Immunoproteomic identification of bovine pericardium xenoantigens

Abstract

Bovine pericardium is an important biomaterial with current application in glutaraldehyde-fixed bioprosthetic heart valves and possible future application as an unfixed biological scaffold for tissue engineering. The importance of both humoral and cell-mediated rejection responses toward fixed and unfixed xenogeneic tissues has become increasingly apparent. However, the full scope and specific identities of bovine pericardium proteins that can elicit an immune response remain largely unknown. In this study, an immunoproteomic approach was used to survey bovine pericardium proteins for their ability to elicit a humoral immune response in rabbits. A two-stage protein extraction protocol was used to separate bovine pericardium proteins into water- and lipid-soluble fractions. Two-dimensional (2-D) gel electrophoresis was performed to separate the proteins from each fraction. Western blots were generated from 2-D gels of both bovine pericardium protein fractions. These blots were probed with serum from rabbits immunized with bovine pericardium and a secondary antibody was used to assess for IgG positivity. Western blots were compared to duplicate 2-D gels and proteins in matched spots were identified by tandem mass spectrometry. Thirty-one putative protein antigens were identified, eight of which are known to be antigenic from previous studies. All of the putative antigens demonstrated progressive staining intensity with increasing days of post-exposure serum. Identified antigenic proteins represented a variety of functional and structural protein types, and included both cellular and matrix proteins. The results of this study have implications for the use of bovine pericardium as a biomaterial in bioprostheses and tissue engineering applications, as well as xenotransplantation in general.

Fig. 1

Workflow scheme for immunoproteomic identification of bovine pericardium xenoantigens.

Fig. 2

One-dimensional western blots of water-soluble (A) and lipid-soluble (B) bovine pericardium protein fractions treated with pre-immune (0-d) and 21-, 42-, and 72-d post-exposure anti-bovine pericardium rabbit serum.

Fig. 3

Two-dimensional gel of bovine pericardium water-soluble fraction proteins identified as antigens. Superscript numbers correspond to spot numbers in Table 1.

Fig. 4

Two-dimensional gel of bovine pericardium lipid-soluble fraction proteins identified as antigens. Superscript numbers correspond to spot numbers in Table 2.

Fig. 5

Sequential 2-D western blot spots of 4 bovine pericardium (BP) putative protein antigens resolved with pre-immune (0-d) and 21-, 56-, and 72-d post-exposure anti-BP rabbit serum.