Molecular cloning and characterization of a C-type lectin from Ancylostoma ceylanicum: evidence for a role in hookworm reproductive physiology.

Lectins comprise a family of related proteins that mediate essential cell functions through binding to carbohydrates. Within this protein family, C-type lectins are defined by the requirement of calcium for optimal biologic activity. Using reverse transcription PCR, a cDNA corresponding to a putative C-type lectin has been amplified from the hookworm parasite Ancylostoma ceylanicum. The 550 nucleotide open reading frame of the A. ceylanicum C-type Lectin-1 (AceCTL-1) cDNA corresponds to a 167 amino acid mature protein (18,706 Da) preceded by a 17 amino acid secretory signal sequence. The recombinant protein (rAceCTL-1) was expressed in Drosophila S2 cells and purified using a combination of affinity chromatography and reverse phase HPLC. Using in vitro carbohydrate binding studies, it was determined that rAceCTL-1 binds N-acetyl-d-glucosamine, a common component of eukaryotic egg cell membranes. Using a polyclonal IgG raised against the recombinant protein, the native AceCTL-1 was identified in sperm and soluble protein extracts of adult male A. ceylanicum by immunoblot. Probing of adult hookworm sections with the polyclonal IgG demonstrated localization to the testes in males, as well as the spermatheca and developing embryos in females, consistent with its role as a sperm protein. Together, these data strongly suggest that AceCTL-1 is a male gender-specific C-type lectin with a function in hookworm reproductive physiology.

Compensatory regulation among ER chaperones in C. elegans.

We have identified a particularly clear case of compensatory transcriptional regulation among ER chaperones in Caenorhabditis elegans using a GFP reporter transgene that is under the control of the promoter of hsp-4, a C. elegans homolog of GRP-78/BiP. Knockdown by RNA interference of 9 known or predicted ER chaperones induced hsp-4 upregulation via the ire-1/xbp-1 signaling cascade employed in the unfolded protein response. We show that this compensatory regulation is specific for ER chaperones, not dependent on RNA interference, and required for maintaining viability in worms containing a deletion of the hsp-3 gene.

Investigating hookworm genomes by comparative analysis of two Ancylostoma species.

BACKGROUND: Hookworms, infecting over one billion people, are the mostly closely related major human parasites to the model nematode Caenorhabditis elegans. Applying genomics techniques to these species, we analyzed 3,840 and 3,149 genes from Ancylostoma caninum and A. ceylanicum. RESULTS: Transcripts originated from libraries representing infective L3 larva, stimulated L3, arrested L3, and adults. Most genes are represented in single stages including abundant transcripts like hsp-20 in infective L3 and vit-3 in adults. Over 80% of the genes have homologs in C. elegans, and nearly 30% of these were with observable RNA interference phenotypes. Homologies were identified to nematode-specific and clade V specific gene families. To study the evolution of hookworm genes, 574 A. caninum/A. ceylanicum orthologs were identified, all of which were found to be under purifying selection with distribution ratios of nonsynonymous to synonymous amino acid substitutions similar to that reported for C. elegans/C. briggsae orthologs. The phylogenetic distance between A. caninum and A. ceylanicum is almost identical to that for C. elegans/C. briggsae. CONCLUSION: The genes discovered should substantially accelerate research toward better understanding of the parasites' basic biology as well as new therapies including vaccines and novel anthelmintics.

Interaction of intracellular beta amyloid peptide with chaperone proteins.

Expression of the human beta amyloid peptide (A beta) in transgenic Caenorhabditis elegans animals can lead to the formation of intracellular immunoreactive deposits as well as the formation of intracellular amyloid. We have used this model to identify proteins that interact with intracellular A beta in vivo. Mass spectrometry analysis of proteins that specifically coimmunoprecipitate with A beta has identified six likely chaperone proteins: two members of the HSP70 family, three alpha B-crystallin-related small heat shock proteins (HSP-16s), and a putative ortholog of a mammalian small glutamine-rich tetratricopeptide repeat-containing protein proposed to regulate HSP70 function. Quantitative reverse transcription-PCR analysis shows that the small heat shock proteins are also transcriptionally induced by A beta expression. Immunohistochemistry demonstrates that HSP-16 protein closely colocalizes with intracellular A beta in this model. Transgenic animals expressing a nonaggregating A beta variant, a single-chain A beta dimer, show an altered pattern of coimmunoprecipitating proteins and an altered cellular distribution of HSP-16. Double-stranded RNA inhibition of R05F9.10, the putative C. elegans ortholog of the human small glutamine-rich tetratricopeptide-repeat-containing protein (SGT), results in suppression of toxicity associated with A beta expression. These results suggest that chaperone function can play a role in modulating intracellular A beta metabolism and toxicity.