Structure-activity relationships of 18 endogenous neuropeptides on the motor nervous system of the nematode Ascaris suum

Neuropeptides play an important role in all nervous systems and structure-activity studies of related peptides is one approach to understanding this role. This study of the motor nervous system of the parasitic nematode Ascaris suum describes the physiological effects of a family of 18 endogenous Ascaris FMRFamide-like peptides (AF peptides) on the membrane potential and input resistance of the dorsal excitatory type 2 (DE2) and dorsal inhibitory (DI) motor neurons. These motor neurons are part of the final common output pathway from the motor nervous system to the somatic muscle cells responsible for locomotion. AF peptide effects on the frequency of excitatory postsynaptic potentials (EPSPs) in DE2 motor neurons were also measured to infer peptide effects on central presynaptic spiking neurons. AF peptide injections into intact worms were made to assess their qualitative effects on behavior, providing a context for interpreting motor neuron data. One category of AF peptides, N-terminally extended -FIRFa peptides (AF5, AF7 and AF1), has pronounced behavioral effects and qualitatively similar, but quantitatively different effects on DE2 and DI motor neurons. A second category of AF peptides (AF2, AF9, and AF8) also produces dramatic behavioral effects and strong electrophysiological effects on DE2 and/or DI motor neurons. A third category of AF peptides, consisting of six members of the -PGVLRFa group (which are encoded by the same gene and have closely related sequences) and peptide AF11, have pronounced behavioral effects, but relatively weak or negligible effects on DE2 and DI motor neurons. A fourth category of AF peptides, also consisting of structurally unrelated members, has pronounced behavioral effects and, as individual peptides, similar effects on both DE2 and DI motor neurons; AF15 is excitatory, while AF17 and AF19 are inhibitory, on both motor neuron types. Finally, two AF peptides (AF6, AF16) are relatively weak or inactive in producing behavioral or motor neuronal effects. Based on comparisons of the effects of AF peptides on DE2 and DI motor neurons, a tentative list of 5 major response-types is proposed as a working hypothesis to guide the search for AF peptide receptors. The findings attest to the potential complexity of neurosignaling in this comparatively simple nervous system.     

Error-tolerant EST database searches by tandem mass spectrometry and multiTag software

The MultiTag method (Sunyaev et al., Anal. Chem. 2003 15, 1307-1315) employs multiple error-tolerant searches with peptide sequence tags (Mann and Wilm, Anal. Chem. 1994, 66, 4390-4399) for the identification of proteins from organisms with unsequenced genomes. Here we demonstrate that the error-tolerant capabilities of MultiTag increased the number of peptide alignments and improved the confidence of identifications in an EST database. The MultiTag outperformed conventional database searching software that only utilizes stringent matching of tandem mass spectra to nucleotide sequences of ESTs.     

DNA synthesis in the early embryo of the nematode Ascaris suum.

We have used microspectrofluorimetry to measure the rate of DNA synthesis in the first two embryonic cell cycles of the parasitic nematode Ascaris suum. The S-phase of the early Ascaris cell cycles occupies at most 1 hr; G2 phase is prominent and occupies approximately 11 hr; no G1 phase could be detected. These results contrast with our previous measurements made with embryos of the free-living nematode Caenorhabditis elegans, in which the earliest cell cycles consist of simple alternations between S and M phases.     

Catalase activity during the development of the parasitic nematode, Ascaris suum

1. Catalase activity was partially purified from body wall muscle of the parasitic nematode, Ascaris suum, and was similar to catalases isolated from mammalian tissues. It exhibited a broad pH optimum and was unaffected by 2 mM ethylenediaminetetra-acetate. In contrast, it was inhibited reversibly by 1 mM cyanide and irreversibly by prior incubation in 40 mM 3-amino-1:2:4-triazole for 1 hr or heating at 80 degrees C for 15 min. 2. Catalase activity was highest in the unembryonated "egg" and decreased dramatically as development proceeded. 3. Catalase activity in adult body wall muscle was similar to that in rat skeletal muscle, but dramatically lower than that in rat liver. Catalase activity was barely detectable in A. suum testis. 4. Cytochrome-c peroxidase activity did not appear to be present in adult A. suum muscle mitochondria.     

Molecular and catalytic properties of an arginine kinase from the nematode Ascaris suum

We amplified the cDNA coding for arginine kinase (AK) from the parasitic nematode Ascaris suum, cloned it in pMAL plasmid and expressed the enzyme as a fusion protein with the maltose-binding protein. The whole cDNA was 1260 bp, encoding 400 amino acids, and the recombinant protein had a molecular mass of 45,341 Da. Ascaris suum recombinant AK showed significant activity and strong affinity ( K(m)(Arg) = 0.126 mM) for the substrate L-arginine. It also exhibited high catalytic efficiency ( k(ca)/K(m)(Arg) = 352) comparable with AKs from other organisms. Sequence analysis revealed high amino acid sequence identity between A. suum AK and other nematode AKs, all of which cluster in a phylogenetic tree. However, comparison of gene structures showed that A. suum AK gene intron/exon organization is quite distinct from that of other nematode AKs. Phosphagen kinases (PKs) from certain parasites have been shown to be potential novel drug targets or tools for detection of infection. The characterization of A. suum AK will be useful in the development of strategies for control not only of A. suum but also of related species infecting humans.     

Molecular symmetry and arrangement of subunits in extracellular hemoglobin from the nematode Ascaris suum

The arrangement of subunits and molecular symmetry of extracellular hemoglobin from the nematode Ascaris suum, an 11.7S molecular of molecular mass 332 kDa and composed of eight identical subunits, was studied. Dissociation of the molecule at alkaline and acid pH yielded 4.6S and 2.7S components, identified as polypeptide-chain dimers and monomers, respectively. Cross-linking with glutardialdehyde followed by SDS/PAGE resulted in a maximum number of eight bands identified in order of decreasing mobility as monomeric and 2-8 cross-linked-polypeptide-chain species. Comparison with values predicted from theory shows that the distribution of protein among the various cross-linked species, obtained after different extents of exposure to cross-linker, is consistent with a two-layered arrangement of subunits involving one type of interaction between subunits from different layers and another between subunits within the same layer. Electron micrographs of the molecule showed two profiles, a square and a rectangle. We propose a model for the molecule which is eight subunits arranged in two layers, stacked in an eclipsed orientation. The proposed model is consistent with the results from sedimentation, cross-linking and electron microscopy. Taken together, our findings indicate D4 symmetry for Ascaris hemoglobin.     

Crystal structure of mitochondrial quinol-fumarate reductase from the parasitic nematode Ascaris suum

In the anaerobic respiratory chain of the parasitic nematode Ascaris suum, complex II couples the reduction of fumarate to the oxidation of rhodoquinol, a reverse reaction catalyzed by mammalian complex II. In this study, the first structure of anaerobic complex II of mitochondria was determined. The structure, composed of four subunits and five co-factors, is similar to that of aerobic complex II, except for an extra peptide found in the smallest anchor subunit of the A. suum enzyme. We discuss herein the structure-function relationship of the enzyme and the critical role of the low redox potential of rhodoquinol in the fumarate reduction of A. suum complex II.     

Extracellular recordings from the motor nervous system of the nematode,Ascaris suum

1. The close association of muscle and neurons in Ascaris suum makes it difficult to determine whether spikes recorded from nerve cords originate in muscle or neurons. We have developed criteria that distinguish muscle and neuronal activity. There are two categories of extracellular spikes. 2. The first category consists of spikes with a wide range of amplitudes, marked by large spikes. These spikes, which can be recorded over lateral muscle and over the dorsal and ventral nerve cords, are abolished when muscle is disrupted or removed, or when curare is applied. Large spikes are relatively infrequent, are correlated with intracellularly recorded muscle events, and respond to polarizations of motor neurons, implying that they originate in muscle. 3. The second spike category, small amplitude spikes, is exclusive to the ventral nerve cord, occurs more frequently than large spikes and displays patterned firing. Small spikes are not affected by muscle removal or by curare, and are correlated with motor neuronal post-synaptic potentials, but not with intracellularly recorded muscle events. We infer that they originate in neurons. 4. Low level activity recorded extracellularly over nerve cords may represent muscle activity due to tonic motor neuronal synaptic transmission. It responds to motor neuronal polarization and is suppressed by curare or muscle removal.     

Crystallization of the NAD-dependent malic enzyme from the parasitic nematode Ascaris suum.

The malic enzyme from muscle mitochondria of the parasitic nematode Ascaris suum is a tetramer of 65 kDa monomers that catalyzes the oxidative decarboxylation of malate to pyruvate and CO2 with NAD cofactor as oxidant. This malic enzyme is critical to the nematode for muscle function under anaerobic conditions. Unlike mammalian versions of the enzyme such as that found in rat liver, which require NADP as cofactor, the nematode version is an NAD-dependent enzyme. We report the crystallization of samples of the nematode enzyme at room temperature from pH 7.5 solutions of polyethylene glycol 4000 containing magnesium sulfate, NAD and sodium tartronate. Immediately upon mixing of protein and precipitant solutions, a marked precipitation of the protein occurs. Out of this precipitate, crystals appear almost immediately, most commonly in a truncated cube form that can grow to 0.5 to 0.7 mm on a cube edge in two to three days. The crystals are trigonal, space group P3(1)21 or its enantiomer, with a = b = 131.2(7) A, c = 152.6(9) A, and two monomers per asymmetric unit. Fresh crystals diffract X-radiation from a synchrotron source (lambda = 0.95 A) to about 3.0 A resolution. Rotational analysis of Patterson functions indicates that the malic enzyme tetramer has 222 symmetry.     

Initial characterization of several actin genes in the parasitic nematode, Ascaris suum.

Southern hybridization data suggest a large actin gene family in Ascaris suum. Our genomic reconstruction experiment indicated that it consists of 50-75 members. Polymorphism was uncovered in the actin genes or in their surrounding sequences. From the genomic library 5 nonoverlapping actin clones were isolated and characterized.     

Cloning, expression, and purification of fumarase from the parasitic nematode Ascaris suum

The cDNA encoding fumarase, an enzyme catalyzing reversible hydration of fumarate to L-malate, from the parasitic roundworm Ascaris suum, has been cloned, sequenced, over-expressed in Escherichia coli, and purified. The single open reading frame translates into a protein of 50,502Da containing 467 amino acids. It shows 82, 77, and 58% identity with Caenorhabditis elegans, human, and E. coli fumC fumarases, respectively. The A. suum fumarase shows the signature sequence motif (GSSIMPGKVNPTQCE), which defines not only the class II fumarase family but also a much broader superfamily of proteins containing GSSxMPxKxNPxxxE motif. The coding region was cloned into pET101D-directional TOPO expression vector and transformed into E. coli BL21 Star (DE3). The protein after induction was expressed at high levels, almost 10% of the soluble protein, purified to near homogeneity, and appears identical to the enzyme purified from Ascaris suum.     

Structure of the polypeptide chain of extracellular hemoglobin from the nematode Ascaris suum.

1. Ascaris suum extracellular hemoglobin is composed of eight identical single polypeptide chain subunits carrying two heme binding sites each. 2. Limited trypsinolysis followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis gave a major band corresponding to half the molecular mass of an intact subunit. 3. Peptide mapping of tryptic hydrolysates yielded 27 to 30 fluorescamine positive spots, about half the number of lysyl and arginyl residues in a polypeptide chain. 4. The findings indicate that a subunit of Ascaris hemoglobin consists of two structural units of roughly equal size, corresponding to two recurring sequences, connected together by the continuity of the polypeptide chain.     

Primary structure of the major pepsin inhibitor from the intestinal parasitic nematode Ascaris suum

The major pepsin inhibitor from Ascaris suum was isolated by affinity chromatography and chromatofocusing. Its amino acid sequence was determined by automated Edman degradation of peptide fragments. Peptides were produced by chemical and enzymatic cleavage of pyridylethylated protein and were purified by reverse-phase high-performance liquid chromatography. The inhibitor consists of 149 residues with the following sequence: QFLFSMSTGP10FICTVKDNQV20FVANLPWTML30EGDDIQVGKE40 FAARVEDCTN50VKHDMAPTCT60KPPPFCGPQD70MKMFNFVGCS80VLGNKLFIDQ90KYVRDLTAK D100 HAEVQTFREK110IAAFEEQQEN120QPPSSGMPHG130AVPAGGLSPP140PPPSFCTVQ149. It has a molecular weight of 16,396. All cysteines are engaged as disulfide bonds: Cys(13)-Cys(59), Cys(48)-Cys(66), and Cys(79)-Cys(146). The protein is probably composed of two domains connected by a short hydrophobic region. This is the first aspartyl protease inhibitor of animal origin that has been sequenced. The sequence has no significant homology with any other known protein.     

AF1, a sequenced bioactive neuropeptide isolated from the nematode Ascaris suum

An FMRFamide-like neuropeptide, named AF1, was isolated from head extracts of the nematode Ascaris suum using five steps of HPLC. AF1 is a heptapeptide with the amino acid sequence Lys-Asn-Glu-Phe-Ile-Arg-Phe-NH2. Synthetic AF1 (10(-9) to 10(-7) M) rapidly and reversibly abolished slow membrane potential oscillations of identified ventral and dorsal inhibitory motoneurons and selectively reduced their input resistances. Synaptic transmission was not blocked. In intact Ascaris, AF1 inhibited locomotory movements. This study indicates a potential physiological role for an endogenous neuropeptide in nematodes.     

Actions of cholinergic drugs in the nematode Ascaris suum. Complex pharmacology of muscle and motorneurons

The cholinergic agonists acetylcholine (ACh), nicotine, and pilocarpine produced depolarizations and contractions of muscle of the nematode Ascaris suum. Dose-dependent depolarization and contraction by ACh were suppressed by about two orders of magnitude by 100 microM d- tubocurarine (dTC), a nicotinic antagonist, but only about fivefold by 100 microM N-methyl-scopolamine (NMS), a muscarinic antagonist. NMS itself depolarized both normal and synaptically isolated muscle cells. The muscle depolarizing action of pilocarpine was not consistently antagonized by either NMS or dTC. ACh receptors were detected on motorneuron classes DE1, DE2, DI, and VI as ACh-induced reductions in input resistance. These input resistance changes were reversed by washing in drug-free saline or by application of dTC. NMS applied alone lowered input resistance in DE1, but not in DE2, DI, or VI motorneurons. In contrast to the effect of ACh, the action of NMS in DE1 was not reversed by dTC, suggesting that NMS-sensitive sites may not respond to ACh. Excitatory synaptic responses in muscle evoked by depolarizing current injections into DE1 and DE2 motorneurons were antagonized by dTC; however, NMS antagonized the synaptic output of only the DE1 and DE3 classes of motorneurons, an effect that was more likely to have been produced by motorneuron conduction failure than by pharmacological blockade of receptor. The concentration of NMS required to produce these changes in muscle polarization and contraction, ACh antagonism, input resistance reduction, and synaptic antagonism was 100 microM, or more than five orders of magnitude higher than the binding affinity for [3H]NMS in larval Ascaris homogenates and adult Caenorhabditis elegans (Segerberg, M. A. 1989. Ph.D. thesis. University of Wisconsin-Madison, Madison, WI). These results describe a nicotinic- like pharmacology, but muscle and motorneurons also have unusual responses to muscarinic agents.     

In vitro induction of crawling in the amoeboid sperm of the nematode parasite, Ascaris suum

In a highly synchronous process, the immotile spermatids of Ascaris suum extend pseudopods and become rapidly crawling sperm when treated with an extract from the glandular vas deferens of the male under strict anaerobic conditions. Within 9-12 min, a pseudopod develops, elongates rapidly, and exhibits a continuous flow of membrane specializations, the villipodia, from tip toward base. When attached to acid-washed glass, the pseudopod pulls the cell body along at speeds exceeding 70 microns/min. The pseudopod length remains constant while retrograde flow of villipodia proceeds at the same rate as the sperm's forward movement. Cohorts of about 15 villipodia form at the leading edge, move rearward together, and disappear at the junction of pseudopod and cell body. These are the terminations of branched, refringent fibers, which extend the length of the pseudopod. The latter are the fiber complexes that form its cytoskeleton (Sepsenwol et al.: Journal of Cell Biology 108:55-66, 1989). Locomoting cells sometimes change direction when another crawls by and follow each other. When cells are exposed to air, forward movement ceases in a predictable pattern: the forward extension of the leading edge ceases, the pseudopod shortens from the base, and the cell body continues to be pulled forward. These data contribute to a model for Ascaris sperm amoeboid motility in which independent processes of continuous extension at the leading edge and continuous shortening at the base of the pseudopod act to propel the cell forward.