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

Program in International Child Health, Department of Pediatrics, Yale University School of Medicine, 464 Congress Avenue, New Haven, CT 06520, USA.
Molecular and Biochemical Parasitology (Impact Factor: 1.79). 03/2007; 151(2):141-7. DOI: 10.1016/j.molbiopara.2006.10.017
Source: PubMed


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.

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Available from: Wadim Kapulkin, Apr 16, 2014
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    • "In recent years, an increasing number of C-type lectins from insects have been reported to be involved in various biological responses (12-14). By now, more than 30 encoded C-type lectin-like domains have been identified in the fruit fly Drosophila melanogaster (15). The first C-type lectin from the silkworm Bombyx mori called LPS-binding protein (BmLBP) was isolated and characterized in 1997 (16) and then its cDNA cloning was described in 1999 (17). "
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    ABSTRACT: In this paper, we firstly reported a C-type lectin cDNA clone of 1029 bps from the larvae of A. Pernyi (Ap-CTL) using PCR and RACE techniques. The full-length cDNA contains an open reading frame encoding 308 amino acid residues which has two different carbohydrate-recognition domains (CRDs) arranged in tandem. To investigate the biological activities in the innate immunity, recombinant Ap-CTL was expressed in E. coli with a 6-histidine at the amino-terminus (Ap-rCTL). Besides acted as a broad-spectrum recognition protein binding to a wide range of PAMPs and microorganisms, Ap-rCTL also had the ability to recognize and trigger the agglutination of bacteria and fungi. In the proPO activation assay, Ap-rCTL specifically restored the PO activity of hemolymph blocked by anti- Ap-rCTL antibody in the presence of different PAMPs or microorganisms. In summary, Ap-rCTL plays an important role in insect innate immunity as an pattern recognition protein. [BMB Reports 2013; 46(7): 358-363].
    BMB reports 07/2013; 46(7):358-63. DOI:10.5483/BMBRep.2013.46.7.009 · 2.60 Impact Factor
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    • "Lectins are single-or multi-domain glycoproteins capable of binding sugar moieties through specific interactions with carbohydrate recognition domains (Brown et al. 2007). Most lectins are multimeric, consisting of non-covalently associated subunits that give lectins their ability to agglutinate cells or form aggregates with glycoconjugates (Mohr and Pommerening 1985). "
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    ABSTRACT: Lectins are a diverse group of carbohydrate binding proteins often involved in cellular interactions. A lectin gene, lec-2, was identified in the mycobiont of the lichen Peltigera membranacea. Sequencing of lec-2 open reading frames from 21 individual samples showed an unexpectedly high level of polymorphism in the deduced protein (LEC-2), which was sorted into nine haplotypes based on amino acid sequence. Calculations showed that the rates of nonsynonymous versus synonymous nucleotide substitutions deviated significantly from the null hypothesis of neutrality, indicating strong positive selection. Molecular modeling revealed that most amino acid replacements were around the putative carbohydrate-binding pocket, indicating changes in ligand binding. Lectins have been thought to be involved in the recognition of photobiont partners in lichen symbioses, and the hypothesis that positive selection of LEC-2 is driven by variation in the Nostoc photobiont partner was tested by comparing mycobiont LEC-2 haplotypes and photobiont genotypes, as represented by the rbcLX region. It was not possible to pair up the two types of marker sequences without conflicts, suggesting that positive selection of LEC-2 was not due to variation in photobiont partners.
    Symbiosis 12/2012; 58(1-3):91-98. DOI:10.1007/s13199-012-0206-y · 1.44 Impact Factor
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    • "An intriguing possibility is that the lectins of gastrointestinal nematodes may interact with the complex mucin oligosaccharides which are known to alter significantly in response to worm infection [48]. It should also be noted that other parasite lectins appear to play physiological roles within the parasite, for example that from Ancylostoma ceylanicum [49], which is a functional Glc-NAc-binding lectin and most highly expressed in male gonads and sperm, indicating that its function is in worm reproduction. "
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    ABSTRACT: The C-type lectin superfamily is highly represented in all metazoan phyla so far studied. Many members of this superfamily are important in innate immune defences against infection, while others serve key developmental and structural roles. Within the superfamily, many proteins contain multiple canonical carbohydrate-recognition domains (CRDs), together with additional non-lectin domains. In this report, we have studied two gastrointestinal nematode parasites which are widely used in experimental rodent systems, Heligmosomoides polygyrus and Nippostrongylus brasiliensis. From cDNA libraries, we have isolated 3 new C-type lectins from these species; all are single-CRD proteins with short additional N-terminal domains. The predicted Hp-CTL-1 protein contains 156 aa, Nb-CTL-1 191 aa and Nb-CTL-2 183 aa; all encode predicted signal peptides, as well as key conserved sequence motifs characteristic of the CTL superfamily. These lectins are most similar to C. elegans CLEC-48, 49 and 50, as well as to the lectin domains of mammalian immune system proteins CD23 and CD206. RT-PCR showed that these H. polygyrus and N. brasiliensis genes are primarily expressed in the gut-dwelling adult stages, although Nb-CTL-2 transcripts are also prominent in the free-living infective larval (L3) stage. Polyclonal antibodies raised to Hp-CTL-1 and Nb-CTL-1 reacted to both proteins by ELISA, and in Western blot analysis recognised a 15-kDa band in secreted proteins of adult N. brasiliensis (NES) and a 19-kDa band in H. polygyrus ES (HES). Anti-CTL-1 antibody also bound strongly to the cuticle of adult H. polygyrus. Hence, live parasites release C-type lectins homologous to some key receptors of the mammalian host immune system, raising the possibility that these products interfere in some manner with immunological recognition or effector function.
    Parasitology International 09/2009; 58(4):461-70. DOI:10.1016/j.parint.2009.08.011 · 1.86 Impact Factor
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