ABSTRACT
BACKGROUND: Cells of the clinically important p histo-blood group phenotype lack P1, P(k) , and P glycosphingolipid antigens. All cases investigated so far are due to alterations in the 4-α-galactosyltransferase-encoding Exon 3 of A4GALT. Repetitive elements in the genome can mediate DNA rearrangements, the most abundant being the Alu family of repeats. STUDY DESIGN AND METHODS: The aim of this study was to determine the genetic basis of three p samples with intact A4GALT open reading frames, using long-range polymerase chain reaction (PCR) and sequencing. In addition, transcript measurements were performed with quantitative PCR. RESULTS: This is the first report of the p phenotype as the result of large deletions in A4GALT, comprising the proposed promoter and noncoding Exons 1 and 2a. The breakpoints were different in all three samples and revealed the presence of Alu or MIRb sequences directly flanking, or in close proximity to, all junctions. Furthermore, no A4GALT transcripts could be detected. CONCLUSION: In summary, our data elucidate a new explanation underlying the p phenotype, implicating the deleted regions of A4GALT as crucial for P1 and P(k) synthesis, possibly due to loss of binding sites for erythroid transcription factors. Furthermore, analysis of these regions will improve genetic blood group prediction.
Subject(s)
Galactosyltransferases/genetics , Gene Deletion , Globosides/deficiency , Regulatory Sequences, Nucleic Acid/genetics , Trihexosylceramides/deficiency , Alleles , Base Sequence , Blood Group Antigens/genetics , Globosides/genetics , Humans , Molecular Sequence Data , Phenotype , Trihexosylceramides/geneticsABSTRACT
Recognition of endogenous lipid Ag(s) on CD1d is required for the development of invariant NKT (iNKT) cells. Isoglobotrihexosylceramide (iGb3) has been implicated as this endogenous selecting ligand and recently suggested to control overstimulation and deletion of iNKT cells in α-galactosidase A-deficient (αGalA(-/-)) mice (human Fabry disease), which accumulate isoglobosides and globosides. However, the presence and function of iGb3 in murine thymus remained controversial. In this study, we generate a globotrihexosylceramide (Gb3)-synthase-deficient (Gb3S(-/-)) mouse and show that in thymi of αGalA(-/-)/Gb3S(-/-) double-knockout mice, which store isoglobosides but no globosides, minute amounts of iGb3 can be detected by HPLC. Furthermore, we demonstrate that iGb3 deficiency does not only fail to impact selection of iNKT cells, in terms of frequency and absolute numbers, but also does not alter the distribution of the TCR CDR 3 of iNKT cells. Analyzing multiple gene-targeted mouse strains, we demonstrate that globoside, rather than iGb3, storage is the major cause for reduced iNKT cell frequencies and defective Ag presentation in αGalA(-/-) mice. Finally, we show that correction of globoside storage in αGalA(-/-) mice by crossing them with Gb3S(-/-) normalizes iNKT cell frequencies and dendritic cell (DC) function. We conclude that, although detectable in murine thymus in αGalA(-/-)/Gb3S(-/-) mice, iGb3 does not influence either the development of iNKT cells or their interaction with peripheral DCs. Moreover, in αGalA(-/-) mice, it is the Gb3 storage that is responsible for the decreased iNKT cell numbers and impeded Ag presentation on DCs.
Subject(s)
Cell Differentiation/immunology , Dendritic Cells/immunology , Globosides/physiology , Natural Killer T-Cells/immunology , Trihexosylceramides , Animals , Carbohydrate Sequence , Dendritic Cells/enzymology , Dendritic Cells/metabolism , Globosides/deficiency , Liver/cytology , Liver/enzymology , Liver/metabolism , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Molecular Sequence Data , Natural Killer T-Cells/enzymology , Natural Killer T-Cells/metabolism , Spleen/cytology , Spleen/enzymology , Spleen/metabolism , Thymus Gland/cytology , Thymus Gland/enzymology , Thymus Gland/metabolism , Trihexosylceramides/deficiency , Trihexosylceramides/physiology , alpha-Galactosidase/genetics , alpha-Galactosidase/physiologyABSTRACT
CD1d-restricted natural killer T (NKT) cells, expressing the invariant T cell antigen receptor (TCR) chain encoded by Valpha14-Jalpha18 gene segments in mice and Valpha24-Jalpha18 in humans [invariant NKT (iNKT) cells], contribute to immunoregulatory processes, such as tolerance, host defense, and tumor surveillance. iNKT cells are positively selected in the thymus by CD1d molecules expressed by CD4(+)/CD8(+) cortical thymocytes. However, the identity of the endogenous lipid(s) responsible for positive selection of iNKT cells remains unclear. One candidate lipid proposed to play a role in positive selection is isoglobotrihexosylceramide (iGb3). However, no direct evidence for its physiological role has been provided. Therefore, to directly investigate the role of iGb3 in iNKT cell selection, we have generated mice deficient in iGb3 synthase [iGb3S, also known as alpha1-3galactosyltransferase 2 (A3galt2)]. These mice developed, grew, and reproduced normally and exhibited no overt behavioral abnormalities. Consistent with the notion that iGb3 is synthesized only by iGb3S, lack of iGb3 in the dorsal root ganglia of iGb3S-deficient mice (iGb3S(-/-)), as compared with iGb3S(+/-) mice, was confirmed. iGb3S(-/-) mice showed normal numbers of iNKT cells in the thymus, spleen, and liver with selected TCR Vbeta chains identical to controls. Upon administration of alpha-galactosylceramide, activation of iNKT and dendritic cells was similar in iGb3S(-/-) and iGb3S(+/-) mice, as measured by up-regulation of CD69 as well as intracellular IL-4 and IFN-gamma in iNKT cells, up-regulation of CD86 on dendritic cells, and rise in serum concentrations of IL-4, IL-6, IL-10, IL-12p70, IFN-gamma, TNF-alpha, and Ccl2/MCP-1. Our results strongly suggest that iGb3 is unlikely to be an endogenous CD1d lipid ligand determining thymic iNKT selection.
