title: Molecular and functional characterization of an Entamoeba histolytica protein (EhMLCI) with features of a myosin essential light chain.
abstract: Entamoeba histolytica, a protozoan parasite of humans, relays on its striking motility to survive and invade host tissues. Characterization of the molecular components involved in motile processes is crucial to understand its pathogenicity. Although protein components of myosin II hexamers have been predicted from E. histolytica genome data, only a heavy chain of myosin, EhmhcA, has been characterized so far. We have cloned an E. histolytica cDNA sequence that best matched Dictyostelium discoideum myosin essential light chain and found that the cloned sequence is transcribed as an mRNA of 0.445 kb which could encode a protein of 16.88 kDa, within the predicted range for a myosin light chain. In silico analyses revealed that the protein sequence, named EhMLCI, shows two consensus domains for binding MHC, but lacks the N-terminal sequence for actin binding, as in A2 type myosin essential light chains. A single EF-hand calcium-binding domain was identified in the C-terminus and several high score predictability sites for serine and tyrosine phosphorylation. Antibodies to recombinant EhMLCI identified two proteins of approximately 17 and 15 kDa in trophozoite extracts, the latter phophorylated in tyrosines. Serine phosphorylation was not detected. Immunomicroscopy revealed EhMLCI cortical and cytoplasmic distribution in trophozoites and true colocalization with EhmhcA determined by PCC. Co-immunoprecipitation corroborated EhMLCI interaction with EhmhcA. EhMLCI was also localized in actomyosin-containing complexes. Differential partition of phospho-tyrosinated EhMLCI into cell fractions containing the soluble form of EhmhcA and its lack of serine phosphorylation suggest its possible participation in a novel down regulatory mechanism of myosin II activity in E. histolytica.
authors.affiliation: Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Apartado 14-740, México DF, 07360, Mexico. imeza@cinvestav.mx
authors.forename: Isaura
authors.initials: I
authors.lastname: Meza
authors.forename: J Daniel
authors.initials: JD
authors.lastname: Díaz-Valencia
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Franco
authors.forename: Nicolás
authors.initials: N
authors.lastname: Villegas-Sepúlveda
authors.forename: Ruth A
authors.initials: RA
authors.lastname: Lezama
authors.forename: Gloria
authors.initials: G
authors.lastname: Benítez-King
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004748:Entamoeba histolytica / Q000235:genetics / Q000502:physiology*; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018994:Myosin Light Chains / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D018076:DNA, Complementary; D018994:Myosin Light Chains; D015800:Protozoan Proteins