RESUMEN

Despite its importance in the regulation of growth and differentiation processes of a variety of organisms, the mechanism of synthesis and degradation of cAMP (cyclic AMP) has not yet been described in Giardia lamblia In this work, we measured significant quantities of cAMP in trophozoites of G. lamblia incubated in vitro and later detected how it increases during the first hours of encystation, and how it then returns to basal levels at 24 h. Through an analysis of the genome of G. lamblia, we found sequences of three putative enzymes - one phosphodiesterase (gPDE) and two nucleotidyl cyclases (gNC1 and gNC2) - that should be responsible for the regulation of cAMP in G. lamblia Later, an RT-PCR assay confirmed that these three genes are expressed in trophozoites. The bioinformatic analysis indicated that gPDE is a transmembrane protein of 154 kDa, with a single catalytic domain in the C-terminal end; gNC1 is predicted to be a transmembrane protein of 74 kDa, with only one class III cyclase homology domain (CHD) at the C-terminal end; and gNC2 should be a transmembrane protein of 246 kDa, with two class III CHDs. Finally, we cloned and enriched the catalytic domain of gNC1 (gNC1cd) from bacteria. After that, we confirmed that gNC1cd has adenylyl cyclase (AC) activity. This enzymatic activity depends on the presence of Mn2+ and Ca2+, but no significant activity was displayed in the presence of Mg2+ Additionally, the AC activity of gNC1cd is competitively inhibited with GTP, so it is highly possible that gNC1 has guanylyl cyclase activity as well.

Asunto(s)

Adenilil Ciclasas/química , AMP Cíclico/química , Giardia lamblia/enzimología , Guanilato Ciclasa/química , Hidrolasas Diéster Fosfóricas/química , Proteínas Protozoarias/química , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Secuencia de Aminoácidos , Calcio/química , Calcio/metabolismo , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , AMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Giardia lamblia/genética , Giardia lamblia/crecimiento & desarrollo , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Cinética , Manganeso/química , Manganeso/metabolismo , Modelos Moleculares , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología Estructural de Proteína , Especificidad por Sustrato , Trofozoítos/enzimología , Trofozoítos/genética , Trofozoítos/crecimiento & desarrollo