Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Altered glycosylation of genetically engineered pigs for successful xenotransfusion using LC-MS/MS approach (#756)

Myung Jin Oh 1 2 , Nari Seo 1 2 , Jinyoung Park 1 2 , Jaekyoung Ko 1 2 , Jjin-dan Kang 3 , Xi-jun Yin 3 , Hyun Joo An 1 2
  1. Chungnam National University, Yuseong-gu,, DAEJEON, South Korea
  2. Asia Glycomics Reference Site, Chungnam National University, Daejeon, Korea
  3. Department of Animal Sciences College of Agriculture, Yanbian University, Yanji City Jilin Pro, China

Xenotransfusion of pig blood has been considered as one of potential solutions to the frequent shortage of human blood. One key bottleneck to xenograft is allogenic immune rejection by human antibodies caused by species-specific glycosylation features. Thus, glycosylation directly related to the immune system has been recognized as a gatekeeper for successful xenotransplantation. The major target antigens of human natural antibodies and complements are non-human glycan moieties containing terminal galactose-a-1-3- galactose (alpha-Gal) and N-glycolyl neuraminic acid (NeuGc), respectively. They have been genetically modified to overcome rejection mechanism and incompatibility in xenotransfusion. In order to investigate whether glycosylation can be altered by destruction of the CMAH and GGTA genes, N‐glycans released from RBC and plasma of WT and double KO pigs (n=8, respectively) were examined by PGC LC/MS and MS/MS approach. Species-specific glycans which are linked to target genotype containing specific glycan moieties such as sialic acids, linkage isomers, and alpha-Gal epitope were preferentially characterized. In particular, NeuGc vs NeuAc-sialylated glycan ratio of plasma and RBC of wild pigs are 1:10 and 1:40, respectively. NeuGc glycan was not detected in KO plasma, but less than 1% in KO RBC. The unique structure of alpha-Gal epitope was confirmed by the simultaneous loss of galactose and HexNAc from the terminal as well as the absence of diagnostic ion indicating the hybrid type glycan in MS/MS spectrum. Alpha-Gal epitopes were discovered with significant quantities (> 10% of total glycan) in WT pigs. Although GGTA gene was destructed in KO pigs, the relative amount of alpha-Gal still remained about 3%. Here, for the first time, we characterized structure- and linkage-specific glycans found in RBC and plasma of WT and genetically modified pigs, respectively.