Adenle, Ademola A.
Genetic analysis of latrophilin
in the toxicity of combined latrotoxins for C. elegans.
PhD thesis, University of Nottingham.
Black widow spider venom (BWSV) contains high molecular weight proteins called latrotoxins (LTX) that induce catastrophic neurotransmitter release from nerve terminals, and one toxin, α-latrotoxin, is known to bind with high affinity to three neural proteins in mammals, including latrophilin (lat-1) a member of the class B family of G-protein coupled receptors.
We have established C.elegans as a model organism to study the function of the binding protein, lat-1 and its role in regulating neurotransmitter release by latrotoxins. However, a lat-1-knockout worm is required for determining the function of the lat-1 gene. The lat-1(ok1465) allele has a deletion of the lat-1 gene, and ~95-98% of lat-1(ok1465) homozygous worms arrest or die before adulthood, with only ~2-5 adult offspring per animal. Micro-injection of the B0457 cosmid, that contains the full sequence of the lat-1 gene, or the lat-1a cDNA rescued the lethality of the lat-1 worms, thereby showing that lat-1 gene is responsible for the developmental lethality in these worms. Expression of the marker, GFP, under the control of the lat-1 promoter showed that there was expression of GFP during epithelial morphogenesis, and strong expression in the gut from the three-fold stage through to larval stages. The concordance between the site of expression of lat-1::gfp, with the sites of embryonic defects (epithelial enclosure defects; defective attachement of gut) in lat-1(ok1465) animals, provides further evidence that lat-1 is essential for embryonic and larval development.
Deletion mutants of lat-1a were constructed to examine the role of domains of this protein. Deletion of sequences after the 4xCys domain of lat-1a did not affect the ability to rescue lethality in the lat-1 worm, while deletion of the C-terminus to the seven transmembrane domain impaired the ability of lat-1a to rescue lat-1 worms, and further deletion of six of seven transmembrane domains (the TM1 construct) yielded a construct that was unable to rescue lat-1 worms.
These data suggest an important role for intracellular sequences and seven transmembrane in lat-1a signalling. It was proposed that TM1 could decoy ligand, without causing intracellular signalling. In agreement, the TM1 construct caused a mild phenocopy of the lat-1(ok1465) mutant in wild-type worms, whereas full-length, or non-ligand binding variants of lat-1a caused no such effect. To investigate the putative ligand-binding domain of lat-1a, deletion of residues 62-147 (ΔGBL), 62-250 (ΔHRM) and 62-487 (ΔN) was investigated; while the ΔN construct was incapable of rescuing lat-1(ok1465) worms, deletion of ΔGBL had a minor effect on the ability of lat-1 to rescue the null worms, while ΔHRM had a more marked effect. These data are consistent with a model whereby residues 147-487 are required for ligand binding, and the seventransmembrane and intracellular domains transmit a signal to the inside of the cell.
Combined latrotoxins was highly toxic to wild-type C.elegans (LD50 ~4ng/ml), whereas the lat-1 worms were highly (>105-fold) resistant to combined latrotoxins. Lat-1 worms that were transgenic for B0457cosmid, or lat-1a cDNA, were as sensitive to combined latrotoxins as wildtype worm. Truncation of the C-terminus of lat-1a to TM1 yielded worms that had 105-fold resistance to combined latrotoxins, compared to wild-type; thus the intracellular domain of lat-1 is required for mediating combined latrotoxins toxicity. The deletion of galatactose-binding lectin (ΔGBL) in N-terminus lat-1a was sensitive as wild-type, but deletion of hormone receptor motif (ΔHRM) in N-terminus lat-1a showed a reduced sensitivity to combined latrotoxins by ~105-fold. These data showed presence of lat-1 gene was responsible for the rescue of lat-1 worms or toxicity of combined latrotoxins in lat-1 worms, and the absence of lat-1 gene was responsible for the lethality of lat-1 worms and resistance to combined latrotoxins in lat-1 worms.
Thesis (University of Nottingham only)
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||06 Oct 2010 08:49
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