Babesia divergens: cloning and biochemical characterization of Bd37

Abstract

The immunoprotective potential of Babesia divergens antigens released in supernatants of in vitro cultures of the parasite is generally known. Among a number of parasite molecules, a 37 kDa protein has been found in the supernatants of Babesia divergens cultures. In this report the cloning and biochemical characterization of this protein, called Bd37, are described. In addition, the processing of the protein was studied in vitro. Results suggest that Bd37 is encoded by a single copy gene. Bd37 appears to be a merozoite-associated molecule attached to the surface by a glycosylphosphatidylinositol moiety containing a palmitate residue attached to the inositol ring. In addition, it is demonstrated that both extremities of the protein are linked by a disulphide bond. Results further indicate that a soluble, hydrophilic form of Bd37 can be released from the merozoite surface by GPI-specific phospholipase D. The potential role the Bd37 protein and the GPI anchor are discussed.

Molecular cloning and genetic polymorphism of Babesia capreoli gene Bcp37/41, an ortholog of Babesia divergens merozoite surface antigen Bd37

“…Bd37 is an immunodominant antigen of 37 kDa, that was first detected by immunoblot with sera from B. divergensinfected cattle and humans (Gorenflot et al, 1991) and able to elicit a protective response against challenge (Carcy et al, 1995;Delbecq et al, 2002Delbecq et al, , 2006Précigout et al, 2004;Hadj-Kaddour et al, 2007). The cell surface localization of this antigen was attested by fluorescence microscopy with a Bd37 specific antiserum (Carcy et al, 1995), together with the GPI anchoring of this protein to the merozoite cell surface (Delbecq et al, 2002).…”

“…The cell surface localization of this antigen was attested by fluorescence microscopy with a Bd37 specific antiserum (Carcy et al, 1995), together with the GPI anchoring of this protein to the merozoite cell surface (Delbecq et al, 2002). Since the purified protein is able to bind to erythrocytes, albeit non-specifically, its role in erythrocyte invasion has been postulated (Delbecq et al, 2008).…”

The Solution Structure of the Adhesion Protein Bd37 from Babesia divergens Reveals Structural Homology with Eukaryotic Proteins Involved in Membrane Trafficking

“…The same 13 C or 15 N-edited NOESY were also used to extract NOE restraints. In addition, a [ 1 H, 13 C] NOESY-HSQC 3D experiment recorded on the selectively labeled [ 13 CH 3 ]Δ-Bd37 sample appears especially helpful to identify NOE connectivities arising from methyl groups. Similarly, the analysis of the aromatic region of the spectrum was considerably facilitated by the analysis of 2D NOESY (and TOCSY) experiments recorded on a Bruker Avance 900 MHz spectrometer in an unlabeled sample dissolved in 2 H 2 O.…”

“…Frequently they contain unique domains like SAG1 (Toxoplasma), merozoite surface protein 1 (MSP-1) (Plasmodium), 11 variable merozoite surface antigen (VMSA) (Babesia bovis) 12 and Bd37 (B. divergens). 13 Whatever the Apicomplexa, the role of GPIanchored proteins in the early step of cell invasion involves their exposition to the extracellular environnement. 14 Such localisation during the extracellular step of the parasite life's cycle place the GPIanchored proteins as antigens potentially recognised by antibodies or other immune effectors.…”

“…17 The characterization of this protein has revealed the presence of an N-terminal secretion signal and C-terminal hydrophobic GPI-anchoring signal, as well as the presence of a disulfide bond linking together the N and C-terminal parts of the protein. 13 The Bd37 protein, when used as a recombinant vaccine, is able to elicit a protective response. 18 Moreover, the analysis of the Bd37 gene in different parasite isolates has revealed a variability of some parts of the molecule as well as conserved regions ( Figure 1).…”