Abstract
Abstract: :
Purpose: The mannose-binding protein (MBP) of Acanthamoeba is thought to play a key role in the pathogenesis of Acanthamoeba keratitis by mediating the adhesion of parasites to host cells. The goal of the present study was to isolate, clone and characterize the amoeba MBP. Methods: The amoeba MBP was isolated by affinity chromatography and was analyzed by PAGE in native and denaturing conditions. The purified MBP was detected as a high molecular weight component (> 400-kD) in native conditions and, a ~130-kD component in denaturing/reducing conditions. Based on the amino acid sequence of the N-terminal and two internal peptides of the amoeba MBP, a PCR probe (585 bp) was generated and used to screen an A. Castellanii genomic library to clone and sequence the amoeba MBP gene. Results: These studies revealed that the amoeba MBP is composed of 5 introns and 6 exons that span 3.65 kb of the amoeba genome. The ORF encodes a protein of 833 amino acids. The analysis of the deduced amino acid sequence revealed that the amoeba MBP is a transmembrane protein constituted of a 712 amino acid long N-terminal extracellular domain (residues 22-733), a 22 amino acid long transmembrane domain (residues 734-755) and a short, 78 amino acid long, C-terminal intracellular domain. While the amino acid sequence of the amoeba MBP did not match with any known protein in the NCBI database, the analysis of its predicted secondary structure revealed similarities with E and P-selectins, fibrillin, and human α2-glycoprotein-I (involved in membrane adhesion). Moreover, in the amoeba MBP, a domain (residues 205-262) that matches the secondary structure of ConA-like lectins/glucanases superfamily was identified. Of interest, the extracellular domain of the amoeba MBP contained multiple CXCXC motifs. The cysteine-rich domain most probably confers protease resistance to the amoeba MBP. The calculated peptide weight of the amoeba MBP was 85-kD which is significantly less than the apparent molecular weight (130-kD) detected by SDS-PAGE, and may suggest extensive post-translational modification. Five targets with high probability of N-glycosylation and 12 targets for O-glycosylation were identified in the extracellular domain. Conclusions: Acanthamoeba-MBP is a novel protein constituted of 85-kD subunits that migrated with an apparent MW of 130-kD on electrophoresis gels, most probably due to post-translational modification. The characterization of the amoeba mannose receptor should lead to a better understanding of the pathogenic mechanisms of Acanthamoeba infections.
Keywords: Acanthamoeba • protein purification and characterization • keratitis