Pig xenogeneic antigen modification with green coffee bean α‐galactosidase

Abstract

Green coffee bean alpha-galactosidase can cleave the terminal alpha-galactose (alphaGal) on oligosaccharides that form the major antigen on pig endothelial cells recognized by primate-specific antibodies. Studies have been made of the conditions under which it is functional (e.g. temperature, pH) and of its biochemical and immunologic effects. Pig-to-rhesus monkey vein transplants were studied to identify the efficiency of the enzyme in delaying hyperacute rejection. When a graft became occluded, biopsies were taken for light microscopy (hematoxylin and eosin), scanning electron microscopy (SEM) and immunostaining with Griffonia simplicifolia IB4 lectin (GSIB4), and for IgM, IgG and C3. alpha-Galactosidase was stable for 72-96 h and was effective at 4 degrees C and pH 6.9 (conditions of human liver graft storage), although better function was obtained at 20 degrees C and pH 6.5. Using the porcine PK15 cell assay, the cytotoxicity of human serum was reduced after treatment of the pig cells with the enzyme. In vitro studies demonstrated that porcine veins treated with alpha-galactosidase lost endothelial expression of the Gal epitope within 30 min. SEM, however, demonstrated endothelial damage beginning within 2 h, probably caused by the alpha-galactosidase, as no damage was found in phosphate-buffered saline-treated veins, where the Gal epitope was preserved for >3 h. No change was found in either group on light microscopy. In vivo studies demonstrated that patency of the alpha-galactosidase-treated veins (mean 2.5 h) was longer than that of untreated veins (0.23 h) (P < 0.01). Biopsies showed no GSIB4 lectin staining for alpha-Gal epitopes and much less IgM and C3 deposition in the treated group. Light microscopy and SEM demonstrated more severe endothelial damage, hemorrhage, and fibrin formation in the untreated group. Galactosidase is effective in removing the terminal alphaGal and delays the onset of hyperacute rejection of pig veins transplanted into monkeys. However, its effect is temporary and, on its own, its use is unlikely to prolong survival of pig organs transplanted into primates sufficiently to be of clinical value.

Identification of a GH110 Subfamily of α1,3-Galactosidases

“…A number of investigators have in the past considered use of ␣-galactosidases for procurement of tissues for use in animal to human xenotransplantation to remove the immunodominant ␣3Gal residues found on many glycoconjugates in animals (34 -36). This approach is particularly attractive for tissues and tissue-derived acellular products with little or no ability to replace the glycoconjugates (36). Family GH110 offers ␣-galactosidases with improved characteristics, and the subfamily GH110b enzymes represented by BfGal110B may substantially enhance our ability to produce animal tissues for use in xenotransplantation.…”

In VivoEfficacy of Alpha-Galactosidase as Possible Promise for Prolonged Durability of Bioprosthetic Heart Valve Using Alpha1,3-Galactosyltransferase Knockout Mouse

“…In our previous study, 2,4,5,8 it was proved that enzymatic treatment using a-galactosidase is an effective method of removing a-Gal epitopes, which is easy to use and inexpensive. [13][14][15] Although a-Gal may reappear in the a-galactosidase treated organs after a certain time, 15 a-Gal would not reappear after enzymatic removal of a-Gal epitopes from cardiac valvular and pericardial tissues. So, enzymatic method was used to remove the a-Gal epitope which have caused a delay in the rejection.…”

“…Therefore, in addition to decellularization, we used enzymatic method using a-galactosidase to remove a-Gal epitopes efficiently from cardiac valvular and pericardial tissues in this study. 2,4,5,8,[13][14][15] To investigate the xenoresponse in immunological environment similar to human, our group 1 have inactivated the a1,3-galactosyltransferase gene in mice by gene targeting, and have generated mice that do not synthesize the Gal epitope. [16][17][18][19] Lee et al 1 proved that bovine pericardium implanted into the a-Gal KO mice caused significant increase in anti-a-Gal antibodies, and showed some histologic evidences of chronic rejection and that a-Gal KO mouse subcutaneous implantation model might be a feasible animal model for testing efficacy of anticalcification treatments incorporating immunologic approach, such as alpha-galactosidase.…”

A solid-phase screening method for identification of glycan-binding cells

“…The dried N-glycans were dissolved by addition of 1 mL of ultra pure water and desalted by a solid-phase extraction using a porous graphitic carbon (PGC) cartridge (Alltech, Associate, Deerfield, IL) [16]. Half of the pig N-glycans were treated with the green coffee bean α-galactosidase (Prozyme, San Leandro, CA) for the non-Gal epitopes [17]. .…”