Identification of a Novel Giardia lamblia Cyst Wall Protein with Leucine-rich Repeats

Abstract

Giardia lamblia trophozoites, like most intestinal parasitic protozoa, undergo fundamental biological changes to survive outside the intestine of their mammalian host by differentiating into infective cysts. This complex process entails the coordinated production, processing, and transport of cyst wall constituents for assembly into a protective cyst wall. Yet, little is known about this process and the identity of cyst wall constituents. We previously identified a 26-kDa cyst wall protein, CWP1. In the present work, using monoclonal antibodies to cyst wall antigens, we cloned the gene that encodes a novel 39-kDa cyst wall protein, CWP2. Expression of CWP1 and CWP2 was induced during encystation with identical kinetics. Soon after synthesis, these two proteins combine to form a stable complex, which is concentrated within the encystation-specific secretory granules before incorporation into the cyst wall. Both proteins contain five tandem copies of a 24-residue leucine-rich repeat, a motif implicated in protein-protein interactions. Unlike CWP1, CWP2 has an extremely basic 121-residue COOH-terminal extension that might be involved in the sorting of these proteins to the secretory granules.

A Novel WRKY-like Protein Involved in Transcriptional Activation of Cyst Wall Protein Genes in Giardia lamblia

“…2A). The products of the cwp1 and ran genes are the component of the cyst wall and the ras-related nuclear protein (6,18). To determine the expression of WRKY protein, we generated an antibody specific to the full-length WRKY.…”

“…Unusually short 5Ј-flanking regions (Ͻ65 bp) with no consensus TATA boxes or other cis-acting elements identified in late branching eukaryotic promoters are sufficient for the expression of some genes (6,7,9,(17)(18)(19)(20). Instead, ATrich sequences have been found around the transcription start sites of many genes, functionally similar to the initiator element in late branching eukaryotes (6,7,9,(17)(18)(19)(20). They are essential for promoter activity and play a predominant role in determining the positions of the transcription start sites (17)(18)(19).…”

Enquistación in vitro de Giardia lamblia: análisis por electroforesis bidimensional de proteínas expresadas diferencialmente.

“…Los genes CWP1 y CWP2 predicen proteínas de naturaleza ácida con masas moleculares de 26 y 39 kDa, respectivamente; tienen una identidad en la secuencia de aminoácidos del 61% en el fragmento de 26 kDa que tienen en común, pero la CWP2 tiene una extensión C-terminal de 121 residuos. Es rica en aminoácidos básicos, lo que hace que esta proteína tenga un pl calculado de 12 (4). Los resultados exhibidos en este estudio muestran que la CWP1 y la CWP2 tienen una masa aproximada de ~60 kDa con pI de ~4,8 y >7,0, respectiva-mente.…”

“…Durante la formación del quiste, los trofozoítos sufren cambios morfológicos y bioquímicos importantes: 1) recepción de los estímulos para la enquistación y la consecuente activación de genes específicos; 2) biogénesis de organelos secretorios y síntesis, empaque, transporte y liberación de materiales para la pared del quiste, y 3) ensamblaje de la pared (4).…”

Proteomic analysis of Giardia: Studies from the pre- and post-genomic era

“…Studies have involved the encystation specific vesicles (ESVs) (Luján, et al, 1995, Stefanic, et al, 2006, the ribosomes (Shirakura, et al, 2001) and the 20S proteasome (Emmerlich, et al, 1999). The ESVs of G. duodenalis trophozoites are produced after induction of encystation and contain the cyst wall proteins (CWP1, CWP2) as well as other proteins (Luján, et al, 1995).…”