slo-1 is the gene annotation for a SLOwpoke potassium channel family member.
The protein SLO-1 is a large conductance calcium-gated potassium channel (BK-type channel). C. elegans slo-1 knockout mutants were observed to be highly resistant to emodepside (personal communication Lindy Holden-Dye, School of Biological Sciences, Southampton). Therefore, a search for potential orthologs of slo-1 in H. contortus, C. oncophora, and O. ostertagi was conducted.
A BLAST search using the sequence of C. elegans slo-1 as input in the database of the Sanger BLAST Server found matches with H. contortus in four short fragments of 83 – 150 bp within the coding sequence and a 599 bp fragment containing the last twenty codons of the coding sequence, the stop codon, and part of the 3’ UTR.
A PCR using primers based on these sequences amplified a 1352 bp fragment in H. contortus and C. oncophora, and a 1355 bp fragment in O. ostertagi. These fragments were elongated by RACE experiments. For H. contortus and C. oncophora slo-1, PCR products of the size of the complete coding sequence were obtained.
A test PCR with internal specific primers confirmed the identity of slo-1.
Routine cloning of these PCR products using the StrataCloneTM PCR Cloning Kit or the TOPO TA Cloning® Kit for Sequencing was unsuccessful: either no colonies grew or the colonies did not contain the full-length slo-1 sequence. For H. contortus slo-1, routine cloning using the TOPO TA Cloning® Kit for Sequencing produced plasmids containing an insertion of 145 bp, similar to an intron, which interrupted all three reading frames. Blunt end cloning was also unsuccessful. Transformation of ligation samples into JM109 competent cells, either after TOPO TA ligation or blunt end ligation, produced few clones containing the complete coding sequence of slo-1. The amplification of a full-length coding sequence of O. ostertagi slo-1 was unsuccessful.
Hence, the contig of O. ostertagi slo-1 should be regarded as preliminary sequence.
The coding sequence of C. oncophora and O. ostertagi slo-1 were found to have six additional N-terminal amino acids compared to H. contortus slo-1. An overview of the sequence traits is presented in Table 5, the UTRs should be regarded as putative UTRs. Accession numbers are given in the appendix (8.2).
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H. contortus slo-1 C. oncophora slo-1 O. ostertagi slo-1
Coding sequence 3315 bp 3333 bp 3378
Amino acids (aa) 1105 1111 1126
5’ UTR (bp) 148 766 411
3’ UTR (bp) 530 529 465
Table 5: Sequence traits of slo-1 of H. contortus, C. oncophora, and O. ostertagi. Coding sequence starts with the start codon and excludes the stop codon. 3’ UTR is given as the number of base pairs (bp) of the untranslated region including the stop codon but excluding the poly-A tail
6.5.1 Identities between slo-1 Sequences
The nucleotide sequences of parasitic slo-1 have 84 – 86 % identity with each other, whereas the amino acid sequences of the translated proteins have 96 – 98 % identity. The percentages of identity are presented in detail in Table 6. H. contortus and O. ostertagi slo-1 have an identity of 71 % with C. elegans slo-1 based on cDNA sequence, whereas C. oncophora slo-1 has 72 %. All three parasitic SLO-1 have 87 % identity in amino acid sequence with C. elegans SLO-1.
cDNA Amino acids
Hc slo-1 / Co slo-1 85 % 98 %
Hc slo-1 / Oo slo-1 84 % 96 %
Co slo-1 / Oo slo-1 86 % 96 %
Table 6: Identity of the coding sequences of slo-1 of H. contortus, C. oncophora, and O. ostertagi, based on cDNA and amino acid sequences
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6.5.2 BLAST Results for slo-1 Sequences
NCBI BLAST blastn (nucleotides vs. nucleotides) searches using H. contortus and C. oncophora slo-1 sequences as input retrieved several BK-type potassium channels from different organisms. The identities of C. elegans slo-1 fragments, mainly 100 – 450 bp in size, with parasitic slo-1 were as high as 85 %. The next best matches were fragments of genes for a potassium channel in Cancer borealis, the Jonah crab, and the fruit fly Drosophila melanogaster. Further matches for H. contortus slo-1 included fragments of calcium-activated potassium channels from the red flour beetle Tribolium castaneum and the wasp Nasonia giraulti, with identities of 78 – 79 %. The blastn search for C. oncophora slo-1 found matches with 80 – 180 bp fragments of genes for calcium-activated potassium channels in the insects Drosophila pseudoobscura and Manduca sexta. These fragments had identities with C. oncophora slo-1 of 80 – 85 %; very short fragments of approx.
25 bp had identities as high as 96 %. The order of the listed matches varied for H. contortus and C. oncophora slo-1. A blastn search with the preliminary O. ostertagi slo-1 found, following C. elegans and C. briggsae slo-1, matches with gene sequences for potassium channels of D. melanogaster. For all three parasitic slo-1 several other sequences of BK-type potassium channels, mainly less than 100 bp in size, matched in addition to the sequences derived from insects and crustaceans listed above. Among these further matches were sequences of molluscs (Aplysia californica), fishes (Danio rerio), birds (Gallus gallus), and mammals (C. familiaris, B. taurus and others).
The NCBI BLAST blastx (translated vs. protein) server retrieved matches with higher identities over larger ranges than the blastn search for all three parasitic slo-1 sequences: The best matches were C. elegans and C. briggsae SLO-1, followed by a D. melanogaster calcium-activated potassium channel and the predicted channel in T. castaneum. Other matches were BK-type potassium channels of various species. All fragments had identities of 57 – 59 %, and 70 – 71 % positives, (amino acids with a similar function) with the parasitic SLO-1. The spectrum of species contained molluscs, insects, turtles, mammals, and birds. The order of the listed species differed slightly between the results for H. contortus, C. oncophora and O. ostertagi SLO-1.
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6.5.3 Comparison of SLO-1 Sequences
Using the ClustalW software to compare sequences of calcium-gated potassium channels, the insect channels were found to have an identity of 63 – 65 %, the molluscan 59 %, chicken potassium channels 54 – 55 %, and mammalian 53 – 55 % with the nematode SLO-1 (including C. elegans). The identity between C. elegans SLO-1 and parasitic SLO-1 was 87 %. A phylogenetic tree based on these data is presented in Figure 11. The sequences used for this analysis were SLO-1 of human (H. sapiens), rat (R. norvegicus), dog (C. familiaris), cattle (B. taurus), chicken (G. gallus), fruit fly (D. melanogaster), tobacco hornworm (M. sexta), red flour beetle (T. castaneum), and seahare (A. californica).
Figure 11: Phylogenetic tree of BK-type potassium channels based on amino acid sequence comparison. The numbers on the branches indicate the bootstrap values (in percent;
1000 replicates), the bar indicates the number of substitutions per site
NCBI accession numbers: H. contortus: translated EF494184; C. oncophora: translated EF494185; O. ostertagi: translated preliminary contig (unpublished); C. elegans: translated NM_001029089; H. sapiens: EAW54600; C. familiaris: Q28265; B. taurus: AAK54354; G. gallus:
AAC35370; D. melanogaster: AAX52990; M. sexta: AAT44358; T. castaneum: XP_968651;
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6.5.4 Prediction of Transmembrane Domains and Signal Peptides
For H. contortus, C. oncophora, and O. ostertagi SLO-1, Phobius predicted seven transmembrane regions, an intracellular N-terminus, and an extracellular C-terminus.
TMMOD predicted only six transmembrane helices with extracellular N- and C-termini. ConPred II also predicted seven transmembrane helices with intracellular N-terminus and extracellular C-terminus. The transmembrane helix not marked by TMMOD was congruently detected by the other programs as the fourth helix. The distance between the third and fourth transmembrane helices was five amino acids predicted by Phobius and was three amino acids in the ConPred II prediction, for all three sequences. Anyhow, TMMOD did not predict the region of this additional TM helix to be included in another predicted TM helix; the respective TM helix was not recognized to be hydrophobic at all. The extracellular C-terminus was predicted by all three programs to be very large, approx. 750 - 770 aa of 1105 - 1126 aa in total. None of the programs detected a signal peptide.
The prediction results were partially unexpected, as in literature BK-type potassium channels were described to have an extracellular N-terminus and an intracellular C-terminus (MEERA et al., 1997). For comparison, the sequences of the human and Drosophila potassium channels were analyzed using the three prediction programs.
Providing the sequence of D. melanogaster, which was cited in the respective paper (NCBI Acc. No. JH0697, record had been discontinued but could still be found as Acc. No. 321029) to the prediction programs, similar results as for the parasitic SLO-1 were achieved: seven TM helices with an intracellular N-terminus and an extracellular C-terminus were predicted by Phobius and ConPred II, six TM helices with extracellular N- and C-termini were predicted by TMMOD. The sequence of the human potassium channel (NCBI Acc. No. U11058) cited in the same paper was predicted to have eight TM helices with extracellular N- and C-terminus by Phobius, eight TM helices with intracellular N- and C-terminus by ConPred II, and six TM helices with extracellular N- and C-termini.
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6.5.5 Prediction of Conserved Domains
The domains predicted by the NCBI CDART program were an ion transport protein domain and a calcium-activated BK-type potassium channel α subunit. A scheme is given in Figure 12.
Figure 12: Conserved domains of parasitic SLO-1.
Legend: ion-trans ion transport protein
BK channel calcium-activated BK potassium channel α subunit