Molecular cloning of an alpha-enolase from the human filarial parasite Onchocerca volvulus that binds human plasminogen.
Enolase represents a multifunctional protein involved in basic energy metabolism and plasminogen binding and activation at the surface of prokaryotic pathogens. A complete cDNA of 1615 bp of an alpha-enolase from Onchocerca volvulus (Ov-ENO) was isolated using a PCR-based approach. The open reading frame encoded for 435 amino acids and the high degree of conservation included the crucial amino acid residues that participate in the formation of the catalytic site, Mg(2+) binding site, and a hydrophobic motif reported to relate to surface expression. A 1089-bp fragment was expressed in a N-terminal 6 x His-tag expression vector in Escherichia coli. By immunohistological analysis using anti-Ov-ENO rabbit antibodies, native enolase could be detected in most tissues of adult O. volvulus, microfilariae, and infective larvae. Intense staining was observed in the muscles, where the energy consumption is high. The purified recombinant protein fragment revealed plasminogen binding activity in a blot-overlay assay employing anti-plasminogen antibodies. In sera from individuals infected with O. volvulus, IgG antibodies reactive with recombinant Ov-ENO were demonstrated by immunoblot and enzyme-linked immunosorbent analyses. The plasminogen-binding property of O. volvulus alpha-enolase may support plasmin-mediated proteolysis including degradation of host's extracellular matrix thereby promoting the migration of larval stages through tissues. The recognition by antibodies in sera of O. volvulus-infected persons indicate an involvement of the protein in the interaction between the parasite and the human host.
Jolodar
Abbas
A
Tropical Medicine Section, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str. 74, 20359 Hamburg, Germany.
Fischer
Peter
P
Bergmann
Simone
S
Büttner
Dietrich W
DW
Hammerschmidt
Sven
S
Brattig
Norbert W
NW
D000595:Amino Acid Sequence; D000818:Animals; D000871:Anthelmintics / Q000627:therapeutic use; D003001:Cloning, Molecular; D003429:Cross Reactions; D004625:Embryo, Nonmammalian / Q000201:enzymology; D005260:Female; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007106:Immune Sera; D007559:Ivermectin / Q000627:therapeutic use; D007814:Larva; D008297:Male; D008969:Molecular Sequence Data; D017181:Onchocerca volvulus / Q000196:embryology / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D009855:Onchocerciasis / Q000188:drug therapy / Q000469:parasitology; D010751:Phosphopyruvate Hydratase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D010958:Plasminogen / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000871:Anthelmintics; D015801:Helminth Proteins; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; D007559:Ivermectin; D010958:Plasminogen; D010751:Phosphopyruvate Hydratase; C476563:alpha-enolase, Onchocerca volvulus