title: Biophysical studies on calcium and carbohydrate binding to carbohydrate recognition domain of Gal/GalNAc lectin from Entamoeba histolytica: insights into host cell adhesion.
abstract: Entamoeba histolytica, an enteric parasite expresses a Gal/GalNAc-specific lectin that contributes to its virulence by establishing adhesion to host cell. In this study, carbohydrate recognition domain of Hgl (EhCRD) was purified and biophysical studies were conducted to understand the thermodynamic basis of its binding to carbohydrate and Ca(++) Here, we show that carbohydrate recognition domain (CRD) of the lectin binds to calcium through DPN motif. To decipher the role of calcium in carbohydrate binding and host cell adhesion, biophysical and cell-based studies were carried out. We demonstrated that the presence of the cation neither change the affinity of the lectin for carbohydrates nor alters its conformation. Mutation of the calcium-binding motif in EhCRD resulted in complete loss of ability to bind calcium but retained its affinity for carbohydrates. Purified EhCRD significantly diminished adhesion of the amebic trophozoites to Chinese Hamster Ovary (CHO) cells as well as triggered red blood cell agglutination. The calcium-binding defective mutant abrogated amebic adhesion to CHO cells similar to the wild-type protein, but it failed to agglutinate RBCs suggesting a differential role of the cation in these two processes. This study provides the first molecular description of the role of calcium in Gal/GalNAc mediated host cell adhesion.
authors.affiliation: Department of Biological Sciences.
authors.forename: Rupali
authors.initials: R
authors.lastname: Yadav
authors.affiliation: Department of Biological Sciences.
authors.forename: Kuldeep
authors.initials: K
authors.lastname: Verma
authors.affiliation: Department of Biological Sciences.
authors.forename: Mintu
authors.initials: M
authors.lastname: Chandra
authors.affiliation: Department of Chemistry, Indian Institute of Science Education & Research, Bhopal, Bhopal, 462066, India.
authors.forename: Madhumita
authors.initials: M
authors.lastname: Mukherjee
authors.affiliation: Department of Biological Sciences sunando@iiserb.ac.in.
authors.forename: Sunando
authors.initials: S
authors.lastname: Datta
mesh_terms: D000371:Agglutination; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004748:Entamoeba histolytica / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004912:Erythrocytes / Q000737:chemistry; D037161:Galectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000072417:Protein Domains; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D037161:Galectins; D015800:Protozoan Proteins
keywords: CRD; Entamoeba histolytica; Gal; GalNAc lectin; Host cell adhesion; thermodynamics