12E7 Antigen Research Articles

Investigation of the biochemical relationship between the blood group antigens Xga and CD99 (12E7 antigen) on red cells.

The biochemical relationship between the red cell antigens Xga and the MIC2 gene product, CD99--previously designated the 12E7 antigen--has been examined by immunoblotting and immunoprecipitation analyses of the protein molecules bearing these antigens. Immunoblotting of membrane components and Xga-immunoprecipitates with anti-Xga has shown that Xga antigen is carried on a broad band of apparent molecular weight (Mr)) 24,500-29,500, which consists of a dark stained component at M(r)24,500 and a more diffusely stained component at approximately M(r) 26,500-29,500. Immunoblotting of membrane components and 12E7-immunoprecipitates with 12E7, and RFB-1 and NaM123 which also recognise CD99, distinguished two bands of M(r) 30,000 and 32,000. A non-radioactive immunoprecipitation technique was employed, which uses chemiluminescence detection of biotin-labelled red cell proteins. The protein of M(r) 32,000, which carries CD99, was identified by this method and the red cell quantitative polymorphism of CD99 was demonstrated. When the Xga protein was precipitated from biotin-labelled red cells, a protein of M(r) 32,000 was coprecipitated. This suggests that the proteins carrying the Xga antigen and CD99 are associated in the membrane.

What is important on the red blood cell surface.

Blood group antigens have provided tools for investigation of the red cell surface and been very useful as genetic markers in family, population and forensic studies. Precise definition of phenotypes is very important. Application of MAIEA (monoclonal antibody-specific immobilisation of erythrocyte antigen), a recently reported technique, to identify antigens and to assign red cell antigens to a particular membrane component is described: location of Knops system antigens on CR1 is confirmed and provisional assignment of Cromer system antigens to the different short consensus regions of decay accelerating factor (DAF) is described. Variability of red cell antigen expression is considered. The possibility is discussed that factors other than alterations in Rh genes may be responsible for some Rh variant phenotypes. Some C variants, two of which are associated with low incidence antigens, are described. The relationship of Xga with the quantitative polymorphism of 12E7 antigen is reconsidered in light of some recent immunochemical studies.

Investigation of the Biochemical Relationship between the Blood Group Antigens Xg^a and CD99 (12E7 Antigen) on Red Cells

The biochemical relationship between the red cell antigens Xg^a and the MIC2 gene product, CD99 - previously designated the 12E7 antigen - has been examined by immunoblotting and immunoprecipitation analyses of the protein molecules bearing these antigens. Immunoblotting of membrane components and Xg^a-immunoprecipitates with anti-Xg^a has shown that Xg^a antigen is carried on a broad band of apparent molecular weight (M(r)) 24,500-29,500, which consists of a darkly stained component at M(1) 24,500 and a more diffusely stained component at approximately M(r) 26,500-29,500. Immunoblotting of membrane components and 12E7-immunoprecipitates with 12E7, and RFB-l and NaM123 which also recognise CD99, distinguished two bands of M(1) 30,000 and 32,000. A non-radioactive immunoprecipitation technique was employed, which uses chemiluminescence detection of biotin-labelled red cell proteins. The protein of M(1) 32,000, which carries CD99, was identified by this method and the red cell quantitative polymorphism of CD99 was demonstrated. When the Xg^a protein was precipitated from biotin-labelled red cells, a protein of M(r) 32,000 was coprecipitated. This suggests that the proteins carrying the Xg^a antigen and CD99 are associated in the membrane.

PBDX is the XG blood group gene.

We have identified the Xga antigen, encoded by the XG blood group gene, by employing rabbit polyclonal and mouse monoclonal antibodies raised against a peptide derived from the N-terminal domain of a candidate gene, referred to earlier as PBDX. In indirect haemagglutination assays, these anti-peptide antibodies react with Xg(a+) but not Xg(a-) erythrocytes. In antibody-specific immobilization of antigen (ASIA) and immunoblot assays, the anti-peptide antibodies react with the same molecule as does human anti-Xga. Therefore, by its identity with PBDX, Xga is identified as a cell-surface protein that is 48% homologous to CD99 (previously designated the 12E7 antigen), the product of MIC2 which is tightly linked to XG. PBDX is renamed here XG.

Regulator Genes Affecting Red Cell Antigens

Several mechanisms may be involved to explain the action of genes that regulate the expression of red cell antigens. When carbohydrate antigens are involved, lack of an enzyme in the biochemical pathway prevents formation of the precursor for the next and following steps of that path, or, alternatively, addition of an extra sugar to the immuno-dominant sugar may produce a new structure in which the expression of the expected antigen is masked. Thinking of genetic rather than biochemical interference, a regulator gene may "switch-off" the action of a structural gene, and this mechanism could involve the upset of repressor and/or derepressor genes. The mechanisms for the regulator genes described in this article are unknown. The effect of XGR is limited to red cells: the expression of 12E7 antigen on other tissues and cells, other than red cells, is invariable. The reported effects of XOr and XQ are for red cells, but it is unlikely that other cells and tissues have been studied intensively; propositi with these regulator genes are much rarer than people informative for XGR and In(Lu). The effects of In(Lu) are not limited to red cells but have been shown to regulate the expression of p80 on some white cells. Most of the abnormalities in Rhnull cells appear to be associated with the lack of the Rh antigens and lack of Rh proteins. The hypothesis of a functional complex involving Rh, lack of which affects incorporation of apparently unrelated proteins into the red cell membrane, is an attractive idea. Studies of the similar phenotype, Rhmod, suggest that some Rh specificities can be present in cells that appear to be as abnormal, serologically and morphologically, as Rhnull cells. Perhaps some polypeptides are functionally more important than others and perhaps all polypeptides required for the functional efficiency of the Rh complex have not yet been identified. Lack of Lutheran antigens is not always accompanied by modification of other red cell antigens. As suggested by Telen and green, if In(Lu) acts via a single mechanism, then that mechanism differs from that of XS2. Certainly the mechanisms of In(Lu) and XS2 differ in their action on the expression of CD44 or p80 antigens. The red cell surface is well charted territory, familiar to serologists, immunologists, biochemists, and geneticists. It still provides an excellent model for study of cell surface antigens and for the regulator genes described above that modify expression of some red cell antigens.(ABSTRACT TRUNCATED AT 400 WORDS)

MIC2: a human pseudoautosomal gene.

MIC2 and XGR are the only known pseudoautosomal genes in man. MIC2 encodes the 12E7 antigen, a human cell-surface molecule of unknown function. XGR regulates, in cis, the expression of the XG and MIC2 genes. DNA probes derived from the MIC2 locus have been used in the construction of a meiotic map of the pseudoautosomal region and a long range restriction map into the X- and Y-specific chromosome domains. MIC2 is the most proximal marker in the pseudoautosomal region and recombination between the sex chromsomes only rarely includes the MIC2 locus. Our long-range restriction maps and chromosome walking experiments have localized the pseudoautosomal boundary within 40 kilobases adjacent to the 3' end of the MIC2 gene. The same maps have been used to predict the chromosomal location of TDF.

Recombination between the X and Y chromosomes: implications for the relationship between MIC2, XG and YG.

Three loci affect the levels of expression of the 12E7 antigen on red blood cells: MIC2, the pseudoautosomal structural gene for the 12E7 antigen; XG, an X-linked red cell blood group locus and YG, a polymorphic Y-linked locus. In this report we describe recombination between XG and MIC2 and an exchange between the X and Y chromosomes which included YG. These results have prompted us to propose a new model describing the genetic relationship between the XGa and 12E7 antigens.

Pseudoautosomal genes in man.

MIC2, which encodes the 12E7 antigen, is the only well-defined pseudoautosomal gene in man. We have isolated cDNA and genomic sequences corresponding to MIC2 and have produced monoclonal antibodies reacting with the 12E7 antigen. These molecular tools have been used to investigate the genetics and biochemistry of the MIC2 system. Recent results suggest that MIC2 is the most proximal of the currently defined pseudoautosomal markers and that the escape of MIC2 from X-inactivation may be intrinsic to an associated HTF island found at the 5' end of the gene. Investigation of the inter-relationship between MIC2 and the XG locus has led us to postulate the existence of a second pseudoautosomal gene in man.

Genetic analysis of the human Y chromosome by chromosome-mediated gene transfer.

Chromosome-mediated gene transfer (CMGT) can be used to segregate fragments of human chromosomes in human-rodent hybrid cells. As with all somatic cell genetics methods, a selection technique is needed to isolate the hybrid cell lines produced by CMGT. Expression of the MIC2 gene product on the cell surface (the 12E7 antigen) provides an endogenous selectable marker for the human Y chromosome. Using chromosome transfer followed by separation of 12E7 antigen-positive cells on the fluorescence-activated cell sorter, a series of cell lines containing segregated fragments of the Y chromosome have been derived. The possibility of using these fragments to derive fine structural mapping data for the Y chromosome is considered in this review.

Cloning an expressed gene shared by the human sex chromosomes.

The existence of genes shared by mammalian sex chromosomes has been predicted on both evolutionary and functional grounds. However, the only experimental evidence for such genes in humans is the cell-surface antigen encoded by loci on the X and Y chromosomes (MIC2X and MIC2Y, respectively), which is recognized by the monoclonal antibody 12E7. Using the bacteriophage lambda gt11 expression system in Escherichia coli and immunoscreening techniques, we have isolated a cDNA clone whose primary product is recognized by 12E7. Southern blot analysis using somatic cell hybrids containing only the human X or Y chromosomes shows that the sequences reacting with the cDNA clone are localized to the sex chromosomes. In addition, the clone hybridizes to DNAs isolated from mouse cells that have been transfected with human DNA and selected for 12E7 expression on the fluorescence-activated cell sorter. We conclude that the cDNA clone encodes the 12E7 antigen, which is the primary product of the MIC2 loci. The clone was used to explore sequence homology between MIC2X and MIC2Y; these loci are closely related, if not identical.

EXPRESSION OF Xg AND THE 12E7 ANTIGEN IN PRIMATES

Tests on cells from various non-human primates showed that expression of the 12E7 antigen is the same on cultured fibroblasts, peripheral blood lymphocytes and red blood cells. No quantitative polymorphism of 12E7 expression was observed in any of the animals tested. Tests on red blood cells of these primates confirm that anti-Xga and the monoclonal antibody 12E7 define different determinants.

Expression of human neuronal antigens in a mouse neuroblastoma x human dorsal root ganglion cell hybrid.

Clonal mouse neuroblastoma cells were fused with cells from human foetal dorsal root ganglia and several continuously-growing hybrid clones isolated. One hybrid cell line (F2.1D1) containing a number of human chromosomes, was shown to retain the ability to extend neurites in response to dibutyryl cyclic AMP and to express various antigens characteristic of human foetal dorsal root ganglion neurons. The X-chromosome-controlled 12E7 antigen, human Thy-1 and the neuron-specific F12.A2B5 antigen were identified as surface components of the hybrid cells. None of these antigens were detected in the parental neuroblastoma cell line. In addition, using a species-specific monoclonal antibody, the hybrid cells were shown to synthesize human neurofilament protein. This is the first demonstration of the continued expression of a human species- and neuron-specific gene product in a human-mouse somatic cell hybrid.

Expression of the 12E7 antigen is controlled independently by genes on the human X and Y chromosomes.

The existence of sequence homology between the mammalian sex chromosomes has been proposed to explain the pairing of the X and Y chromosomes at meiosis [7, 26, 34] and is believed to reflect the evolutionary origin of these chromosomes [24]. Consideration of patients with sex chromosome abnormalities has also led to the suggestion that the X and Y chromosomes might share some sequences [3, 9]. However, direct evidence to support this hypothesis did not appear until the advent of the techniques of molecular biology. At least three sets of DNA sequences have been shown to be shared between X and Y chromosomes. Phillips et al. [28] have described the presence of retrovirus-related sequences integrated into the mouse X and Y chromosomes as well as the autosomes. In man sequences related to the argininosuccinate synthetase gene have also been found associated with autosomes and X and Y chromosomes [6]. Most recently, a third set of sequences have been described which are restricted to the human X and Y chromosomes and are absent from the autosomes [25]. No evidence has been presented to suggest that any of these X and Y chromosome-shared sequences are functional coding sequences.

A human quantitative polymorphism related to Xg blood groups.

A monoclonal antibody, 12E7, raised against lymphocytes from a patient with a T-cell acute lymphocytic leukaemia reacts strongly with cortical thymocytes and, to a lesser extent, with other human cells. The antigen defined by 12E7 is not expressed on mouse or hamster cells; this species specificity allowed us to investigate the genetics of the expression of the 12E7 antigen using human--rodent somatic-cell hybrids. Hybrid cells which contain the human X chromosome as the only human genetic contribution react with 12E7. The X-linkage of the gene, or genes, controlling 12E7 antigen expression and the fact that 12E7 antigen is also expressed on red blood cells led us to investigate the possibility of a relationship with the X-linked Xg blood group system. Although the 12E7 and Xga antigens seem to be different antigenic determinants specified by different loci, we report here a sex-limited effect involving the Xg phenotype on the expression of 12E7 antigen.