Genetic knockout of porcine GGTA1 or CMAH/GGTA1 is associated with the emergence of neo-glycans.

BACKGROUND: Pig-derived tissues could overcome the shortage of human donor organs in transplantation. However, the glycans with terminal α-Gal and Neu5Gc, which are synthesized by enzymes, encoded by the genes GGTA1 and CMAH, are known to play a major role in immunogenicity of porcine tissue, ultimately leading to xenograft rejection. METHODS: The N-glycome and glycosphingolipidome of native and decellularized porcine pericardia from wildtype (WT), GGTA1-KO and GGTA1/CMAH-KO pigs were analyzed by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection. RESULTS: We identified biantennary and core-fucosylated N-glycans terminating with immunogenic α-Gal- and α-Gal-/Neu5Gc-epitopes on pericardium of WT pigs that were absent in GGTA1 and GGTA1/CMAH-KO pigs, respectively. Levels of N-glycans terminating with galactose bound in ß(1-4)-linkage to N-acetylglucosamine and their derivatives elongated by Neu5Ac were increased in both KO groups. N-glycans capped with Neu5Gc were increased in GGTA1-KO pigs compared to WT, but were not detected in GGTA1/CMAH-KO pigs. Similarly, the ganglioside Neu5Gc-GM3 was found in WT and GGTA1-KO but not in GGTA1/CMAH-KO pigs. The applied detergent based decellularization efficiently removed GSL glycans. CONCLUSION: Genetic deletion of GGTA1 or GGTA1/CMAH removes specific epitopes providing a more human-like glycosylation pattern, but at the same time changes distribution and levels of other porcine glycans that are potentially immunogenic.

Generation of glycans depleted decellularized porcine pericardium, using digestive enzymatic supplements and enzymatic mixtures for food industry.

BACKGROUND: Xenogeneic pericardium has been used largely for various applications in cardiovascular surgery. Nevertheless, xenogeneic pericardial patches fail mainly due to their antigenic components. The xenoantigens identified as playing a major role in recipient immune response are the Galα1-3Gal (α-Gal) epitope, the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc), and the porcine SDa antigen, associated with both proteins and lipids. The reduction in glycans from porcine pericardium might hinder or reduce the immunogenicity of xenogeneic scaffolds. METHODS: Decellularized porcine pericardia were further treated at different time points and dilutions with digestive enzymatic supplements and enzymatic mixtures applied for food industry, for the removal of potentially immunogenic carbohydrates. Carbohydrates removal was investigated using up to 8 different lectin stains for the identification of N- and O-glycosylations, as well as glycolipids. Histoarchitectural changes in the ECM were assessed using Elastica van Gieson stain, whereas changes in mechanical properties were investigated via uniaxial tensile test and burst pressure test. RESULTS: Tissues after enzymatic treatments showed a dramatic decrease in lectin stainings in comparison to tissues which were only decellularized. Histological assessment revealed cell-nuclei removal after decellularization. Some of the enzymatic treatments induced elastic lamellae disruption. Tissue strength decreased after enzymatic treatment; however, treated tissues showed values of burst pressure higher than physiological transvalvular pressures. CONCLUSIONS: The application of these enzymatic treatments for tissue deglycosylation is totally novel, low cost, and appears to be very efficient for glycan removal. The immunogenic potential of treated tissues will be further investigated in subsequent studies, in vitro and in vivo.