exclusively exists as a dimer with a molecular mass of approximately 60 kDa, even if incubated with ATP. Incubation with ATP induces a transition into a more active enzyme with a higher kcat but unchanged Km. This activation only occurs at an enzyme concentration in the incubation buffer of 0.5 micro g/ml (8.42 nM) or higher. C-terminal deletion of the enzyme results in lower catalytic efficiency and stability.     

Cuticle of Caenorhabditis elegans: its isolation and partial characterization

The adult cuticle of the soil nematode, Caenorhabditis elegans, is a proteinaceous extracellular structure elaborated by the underlying layer of hypodermal cells during the final molt in the animal's life cycle. The cuticle is composed of an outer cortical layer connected by regularly arranged struts to an inner basal layer. The cuticle can be isolated largely intact and free of all cellular material by sonication and treatment with 1% sodium dodecyl sulfate (SDS). Purified cuticles exhibit a negative material in the basal cuticle layer. The cuticle layers differ in their solubility in sulfhydryl reducing agents, susceptibility to various proteolytic enzymes and amino acid composition. The struts, basal layer, and internal cortical layer are composed of collagen proteins that are extensively cross-linked by disulfide bonds. The external cortical layer appears to contain primarily noncollagen proteins that are extensively cross-linked by nonreducible covalent bonds. The collagen proteins extracted from the cuticle with a reducing agent can be separated by SDS-polyacrylamide gel electrophoresis into eight major species differing in apparent molecular weight.     

Purification and characterization of a carboxylesterase from the intestine of the nematode Caenorhabditis elegans

The major intestinal esterase from the nematode Caenorhabditis elegans has been purified to essential homogeneity. Starting from whole worms, the overall purification is 9000-fold with a 10% recovery of activity. The esterase is a single polypeptide chain of Mr 60,000 and is stoichiometrically inhibited by organophosphates. Substrate preferences and inhibition patterns classify the enzyme as a carboxylesterase (EC 3.1.1.1), but the physiological function is unknown. The sequence of 13 amino acid residues at the esterase N-terminus has been determined. This partial sequence shows a surprisingly high degree of similarity to the N-terminal sequence of two carboxylesterases recently isolated from Drosophila mojavensis [Pen, J., van Beeumen, J., & Beintema, J. J. (1986) Biochem. J. 238, 691-699].     

Functional characterization of SR and SR-related genes in Caenorhabditis elegans

The SR proteins constitute a family of nuclear phosphoproteins, which are required for constitutive splicing and also influence alternative splicing regulation. Initially, it was suggested that SR proteins were functionally redundant in constitutive splicing. However, differences have been observed in alternative splicing regulation, suggesting unique functions for individual SR proteins. Homology searches of the Caenorhabditis elegans genome identified seven genes encoding putative orthologues of the human factors SF2/ASF, SRp20, SC35, SRp40, SRp75 and p54, and also several SR-related genes. To address the issue of functional redundancy, we used dsRNA interference (RNAi) to inhibit specific SR protein function during C.elegans development. RNAi with CeSF2/ASF caused late embryonic lethality, suggesting that this gene has an essential function during C.elegans development. RNAi with other SR genes resulted in no obvious phenotype, which is indicative of gene redundancy. Simultaneous interference of two or more SR proteins in certain combinations caused lethality or other developmental defects. RNAi with CeSRPK, an SR protein kinase, resulted in early embryonic lethality, suggesting an essential role for SR protein phosphorylation during development.     

pat-4/ILK and unc-112/Mig-2 are required for gonad function in Caenorhabditis elegans

Tissue morphogenesis requires proper interaction between cells and the extracellular matrix (ECM), which is mediated by alphabeta heterodimeric receptor integrin. In Caenorhabditis elegans, integrin signaling is essential for formation of gonad. Here, we probe the role of several integrin-associated molecules in ovulation and cell migration. Function of pat-4/integrin-linked kinase (ILK) and unc-112/Mig-2 was examined using RNA-mediated interference (RNAi). Depletion of these messages caused oocyte accumulation in the proximal gonad and distal tip cells (DTC) migration defects. It was further determined that failed ovulation was due to defective contraction and dilation of somatic gonad structures, including spermatheca and gonad sheath. Actin cytoskeleton in the proximal gonad of RNAi animals appeared disorganized, indicating that RNAi of pat-4 or unc-112 inhibited the overall assembly of actin cytoskeleton in somatic gonad. Taken together, our analysis confirms the role of integrin and integrin-associated proteins in gonad function.     

Essential kinesins: characterization of Caenorhabditis elegans KLP-15

Kinesins form a superfamily of molecular motors involved in cell division and intracellular transport. Twenty kinesins have been found in the Caenorhabditis elegans genome, and four of these belong to the kinesin-14 subfamily, i.e., kinesins with C-terminal motor domains. Three of these kinesin-14s, KLP-15, KLP-16, and KLP-17, form a distinct subgroup in which KLP-15 and KLP-16 are more than 90% identical and appear to be related by a relatively recent gene duplication. They are essential for meiotic spindle organization and chromosome segregation, and are mostly expressed in the germline. With 587 amino acids each, they are among the smallest kinesins known. Using bacterially expressed KLP-15 constructs with different length extensions preceding the motor domain, we have determined in vitro the following characteristic properties: ATPase activity, microtubule binding, oligomeric state, microtubule gliding activity, and direction of movement. The constructs exhibit a monomer-dimer equilibrium that depends on the length of the predicted alpha-helical coiled-coil region preceding the motor domain. The longest construct with the complete coiled-coil domain is a stable dimer, and the shortest construct with only seven amino acids preceding the motor domain is a monomer. In microtubule gliding assays, the monomer is immobile whereas the fully dimeric KLP-15 construct supports gliding at 2.3 microm/min and moves toward microtubule minus ends, like other members of the kinesin-14 subfamily studied to date.     

Functional characterization of Caenorhabditis elegans heteromeric amino acid transporters

Mammalian heteromeric amino acid transporters (HATs) are composed of a multi-transmembrane spanning catalytic protein covalently associated with a type II glycoprotein (e.g. 4F2hc, rBAT) through a disulfide bond. Caenorhabditis elegans has nine genes encoding close homologues of the HAT catalytic proteins. Three of these genes (designated AAT-1 to AAT-3) have a much higher degree of similarity to the mammalian homologues than the other six, including the presence of a cysteine residue at the position known to form a disulfide bridge to the glycoprotein partner in mammalian HATs. C. elegans also has two genes encoding homologues of the heteromeric amino acid transporter type II glycoprotein subunits (designated ATG-1 and ATG-2). Both ATG, and/or AAT-1, -2, -3 proteins were expressed in Xenopus oocytes and tested for amino acid transport function. This screen revealed that AAT-1 and AAT-3 facilitate amino acid transport when expressed together with ATG-2 but not with ATG-1 or the mammalian type II glycoproteins 4F2hc and rBAT. AAT-1 and AAT-3 covalently bind to both C. elegans ATG glycoproteins, but only the pairs with ATG-2 traffic to the oocyte surface. Both of these functional, surface-expressed C. elegans HATs transport most neutral amino acids and display the highest transport rate for l-Ala and l-Ser (apparent K(m) 100 microm range). Similar to their mammalian counterparts, the C. elegans HATs function as (near) obligatory amino acid exchangers. Taken together, this study demonstrates that the heteromeric structure and the amino acid exchange function of HATs have been conserved throughout the evolution of nematodes to mammals.     

Molecular cloning and characterization of the Caenorhabditis elegans elongation factor 2 gene (eft-2)

A Caenorhabditis elegans lambda ZAP cDNA library was screened using a fragment amplified from highly conserved regions of the mammalian and Drosophila elongation factor 2 (EF-2). Two types of cDNA clones were obtained, corresponding to two mRNA species with 3'-untranslated regions of 60 and 115 nucleotides, both encoding identical polypeptides. Sequence analysis of these clones and comparisons with hamster and Drosophila EF-2 sequences suggests that they encode C. elegans EF-2. Clone pCef6A, encoding the entire C. elegans EF-2 mRNA sequence including 45 nucleotides of 5'-untranslated region, contains a 2,556-bp open reading frame which predicts a polypeptide of 852 amino acid residues (Mr 94,564). The deduced amino acid sequence is greater than 80% identical to that of mammalian and Drosophila EF-2. Conserved sequence segments shared among a variety of GTP-binding proteins are found in the amino-terminal region. The carboxy-terminal half contains segments unique to EF-2 and its prokaryotic homolog, EF-G, as well as the histidyl residue which is ADP-ribosylated by diphtheria toxin. The C. elegans protein contains a 12-amino-acid insertion between positions 90 and 100, and a 13-amino-acid deletion between positions 237 and 260, relative to hamster EF-2. Partial sequencing of a genomic clone encoding the entire C. elegans EF-2 gene (named eft-2) has so far revealed two introns of 48 and 44 bp following codons Gln-191 and Gln-250, respectively. Southern and Northern blot analyses indicate that eft-2 is a single-copy gene and encodes a 3-kb mRNA species which is present throughout nematode development.     

Protein glycosylation lessons from Caenorhabditis elegans

From observations on human diseases and mutant mice, it has become clear that glycosylation plays a major role in metazoan development. Caenorhabditis elegans provides powerful tools to study this problem that are not available in men or mice. The worm has many genes homologous to mammalian genes involved in glycosylation. Glycobiologists have, in recent years, cloned and expressed some of these genes and studied the effects of mutations on worm development. Recent studies have focused on N-glycosylation, lumenal nucleoside diphosphatases, the resistance of C. elegans to a bacterial toxin and infections, fucosylation and proteoglycans.