Mass spectrometric analysis of the glycosphingolipid‐derived glycans from miniature pig endothelial cells and islets: identification of NeuGc epitope in pig islets

Abstract

Glycosphingolipid (GSL) is a major component of the plasma membrane in eukaryotic cells that is involved directly in a variety of immunological events via cell-to-cell or cell-to-protein interactions. In this study, qualitative and quantitative analyses of GSL-derived glycans on endothelial cells and islets from a miniature pig were performed and their glycosylation patterns were compared. A total of 60 and 47 sialylated and neutral GSL-derived glycans from the endothelial cells and islets, respectively, were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and collision-induced fragmentation using positive-ion electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS). In accordance with previous immunohistochemistry studies, the alpha-Gal-terminated GSL was not detected but NeuGc-terminated GSLs were newly detected from miniature pig islets. In addition, the neutral GSL-derived glycans were relatively quantified by derivatization with carboxymethyl trimethylammonium hydrazide (so called Girard's T reagent) and MALDI-TOF MS. The structural information of the GSL-derived glycans from pig endothelial cells and islets suggests that special attention should be paid to all types of glycoconjugates expressed on pig tissues or cells for successful clinical xenotransplantation.

Human antibody reactivity against xenogeneic N‐glycolylneuraminic acid and galactose‐α‐1,3‐galactose antigen

“…There are considerable differences in the expression of Neu5Gc across pig cells and tissues . It is clear that Neu5Gc is highly expressed on the surface of endothelial cells and pancreatic islet cells .…”

Differential Expression Patterns of Gangliosides in the Liver and Heart of NIH-miniature Pigs

“…Kim et al [12] investigated several glycan-binding proteins that were involved in xenograft rejection. Additionally, Kim et al [10,11] showed that 60 and 47 glycosphingolipids derived glycans and N-glycans were present in miniature pig endothelial cells and islets. In summary, expression of various gangliosides in the liver and heart of NIH-miniature pigs can activate both the innate and adaptive immune response; therefore, the targeting of these gangliosides will be an attractive avenue in the development of techniques to control graft rejection.…”

The sweets standing at the borderline between allo‐ and xenotransplantation

“…Kim et al. reported comprehensive porcine glycomes from various sources using mass spectrometry . The study included the N‐glycome from a specific pathogen‐free Chicago Medical School miniature porcine kidney as a model of porcine endothelial cells.…”

“…Kim et al. identified 62 glycolipid‐derived glycans from porcine endothelial cells, and 48 glycans were detected from porcine islet cells . A potential α‐Gal neolactose sequence (Hex 4 HexNAc 1 ) was quantified by mass spectrometry in both cells (Table S4).…”

“…Such databases could be used to predict possible glycan structures of specific cells from the expression profile of GT genes . However, a porcine glycome database has not been established, despite many porcine glycan structures from various samples having been qualitatively and quantitatively investigated . The creation of an interactive porcine glycome database would greatly aid the development of xenotransplantation.…”