The xenograft antigen in complex with GS-1-B4 lectin: crystallization and preliminary X-ray analysis

Abstract

The implantation of animal organs is one approach to overcoming the shortage of human donor organs for medical transplantation. Although readily available, non-primate tissues are subject to hyperacute rejection wherein human anti-Galalpha(1-3)Gal antibodies react with haptens present on the transplanted cells' surfaces. The understanding of this interaction on a molecular level will further the development of a strategy for the prevention of hyperacute rejection in xenotransplantation. The Galalpha(1-3)Gal hapten ('xenograft antigen') has been cocrystallized with the Gal-specific B(4) isolectin of Griffonia simplicifolia lectin-1. Crystals were analyzed by cryocrystallography and were found to diffract to moderately high resolution on a rotating-anode X-ray source. They belong to the P2(1)2(1)2 space group, with unit-cell parameters a = 111.0, b = 51.3, c = 76.9 A, and contain two molecules per asymmetric unit.

Figure 1

Typical 1° oscillation image obtained during data collection. In the zoomed image (right), a reflection at 2.65Å and I/σ of 2.4 is marked by the cross hair. I/σ for this reflection is also illustrated by the intensity profiles at the lower and right-hand side margins of the images.

Figure 2

For the purpose of this study, the xenograft antigen was presented as the β-methyl glycoside.

Figure 3

Crystal of GS-B₄ grown in the presence of Galα(1–3)Galβ-OMe.

Figure 4

Representation of crystal packing: the Cα trace of PDB file 1gsl is placed in the unit cell according to the structure solution by molecular replacement. For each of the four asymmetric units shown, subunit 1 is drawn in yellow, while subunit 2 can be seen in blue. For clarity, the cell origin was shifted along y by one half of the unit-cell edge. The contents of the two asymmetric units in the center of the unit cell constitute the biologically active tetramer. This image was generated using the program RasMol (Bernstein, 2001).

Figure 5

The images depict a region of the asymmetric unit where specific binding of a carbohydrate ligand by the lectin is expected to occur by homology with the GS-4 complex in 1gsl. In (a) a 2F_o – F_c difference map is shown at 1.3σ (blue); in (b) a F_o – F_c map at 2.7σ can be seen in red for the same region. Ligand coordinates were not included for the map calculation but are shown here for clarity as a ball-and-stick model. The numbering of peptide side chains conforms to that used in 1gsl. The images were generated using the program O (Jones et al., 1991).