Sd Antigen Research Articles

The study of variant s antigen expression revealing a novel c.160C>T (p.Arg54Cys) variant on GYPB*s allele associated with partial s phenotype.

Little s antigen is mainly defined by a single nucleotide polymorphism at c.143C (p.Thr48) on the GYPB gene. Several variants on GYPB can alter the expression of s antigen. The aim of this study was to investigate the molecular basis of variant s antigen expression in the Chinese population. A total of 4983 whole blood samples were collected to screen the individuals with discrepant s typing results using two different monoclonal anti-s. Then, the sequence of GYPB exon 4 was analyzed by Sanger sequencing. Flow cytometry analysis was performed to quantify s antigen expression on red blood cells (RBCs). In vitro expression study was performed to verify the effect of the GYPB variants identified on the expression of s antigen. Four donors were identified to have discrepant s typing results. Sanger sequencing showed that three donors carried the c.173C > G variant (p.Pro58Arg) specific for sD antigen, the other one carried a novel GYPB (c.160C > T, p.Arg54Cys) variant. Flow cytometry identified a partial and weak expression of s antigen on the RBCs of the four donors. Furthermore, in vitro expression study confirmed the effect of the two variants on the s antigen expression. The results demonstrated that in addition to p.Thr48, the two extra amino acids p.Arg54 and p.Pro58 are also important for full expression of s antigen. Since the individuals with partial s antigen are at risk for the development of alloanti-s, it is important to select at least two different monoclonal anti-s for correct s typing.

Enzymatic comparison and expression pattern of pig B4GALNT2 and B4GALNT2-like proteins

Abstract Objectives The final step in the production of the human Sd(a) antigen is catalyzed by beta-1,4-N-acetyl-galactosamine transferase 2 (B4GALNT2). This is done by adding a N-acetylgalactosamine residue via a beta-1,4 linkage to a subterminal galactose residue that has been substituted with an alpha-2,3-linked sialic acid. The final stage of the production of the Cad antigen is also catalyzed by B4GALNT2. Knocking out pig B4GALNT2 gene decreased human serum antibodies binding to pig cells, which greatly reduces the immunological rejection in clinical xenotransplantation trials. Interestingly, gene region LOC110255214 (hereafter named B4GALNT2-like) showed high similarity with the B4GALNT2 gene in the pig genome in our previous work, but whether B4GALNT2-like shares similar biological properties like B4GALNT2 remains to be elucidated, whether B4GALNT2-like is a potential immune gene in xenotransplantation remains to be determined. Methods In this study, we compared the tissue expression pattern of B4GALNT2-like and B4GALNT2 in Bama pigs. Results We found the expression of B4GALNT2-like was significantly higher in the duodenum, but lower in the heart, spleen, lung, kidney, comparing to B4GALNT2. Applied the Escherichia coli recombinant expression, we obtained 768 and 1,300 μg protein for B4GALNT2 and B4GALNT2-like from 1 L culture, respectively. Using the expressed recombinant proteins, the enzymatic activity of the two proteins was determined and compared. Conclusions The enzymatic assay showed that B4GALNT2-like has comparable catalytic activity with B4GALNT2 (58.7 % of B4GALNT2), addressing an important question whether B4GALNT2-like is a new immunological rejection gene.

Modifying the sugar icing on the transplantation cake.

As a transplant surgeon, my interest in glycobiology began through my research into ABO-incompatible allotransplantation, and grew when my goal became overcoming the shortage of organs from deceased human donors by the transplantation of pig organs into patients with terminal organ failure (xenotransplantation/cross-species transplantation). The major target for human "natural" (preformed) anti-pig antibodies is galactose-α(1,3)-galactose (the "Gal" epitope), which is expressed on many pig cells, including the vascular endothelium. The binding of human IgM and IgG antibodies to Gal antigens initiates the process of hyperacute rejection, resulting in destruction of the pig graft within minutes or hours. This major barrier has been overcome by the production of pigs in which the gene for the enzyme α(1,3)-galactosyltransferase (GT) has been deleted by genetic engineering, resulting in GT knockout (GTKO) pigs. The two other known carbohydrate antigenic targets on pig cells for human anti-pig antibodies are (i) the product of the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene, i.e., N-glycolylneuraminic acid, and (ii) the product of the β1,4 N-acetylgalactosaminyltransferase gene, i.e., the Sd(a) antigen. Expression of these two has also been deleted in pigs. These genetic manipulations, together with others directed to overcoming primate complement and coagulation activation (the latter of which also relates to glycobiology) have contributed to the prolongation of pig graft survival in nonhuman primate recipients to many months rather than a few minutes. Clinical trials of the transplantation of pig cells are already underway and transplantation of pig organs may be expected within the relatively near future.

Expression of the Human Sda -1,4-N-Acetylgalactosaminyltransferase II Gene is Dependent on the Promoter Methylation Status

It has been noted that the expression of Sd(a), including its antigenic structure, the beta-1,4-N-acetylgalactosyltransferase II (beta4GalNAcT-II) activity responsible for its formation, and the Sd(a) beta4GalNAcT-II mRNA transcript, is drastically reduced in oncogenetic processes in gastrointestinal tissues. Markedly reduced metastatic potential has been demonstrated in colon and gastric cancer cells transfected with the Sd(a) beta4GalNAcT-II gene. In this study, a putative CpG island encompassing the promoter and exon 1 regions in the human Sd(a) beta4GalNAcT-II gene was identified, and the investigation of DNA methylation of the Sd(a) gene promoter region demonstrated a clear association between the methylation status of the CpG island promoter and expression of the Sd(a) gene in gastrointestinal cancer cell lines. Hypomethylation of the promoter region of the Sd(a) gene was shown in cells where this gene was expressed. By contrast, there was significant hypermethylation of the Sd(a) gene promoter in cells that did not express the gene. A specific methylation profile in the Sd(a) gene CpG island was demonstrated in KATO III gastric cancer cells. In colon cancer cells with the hypermethylated Sd(a) gene promoter, treatment with the DNA methylation inhibitor, 5-aza-2'-deoxycytidine, resulted in demethylation of the promoter region and substantially induced the expression of the Sd(a) gene and the Sd(a) antigenic structure. These results strongly suggest that promoter DNA methylation plays a crucial role in the regulation of the Sd(a) beta4GalNAcT-II gene and Sd(a) antigen expression.

Biosynthesis and expression of the Sda and sialyl Lewis x antigens in normal and cancer colon

The carbohydrate determinants Sd(a) and sialyl Lewis x (sLex) both result from substitution of an alpha2,3-sialylated type 2 chain: the first with an N-acetylgalactosamine (GalNAc) beta1,4-linked to Gal and the second by an alpha1,3-linked fucose on N-acetylglucosamine. The Sd(a) antigen is synthesized by Sd(a) beta1,4-N-acetylgalactosaminyltransferase II (beta4GalNAcT-II), which is downregulated in colon cancer, whereas sLex is a cancer-associated antigen. In view of the possible competition between beta4GalNAcT-II and the fucosyltransferases (FucTs) synthesizing the sLex antigen, we investigated whether beta4GalNAcT-II acts as a negative regulator of sLex expression in colon cancer. beta4GalNAcT-II cDNA, when expressed in LS174T colon cancer cells, induces the expression of the Sd(a) antigen, a dramatic inhibition of sLex expression on cell membranes, and the replacement of sLex with the Sd(a) antigen on 290 kDa glycoproteins. Unexpectedly, in colorectal cancer specimens, beta4GalNAcT-II and sLex show a direct relation. The reasons appear to be (i) Sd(a) and sLex antigens are expressed by different glycoproteins of 340 and 290 kDa, respectively; (ii) the activity of alpha1,3-FucTs on 3'-sialyllactosamine parallels that of beta4GalNAcT-II; and (iii) both beta4GalNAcT-II and FucT activities parallel sLex expression. Quantitative reverse transcription-polymerase chain reaction analysis reveals that the transcripts of beta4GalNAcT-II and those of FucT-III and FucT-VII are positively correlated. These data indicate that in colon cancer tissues, the sLex antigen is regulated mainly by the total FucT activity on 3'-sialyllactosamine acceptors and that beta4GalNAcT-II can inhibit sLex expression in an experimental model, although not in colon cancer tissues.

Structural Analysis of Murine Zona Pellucida Glycans: EVIDENCE FOR THE EXPRESSION OF CORE 2-TYPE O-GLYCANS AND THE Sda ANTIGEN

Murine sperm initiate fertilization by binding to specific oligosaccharides linked to the zona pellucida, the specialized matrix coating the egg. Biophysical analyses have revealed the presence of both high mannose and complex-type N-glycans in murine zona pellucida. The predominant high mannose-type glycan had the composition Man(5)GlcNAc(2), but larger oligosaccharides of this type were also detected. Biantennary, triantennary, and tetraantennary complex-type N-glycans were found to be terminated with the following antennae: Galbeta1-4GlcNAc, NeuAcalpha2-3Galbeta1-4GlcNAc, NeuGcalpha2-3Galbeta1-4GlcNAc, the Sd(a) antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc, NeuGcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc), and terminal GlcNAc. Polylactosamine-type sequence was also detected on a subset of the antennae. Analysis of the O-glycans indicated that the majority were core 2-type (Galbeta1-4GlcNAcbeta1-6[Galbeta1-3]GalNAc). The beta1-6-linked branches attached to these O-glycans were terminated with the same sequences as the N-glycans, except for terminal GlcNAc. Glycans bearing Galbeta1-4GlcNAcbeta1-6 branches have previously been suggested to mediate initial murine gamete binding. Oligosaccharides terminated with GalNAcbeta1-4Gal have been implicated in the secondary binding interaction that occurs following the acrosome reaction. The significant implications of these observations are discussed.

QUANTITATIVE STUDIES ON THE TISSUE DISTRIBUTION OF Ia AND SD ANTIGENS IN THE DA AND LEWIS RAT STRAINS

The tissue distribution of major histocompatibility complex (MHC) antigens in the DA and Lewis (LEW) strains was studied using LEW anti-DA and DA anti-LEW alloantisera. Quantitative absorption analyses were used with quantitative binding assays for SD and Ia antigens. Initial screening showed that the LEW anti-DA serum contained significant amounts of antibodies against both Ia and SD antigens. On the other hand, the DA anti-LEW serum seemed to be directed almost entirely against Ia antigens, and it was not possible to set up assays for SD antigens in the LEW strain. The most surprising finding was the presence of large amounts of Ia antigen on the kidneys of both the DA and LEW strains, one kidney containing as much Ia antigen as half a spleen. Kidney also contained large amounts of SD antigen. Liver had large amounts of SD, but very little Ia. Heart had only small amounts of both SD and Ia. The relevance of these findings to transplantation of the kidney, liver, and heart are discussed. The other tissues studied were brain, spleen, lymph node, thoracic duct lymphocytes, bone marrow, thymus, RBC, and platelets. The most interesting findings were the presence of relatively large amounts of SD antigens on DA RBC, and small amounts of Ia on the thymus and bone marrow.

Genetic basis of memory in cell-mediated immune response.

The present experiments were undertaken to delineate the role of LD and SD antigens in terms of memory in cell-mediated immune response (CML). Using recombinants that differ for either the H-2K or H-2D determinants (SD-different) and other recombinants differing for the central region of H-2 (LD-different), we have investigated this question. The results indicate that priming to SD determinants leads to a more rapid and higher response when cells are restimulated in vitro with either the same SD antigen alone or the same SD plus an LD antigen and tested on target cells that possess either the SD antigen alone or both the LD and SD antigen. Antigens in the central regions of H-2, which includes LD, serve as the stimulus both for the helper T cells and for the cytotoxic T lymphocytes. Priming with a high dose of LD antigens leads to memory in the cytotoxic T lymphocyte recognizing the cytotoxic target antigens coded by the central region of H-2. Nevertheless, memory could not be detected when priming was done under the same conditions as for SD antigens. Moreover, a primed helper T-cell response does not facilitate the development of increased CML on restimulation in vitro with the LD antigen used for priming and a new SD antigen. Thus, it seems that memory in CML is in the cytotoxic T lymphocyte and not in the proliferating helper cells.

LD antigens associated with HL-A8 and myasthenia gravis.

The finding that the SD antigen HL-A8 is associated with myasthenia gravis has raised the question as to whether or not there is also an association between some specific LD antigen and myasthenia gravis and/or HL-A8. MLC technique was used to study LD antigens in 33 myasthenic patients. The cells of a myasthenic patient who was homozygous for both HL-A and LD products were used in MLC tests as stimulators with HL-A8 bearing cells from 24 myasthenics, their 17 relatives and 16 controls and to non-HL-A8 cells from nine myasthenics and 16 controls. At least three different LD genes were found to be associated with HL-A8. One of them, called LDm' was present in 17 (63 %) of the 27 HL-A8-bearing haplotypes of myasthenics, and in nine (47 %) of the 19 HL-A8-bearing haplotypes of controls. In our study LDm was not found in the 84 haplotypes devoid of HL-A8. LDm is strongly associated with HL-A8 and through this also with myasthenia. Calculated from phenotype frequencies for HL-A8 and its association to LDm' the LDm frequencies are 9 % in the control population, 30 % in myasthenics and 48 % in young females with onset of myasthenia below 35 years. LDm had no indendent correlation with any of the clinical parameters of myasthenia gravis, even though is was found more often in HL-A8+ females with the onset of myasthenia before 35 years than in the whole myasthenic group. LDm appears to be similar to LD8a antigen.