Entamoeba histolytica : a novel cysteine protease and an adhesin form the 112 kDa surface protein.
Here, we present evidence that a cysteine protease (EhCP112) and a protein with an adherence domain (EhADH112) form the Entamoeba histolytica 112 kDa adhesin. Immunoelectron microscopy and immunofluorescence assays using monoclonal antibodies (mAbAdh) revealed that, during phagocytosis, the adhesin is translocated from the plasma membrane to phagocytic vacuoles. mAbAdh inhibited 54% adherence, 41% phagocytosis, and 35% and 62% destruction of MDCK cell monolayers by live trophozoites and their extracts respectively. We cloned a 3587 bp DNA fragment (Eh112 ) with two open reading frames (ORFs) separated by a 188 bp non-coding region. The ORF at the 5' end (Ehcp112 ) encodes a protein with a cysteine protease active site, a transmembranal segment and an RGD motif. The second ORF (Ehadh112 ) encodes a protein recognized by mAbAdh with three putative transmembranal segments and four glycosylation sites. Northern blot, primer extension and Southern blot experiments revealed that Ehcp112 and Ehadh112 are two adjacent genes in DNA. Ehcp112 and Ehadh112 genes were expressed in bacteria. The recombinant peptides presented protease activity and inhibited adherence and phagocytosis, respectively, and both were recognized by mAbAdh. The EhCP112 and EhADH112 peptides could be joined by covalent or strong electrostatic forces, which are not broken during phagocytosis.
Centro de Investigación en Ciencia Aplicada y Tecnología Avanzada del Instituto Politécnico Nacional (CICATA-IPN), Legaria 694, Col. Irrigación, México, DF 11500.
G
G
García-Rivera
M A
MA
Rodríguez
R
R
Ocádiz
M C
MC
Martínez-López
R
R
Arroyo
A
A
González-Robles
E
E
Orozco
D018829:Adhesins, Bacterial; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001426:Bacterial Proteins; D002448:Cell Adhesion; D002460:Cell Line; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics*; D004285:Dogs; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D037102:Lectins; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics*; D008565:Membrane Proteins / Q000737:chemistry; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology; D010587:Phagocytosis; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D016415:Sequence Alignment
D018829:Adhesins, Bacterial; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; C122005:EhADH112 protein, Entamoeba histolytica; D037102:Lectins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D015800:Protozoan Proteins; D012333:RNA, Messenger; C052452:galactose inhibitable adherence protein, Entamoeba histolytica; D003546:Cysteine Endopeptidases; C122004:EhCP112 protein, Entamoeba histolytica