Physical,structural,and functional properties of the b1 integrin-like fibronectin receptor ( b1EhFNR) in Entamoeba histolytica Krishanu Sengupta a,1 , Vero ´nica Ivonne Herna ´ndez-Ramı ´rez a , Jose ´ Luis Rosales-Encina a , Ricardo Mondrago ´n b , Olga Lilia Garibay-Cerdenares a , Donaciano Flores-Robles a,2 , Rosario Javier-Reyna a , Silvana Pertuz a , Patricia Talama ´s-Rohana a, * a Departamento de Infecto ´ mica y Patoge ´ nesis Molecular, Centro de Investigacio ´ n y de Estudios Avanzados del IPN, Me ´ xico,DF,Mexico b Departamento de Bioquı ´mica,Centro de Investigacio ´n y de Estudios Avanzados del IPN, Me´xico,DF,Mexico 1. Introduction During amebiasis, Entamoeba histolytica celladhesion and motility are key features required for it to achieve progression across tissues. The pathogenicity of E. histolytica includesits capacity to adhere,destroy and phagocytose human cells and degrade extracellular matrix components. These processes depend on the actin cytoskeleton and actin-associated proteins ( Voit and Guille´n, 1999). Integrins,a family of glycosylated,heterodimeric transmem- brane adhesion receptors, are involved in a wide range of cellular processes thatrequire cytoskeletalremodeling and cell shape changes (van der Flier and Sonnenberg, 2001).These transmem- brane molecules are composed of non-covalently bound a and b subunits that bind to a variety of extracellular matrix (ECM) proteins and act as signaling receptors (Harburger and Calder- wood, 2009). Both vertebrate and invertebrate integrins show conserved structural and functional features (Johnson et al., 2009). Following ligand binding, integrins cluster into different types of adhesion structures such as focal complexes,focal adhesions, fibrillar adhesions and podosomes (Wiesner et al., 2005).Each of these adhesion structures contains many different structural actin- associated proteins (such as a-actinin, vinculin, tensin, and paxillin) that link the integrin to the cytoskeleton.Integrin subunits can undergo extensive post-translational modifications (phosphorylation,alternative splicing, proteolytic cleavage of cytoplasmic tails) that affect their function; glycosylation of the extracellular domains of a and b subunits is also likely to regulate Infection, Genetics and Evolution 9 (2009) 962–970 A R T I C L E I N F O Article history: Received 14 February 2009 Received in revised form 20 June 2009 Accepted 24 June 2009 Available online 2 July 2009 Keywords: Cell adhesion molecule Entamoeba histolytica Endo/exocitosis Fibronectin receptor Hydrodynamic properties Integrins Phagocytosis A B S T R A C T The presence in Entamoeba histolytica of a fibronectin (FN) receptor, which is antigenically related to b1 integrin-like molecules and shows 99% homology with the intermediate subunit-2 of the Gal/GalNAc- specific lectin has been described. The E. histolytica genome has been sequenced, and its analysis shows no integrin sequences. Here we provide further evidence to demonstrate that this molecule behaves as integrin-like in its physical, structural and functional properties.The purified b1EhFNR complex is resolved into three polypeptides of 150, 140,and 130 kDa.Transmission electron microscopy showed individual complexes consisting of oblong heads of 3 nm 4 nm and two projecting arms 6–7 nm in length. In the absence of detergent, these complexes formed aggregates that were composed of clusters or ‘‘rosettes’’ of between two and six or more b1EhFNR complexes. The physical properties of the purified b1EhFNR complexes were: R S = 5.8 nm, S 20 W = 8.3, f/f 0 = 1.4.This complex was seen in close physical association with adhesion plates and phagocytic invaginations, using confocal microscopy and the 3C10 mAb that recognizes these three subunits complex. Regulation of its surface expression is not dependent on protein synthesis; rather it is regulated by inward and outward mobilization of the molecules. The presence and antigenic similarity of putative b1EhFNRs in different strains and species of Entamoeba was analyzed using the 3C10 mAb; this mAb recognized the complex in all E. histolytica species, however there was no recognition in E. dispar, E. invadens, and Laredo strains. Finally, evidence is provided about post-translationalmodifications such as tyrosine phosphorylation and glycosylation suffered by the b1EhFNR complex. ß 2009 Elsevier B.V. All rights reserved. * Corresponding author at: CINVESTAV-IPN, Ave. IPN No. 2508,Col. San Pedro Zacatenco, Del. G.A. Madero, Me ´xico 07360, DF, Mexico. Tel.: +52 5747 3800x3351; fax: +52 5747 3377. E-mail address: ptr@cinvestav.mx (P. Talama ´s-Rohana). 1 Present address: Cellular & Molecular Biology Division, Laila Impex R&D Center, Unit 1 Phase III Jawahar Autonagar, Vijayawada 520007, Andhra Pradesh, India. 2 Present address: Unidad de Investigacio ´n especializada en Microbiologı ´a, Universidad Auto ´noma de Guerrero, Chilpancingo,Gro., Mexico. Contents lists available at ScienceDirect Infection,Genetics and Evolution j o u r n a l h o m e p a g e: w w w . e l s e v i e r . c o m / l o c a t e / m e e g i d 1567-1348/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2009.06.020