S-Space College of Engineering/Engineering Practice School (공과대학/대학원) Dept. of Chemical and Biological Engineering (화학생물공학부) Theses (Ph.D. / Sc.D._화학생물공학부)
Mass spectrometry-based glycomic profiling and its applications to α1,3-galactosyltransferase gene-knockout pig in xenotransplantation
- 공과대학 화학생물공학부
- Issue Date
- 서울대학교 대학원
- Xenotransplantation; GalT-KO pig; N-linked glycan; NeuGc xenoantigen; Pig glycome database; Human immune cell; Mass spectrometry
- 학위논문 (박사)-- 서울대학교 대학원 : 화학생물공학부, 2014. 8. 김병기.
- Glycans are expressed on surface of mammalian cells and tissues, and play important roles in diverse biological functions including cell-cell interaction, signal transduction, metastasis, etc. In xenotransplantation, a galactose-alpha-1,3-galactose epitope, namely α-Gal, is the major carbohydrate xenoantigen that triggers hyperacute rejection (HAR) by the interaction of natural antibodies. Nevertheless, despite of production of α1,3-galactosyltransferase gene-knock out (GalT-KO) pigs as donors, there are still other antigens (e.g., non-Gal antigens) such as Hanganutziu-Deicher (H-D) antigen that induce acute vascular rejection (AVR) or delayed xenograft rejection (DXR) and subsequence xenograft failure.
Mass spectrometry (MS) is a powerful tool for identification and structural elucidation of diverse and complex oligosaccharides. In addition, MS-based quantitative technologies for the oligosaccharide analysis have been developed in recent years. More recently, the improvement of mass spectrometry-based glycomics techniques (i.e. highly sensitive, quantitative and high-throughput analytical tools) has enabled us to obtain a large dataset of glycans.
Cell-based glycomic analysis of WT and GalT-KO pig fibroblasts was performed by using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and MALDI- quadrupole ion trap (QIT)-TOF MS/MS. Total 47 N-glycans from WT and GalT-KO pig fibroblasts were identified and quantified. These results revealed that the level of overall sialylated glycans was higher in GalT-KO pig fibroblasts than WT, and relative quantity of N-glycolylneuraminic acid (NeuGc) antigen, which has been considered as one of non-Gal antigens, slightly increased in GalT-KO. The result proposed that the targeted knockout of α1,3-galactosyltransferase gene not only result in the removal of Galα1,3Gal residue but also influence sialylated glycan expression including NeuGc antigen.
In the case of the pig tissue, the integrated MS-based glycomic analysis of GalT-KO pig kidney was carried out by MALDI-TOF MS, MALDI-QIT-TOF MS/MS, and electrospray ionization (ESI)-MS and MS/MS. The N-glycome derived from GalT-KO pig kidney was identified and quantified, and the result allowed an in-depth comparison of the total N-glycomic profiles between wild-type (WT) and GalT-KO. MS analysis revealed that the relative abundance of sialic acid-containing glycans increased in GalT-KO. The current analysis also enabled a priori identification of N-glycolylneuraminic acid (NeuGc) regarded as one of non-Gal antigens in both WT and GalT-KO, and showed that the NeuGc level was higher in GalT-KO pig relative to WT.
In addition, based on these glycome profiles of pig cells and tissues, a pig glycome database was developed here. The database contains cell- or tissue-specific pig glycomes analyzed with mass spectrometry-based techniques, including the comprehensive pig glycan information on chemical structures, mass values, types and relative quantities. It was designed as a user-friendly web-based interface that allows users to query the database according to pig tissue/cell types or glycan masses. This database provides qualitative and quantitative information on glycomes characterized from various pig cells/organs in xenotransplantation.
Finally, the study on pig glycome-human immune cell interactions was performed by fluorescence-activated cell sorting (FACS) and LC-MS/MS analysis. The in-vitro experiment of human immune cell activation against pig glycans, and the screening and identification of their surface proteins using glycan-immobilized beads were carried out. The results showed no difference associated with human immune cells between WT and GalT-KO pig. However, the more complete characterization of the interaction between pig glycans and human immune cells remained to be improved for the xenotransplantation research.