Alpha 1,3-galactosyltransferase deficiency in pigs increases sialyltransferase activities that potentially raise non-gal xenoantigenicity

Abstract

We examined whether deficiency of the GGTA1 gene in pigs altered the expression of several glycosyltransferase genes. Real-time RT-PCR and glycosyltransferase activity showed that 2 sialyltransferases [α2,3-sialyltransferase (α2,3ST) and α2,6-sialyltransferase (α2,6ST)] in the heterozygote GalT KO liver have higher expression levels and activities compared to controls. Enzyme-linked lectin assays indicated that there were also more sialic acid-containing glycoconjugate epitopes in GalT KO livers than in controls. The elevated level of sialic-acid-containing glycoconjugate epitopes was due to the low level of α-Gal in heterozygote GalT KO livers. Furthermore, proteomics analysis showed that heterozygote GalT KO pigs had a higher expression of NAD+-isocitrate dehydrogenase (IDH), which is related to the CMP-N-acetylneuraminic acid hydroxylase (CMAH) enzyme reaction. These findings suggest the deficiency of GGTA1 gene in pigs results in increased production of N-glycolylneuraminic acid (Neu5Gc) due to an increase of α2,6-sialyltransferase and a CMAH cofactor, NAD+-IDH. This indicates that Neu5Gc may be a critical xenoantigen. The deletion of the CMAH gene in the GalT KO background is expected to further prolong xenograft survival.

Figure 1

Expression of sialyltransferases in control and heterozygote α1,3-galactosyltransferase gene (GGTA1) knockout (GalT KO) liver. (a) Electrophoretic analysis of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in control and heterozygote GalT KO liver. (b) Quantification of real-time RT-PCR analysis in control and heterozygote GalT KO liver. All RT-PCR reactions were conducted in triplicate and normalized with pig H2A histone family, member Z gene (pH2AFZ). Each of the GalT KO relative values is presented as an n-fold expression difference compared to the control, which was set as 1. *P < 0.05, ***P < 0.001.

Figure 2

Comparison of α1,3-galactosyltransferase (GalT), α2,3- and α2,6-sialyltransferase (α2,3ST and α2,6ST) activity between control and heterozygote GalT KO liver. From left, GalT, α2,3ST, and α2,6ST activity. Each value is the mean ± standard deviation (SD) of triplicate determinations. ***P < 0.001.

Figure 3

Expression of full-length CMAH and variants in control and heterozygote α1,3-galactosyltransferase gene (GGTA1) knockout (GalT KO) liver. (a) Electrophoretic analysis of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in control and heterozygote GalT KO liver. (b) Quantification of real-time RT-PCR analysis in control and heterozygote GalT KO liver. All RT-PCR reactions were conducted in triplicate and normalized for pig H2A histone family, member Z gene (pH2AFZ). Each of the GalT KO relative values is presented as an n-fold expression difference compared to the control, which was set as 1. ***P < 0.001.

Figure 4

2-D gel electrophoresis analysis of control and heterozygote GalT KO liver. Numbered spots were selected due to variations between the gels or overexpression in heterozygote GalT KO liver. Quantitative data were obtained from triplicate experiments. Identified spot numbers correspond to those in Table 3.