MOLECULAR CLONING OF A MEMBER OF THE FASCIOLA HEPATICA SAPOSIN-LIKE PROTEIN FAMILY

• Published in: The Journal of parasitology 2003-06, Vol.89 (3), p.545-552
• Main Authors: Espino, Ana M, Hillyer, George V
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; Amino acids; animal proteins; Animals; antigen-antibody reactions; Antigens; antiserum; Biochemistry. Physiology. Immunology. Molecular biology; Biological and medical sciences; Blotting, Northern; Blotting, Western; Cloning; Cloning, Molecular; Complementary DNA; cytolysis; DNA; DNA, Complementary - chemistry; DNA, Helminth - chemistry; E coli; Enzyme-Linked Immunosorbent Assay; Erythrocytes; Erythrocytes - parasitology; Fasciola hepatica; Fasciola hepatica - genetics; Fasciola hepatica - immunology; Fasciola hepatica - metabolism; Fascioliasis; Fascioliasis - parasitology; Fundamental and applied biological sciences. Psychology; Gene expression; Glycoproteins - chemistry; Glycoproteins - genetics; Glycoproteins - immunology; Helminth Proteins - chemistry; Helminth Proteins - genetics; Helminth Proteins - immunology; Homology; Humans; Hydrophobicity; Immune Sera - immunology; Immunization; Infections; Invertebrates; Leukocytes (mononuclear); Leukocytes, Mononuclear - parasitology; Liver; liver flukes; Lysis; Mammals; Messenger RNA; Molecular Sequence Data; MOLECULAR-CELL BIOLOGY; monocytes; Nemathelminthia. Plathelmintha; nucleotide sequences; Parasites; Parasitology; Peptides; Peripheral blood mononuclear cells; Physiology. Development; Polypeptides; Protein families; Proteins; Proteolipids - chemistry; Proteolipids - genetics; Proteolipids - immunology; Pulmonary Surfactants - chemistry; Pulmonary Surfactants - immunology; Rabbits; recombinant fusion proteins; Residues; RNA, Helminth - genetics; RNA, Messenger - genetics; RNA, Messenger - metabolism; Saposins; Sequence Alignment; sequence homology; Sequence Homology, Amino Acid; Sphingolipid Activator Proteins
• ISSN: 0022-3395; 1937-2345
• DOI: 10.1645/GE-3113

A 436-bp complementary DNA (cDNA) was isolated from an adult Fasciola hepatica cDNA expression library by screening with the serum from a rabbit infected with F. hepatica for 4 wk. The deduced amino acid sequence encoded by this cDNA is an 11.5-kDa polypeptide that has significant homology to F. hepatica NK-lysin protein, to several members of saposin-like or NK-lysin protein families, as well as 3 amoebapore precursors of Entamoeba histolytica. The most striking feature observed within this protein, denoted FhSAP-2, is the presence of 6 conserved cysteine residues arranged within 5 amphipathic α-helical domains and the presence of 7 hydrophobic residues in strictly conserved positions. Using enzyme-linked immunosorbent assay it was found that rFhSAP-2 is highly reactive with sera from rabbits infected with F. hepatica for 2–14 wk as well as with sera from humans with chronic fascioliasis. An anti-rFhSAP-2 rabbit antiserum reacted with F. hepatica excretory–secretory antigens by Western blot, revealing a major 11.5-kDa and 2 minor 46- and 67-kDa antigenic polypeptides. This suggests that FhSAP-2 may be an antigen released from cytoplasmic storage granules present within F. hepatica parasites. rFhSAP-2 also exhibits a strong lytic activity on human erythrocytes and peripheral blood mononuclear cells. This suggests that cell lysis could be 1 of the biological functions of this protein.