Isolation and molecular cloning of a secreted hookworm platelet inhibitor from adult Ancylostoma caninum

Vallé, Antonio Del; Jones, Brian F.; Harrison, Lisa M.; Chadderdon, Robert C.; Cappello, Michael

doi:10.1016/s0166-6851(03)00121-x

Cited by 92 publications

(73 citation statements)

References 41 publications

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“…Bound protein was eluted from the column with imidazole, and the fractions containing recombinant protein were pooled. Following affinity chromatography, the partially purified recombinant protein was subjected to reverse-phase high-pressure liquid chromatography by using a C 18 column (Vydac, Hesperia, Calif.) (10,19,48). The relative purity and molar concentration of the recombinant protein were determined by electrospray ionization mass spectrometry and quantitative amino acid analysis as previously described (12,26,48).…”

Section: Methodsmentioning

confidence: 99%

“…All animal immunizations were carried out by certified personnel from the Yale Animal Resources Center by using protocols approved by the Yale Animal Care and Use Committee. Serum was collected following the second boost, and the immunoglobulin G (IgG) fraction was purified by protein G affinity chromatography as previously described (19,25). Protein concentrations in the fractions containing the purified IgG were determined by using reagents from a bicinchoninic acid kit (Pierce, Rockford, Ill.).…”

Section: Methodsmentioning

confidence: 99%

“…The stage specificity of AceKI RNA expression was evaluated by reverse transcription (RT)-PCR. Briefly, total RNA was isolated from 5,000 A. ceylanicum third-stage larvae (L3) or 30 adult parasites by homogenizing whole worms in Trizol (Life Technologies) (5,19,26). RNA was also isolated from 5,000 L3 that had been activated by incubating them in 50% fetal bovine serum for 2 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

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Molecular Characterization ofAncylostoma ceylanicumKunitz-Type Serine Protease Inhibitor: Evidence for a Role in Hookworm-Associated Growth Delay

Chu¹,

Bungiro²,

Ibanez³

et al. 2004

Infect Immun

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Hookworm infection is a major cause of iron deficiency anemia and malnutrition in developing countries. The Ancylostoma ceylanicum Kunitz-type inhibitor (AceKI) is a 7.9-kDa broad-spectrum inhibitor of trypsin, chymotrypsin, and pancreatic elastase that has previously been isolated from adult hookworms. Site-directed mutagenesis of the predicted P1 inhibitory reactive site amino acid confirmed the role of Met 26 in mediating inhibition of the three target serine proteases. By using reverse transcription-PCR, it was demonstrated that the level of AceKI gene expression increased following activation of third-stage larvae with serum and that the highest level of expression was reached in the adult stage of the parasite. Immunohistochemistry studies performed with polyclonal immunoglobulin G raised against recombinant AceKI showed that the inhibitor localized to the subcuticle of the adult hookworm, suggesting that it has a potential in vivo role in neutralizing intestinal proteases at the surface of the parasite. Immunization with recombinant AceKI was shown to confer partial protection against hookworm-associated growth delay without a measurable effect on anemia. Taken together, the data suggest that AceKI plays a role in the pathogenesis of hookworm-associated malnutrition and growth delay, perhaps through inhibition of nutrient absorption in infected hosts.Hookworm infection remains a major global health problem, and over one billion people are reportedly infected in developing countries (9, 14). Hookworms, which are bloodfeeding intestinal nematodes, are a major cause of iron deficiency anemia and malnutrition (15,20,(59)(60)(61)64). While the anemia is presumably due to the cumulative effect of chronic intestinal blood loss, the molecular mechanisms underlying the pathogenesis of hookworm malnutrition remain unknown. Although it has been suggested that hookworm malnutrition and growth delay occur secondary to chronic iron deficiency, particularly in children, evidence from prior clinical studies suggests that hookworm infection is also associated with various degrees of intestinal malabsorption (18,35,54,57,62). It has been hypothesized that this hookworm malabsorption syndrome might occur secondary to mucosal inflammation triggered by the adult worm attached to the intestinal epithelium or might be a result of secretion of parasite inhibitors of host digestive enzymes (18).As part of a series of ongoing studies aimed at characterizing adult hookworm secretory proteins, a cDNA corresponding to the gene encoding a putative Kunitz-type serine protease inhibitor was previously identified from adult Ancylostoma ceylanicum RNA by using a PCR-based approach (48). The A. ceylanicum Kunitz-type inhibitor (AceKI) cDNA was expressed in Escherichia coli, and the recombinant protein was found to inhibit the pancreatic enzymes chymotrypsin, pancreatic elastase, and trypsin in vitro, with equilibrium inhibitory dissociation constant (K i ) values ranging from picomolar levels to low nanomolar levels. The native AceKI prote...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Molecular Characterization ofAncylostoma ceylanicumKunitz-Type Serine Protease Inhibitor: Evidence for a Role in Hookworm-Associated Growth Delay

Chu¹,

Bungiro²,

Ibanez³

et al. 2004

Infect Immun

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“…[68] Less is known for another A. caninum ASP termed hookworm platelet inhibitor (HPI), which acts as an antagonistic ligand of cell surface integrins ( IIb b 3 and  2 b 1 ), resulting in the inhibition of the aggregation and adhesion of platelets. [69] To date, the interaction of Ac-NIF with complement receptor 3 constitutes the only experimentally proven interaction of an ASP with a human receptor. As a member of the complement system, complement receptor 3 (a heterodimeric complex consisting of  M -and b2-integrin) is a cell surface receptor on human leukocytes and macrophages that constitutes the innate immune system and helps to clear pathogens.…”

Section: Excreted/secreted Products: Scp/taps Proteinsmentioning

confidence: 99%

The Relevance of Structural Biology in Studying Molecules Involved in Parasite–Host Interactions: Potential for Designing New Interventions

et al. 2014

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1Mason et al. Structures of parasite proteinsNew interventions against infectious diseases require a detailed knowledge and understanding of pathogen-host interactions and pathogeneses at the molecular level. The combination of the considerable advances in systems biology research with methods to explore the structural biology of molecules is poised to provide new insights into these areas. Importantly, exploring threedimensional structures of proteins is central to understanding disease processes, and establishing structure-function relationships assists in identification and assessment of new drug and vaccine targets. Frequently, the molecular arsenal deployed by invading pathogens, and in particular parasites, reveals a common theme whereby families of proteins with conserved three-dimensional folds play crucial roles in infectious processes, but individual members of such families show high levels of specialisation which is often achieved through grafting particular structural features onto the shared overall fold. Accordingly, the applicability of predictive methodologies based on the primary structure of proteins or genome annotations is limited, particularly when thorough knowledge of molecular level mechanisms is required. Such instances exemplify the need for experimental threedimensional structures provided by protein crystallography, which remain an essential component of this area of research. In the present article, we review two examples of key protein families recently investigated in our laboratories, which could represent intervention targets in the metabolome or secretome of parasites. IntroductionInfectious diseases caused by eukaryotic pathogens represent a major disease burden to humans world-wide. In the absence of effective preventative approaches and new intervention strategies, this burden is likely to increase further, [1,2] compounded by food and water shortages, economic crises, wars and climate change, particularly in disadvantaged countries. In addition, there is evidence of increasing problems with resistance in pathogens against a broad range of chemotherapeutic agents, compromising the treatment of infectious diseases. [3] Moreover, in spite of major efforts, there are very few examples of success in developing new drugs and vaccines against eukaryotic pathogens, [4,5] indicating that alternative methods are needed at a time of major advances in molecular and computer technologies. In our opinion, the development of new treatments requires a detailed understanding of pathogens, host interactions and the molecular processes of disease. For instance, knowledge of the three-dimensional structures of proteins with pivotal roles in disease, and the probing of their underlying biology are crucial for understanding the pathogenesis. Through establishing the relevant structure-function relationships, one can elucidate how individual proteins function, so that new ways of disrupting relevant pathways can be found, in order to facilitate the identification of new drug and vaccine targets. ...

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“…Transfected cells were then selected by the addition of blasticidin (final concentration 25 μg/ml), and were subsequently established in a suspension culture of Drosophila Serum Free Media (Invitrogen). Recombinant protein expression was induced by the addition of CuSO 4 and the recombinant AceCTL-1 protein (rAceCTL-1) was purified using nickel resin affinity chromatography and reverse phase HPLC as previously described for hookworm secretory proteins [19,24,25]. The protein concentration was estimated using the BCA assay.…”

Section: Eukaryotic Expression Of Recombinant Proteinmentioning

confidence: 99%

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

Brown

Harrison

Kapulkin

et al. 2007

Molecular and Biochemical Parasitology

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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 Ancylostoma ceylanicum C-type Lectin-1 (AceCTL-1) cDNA corresponds to a 167 amino acid mature protein (18706 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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Isolation and molecular cloning of a secreted hookworm platelet inhibitor from adult Ancylostoma caninum

Cited by 92 publications

References 41 publications

Molecular Characterization ofAncylostoma ceylanicumKunitz-Type Serine Protease Inhibitor: Evidence for a Role in Hookworm-Associated Growth Delay

Molecular Characterization ofAncylostoma ceylanicumKunitz-Type Serine Protease Inhibitor: Evidence for a Role in Hookworm-Associated Growth Delay

The Relevance of Structural Biology in Studying Molecules Involved in Parasite–Host Interactions: Potential for Designing New Interventions

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

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