Organization and expression of thirteen alternatively spliced exons in catfish CD45 homologs

CD45, also known as LCA, is a transmembrane protein tyrosine phosphatase encoded by the PTPRC gene. In mammals, it plays an important role in T and B cell receptor and cytokine signaling by maintaining receptor associated kinases in an active state. A prominent CD45 feature is alternative splicing of exons encoding the N-terminus, resulting in the generation of several isoforms. The expression of isoforms is tightly regulated and dependent on the developmental/activation state of the lymphocyte. Nevertheless, the significance of these multiple isoforms in mammals is poorly understood. In this study, the channel catfish CD45 homolog was sequenced and found to be similar to CD45 of other species. However, unlike mammalian CD45, it appears that up to 13 exons are used in producing multiple alternatively spliced CD45 variants in catfish cells. These 13 alternatively spliced exons variably encode for O-linked glycosylation sites. Several of the exons are identical or very similar, suggesting gene duplication of a block of four exons. As demonstrated by RT-PCR, many of the alternatively spliced forms of catfish CD45 are differentially expressed in lymphoid cell lines with B cells expressing larger isoforms than do T cells. Furthermore, immunoprecipitation experiments utilizing anti-catfish CD45 mAbs substantiated that different size CD45 isoforms are expressed at the protein level on catfish T and B cells.     

Does 'soluble' HLA-G really exist? Another twist to the tale

HLA-G is thought to play a key role in implantation by modulating cytokine secretion to control trophoblast invasion and to maintain a local immunosuppressive state. It differs from other class I molecules in that the gene can be alternatively spliced to produce four membrane-bound (G1, G2, G3 and G4) and three soluble isoforms (G5, G6 and G7). The soluble isoforms have recently attracted attention as their levels may be diagnostic of poor trophoblast invasion in miscarriage or pre-eclampsia and the implantation potential of IVF embryos. Although the expression and function of HLA-G2, G3, G4 and G7 has previously been a matter of debate, until now it has been generally accepted that soluble HLA-G5 and HLA-G6 are both expressed and secreted by trophoblast. However, Blaschitz et al. (2005) have reinvestigated this question and come to the surprising conclusion that they are not. They have shown that trophoblast only expresses the membrane-bound HLA-G1 isoform and not soluble HLA-G5 and -G6. Furthermore, although soluble HLA-G could be found in trophoblast culture supernatants, it appears not to be derived by alternative splicing but by the cleavage of HLA-G1. The source of the soluble HLA-G may not matter from a diagnostic perspective, but these findings, if confirmed, have important implications for our understanding of the biology of HLA-G.     

Molecular cloning, functional characterization and genomic organization of four alternatively spliced isoforms of the human organic cation transporter 1 (hOCT1/SLC22A1)

In this study we report the cloning of four human OCT1 ( hOCT1 / SLC22A1 ) isoforms: a long form, hOCT1G/L554, and three shorter forms (hOCT1G/L506, hOCT1G483 and hOCT1G353). All four variants could be identified in the human glioma cell line SK-MG-1, whereas only two isoforms (hOCT1G/L554 and hOCT1G/L506) were found in human liver cDNA. The hOCT1G/L554 represents the full length hOCT1 since the sequence of this clone is more than 99% identical to previously cloned hOCT1 cDNAs. Elucidation of the gene structure of human OCT1 demonstrated that the other isolated isoforms are alternatively spliced variants. The hOCT1 gene consists of 7 exons and 6 introns. When stably expressed in human embryonic kidney (HEK293) cells, only the full length hOCT1 cDNA mediated decynium-22 (D22)-sensitive uptake of tritiated 1-methyl-4-phenylpyridinium ([³H]-MPP⁺).     

Lack of HLA-G soluble isoforms in Graves-Basedow thyrocytes and complete cDNA sequence of the HLA-G*01012 allele

The presence of HLA-G mRNA has been studied in thyroid follicular cells from autoimmune patients with Graves’ disease. Investigating the possible role of the expression of the HLA-G gene in tissue inflammation, we have found four of the six HLA-G mRNA isoforms described: G1, G2, G3 and G4, but not the soluble ones G5 and G6. Soluble G isoforms may be responsible for inducing tolerance and inflammation control and their absence in autoimmune thyroid follicular cells may induce failure of such control. In addition, the complete coding sequence of HLA-G*01012 has been obtained from thyrocytes and it shows only four synonymous changes with respect to the HLA-G*01011 allele; this further supports the existence of an evolutionary pressure for invariance on HLA-G genes.     

Splenic γδ T cell subsets can be separated by a novel mab specific for two CD45 isoforms

CD45 isoforms have been identified in a variety of different species and mab against various isoforms have been instrumental to define cellular subsets. In the process of generating novel mab against chicken γδ T cells two mab with specificity for CD45 were identified and characterized. The analysis of the chicken CD45 genomic structure suggested three exons being involved in alternative splicing. We cloned and expressed the full length CD45 isoform and three shorter isoforms. While the 7D12 mab reacted with all of these isoforms, the 8B1 mab selectively reacted with two short isoforms lacking either exons 3 and 5 or exons 3, 5 and 6. As expected, the reactivity of 7D12 included all leukocyte subsets, also including thrombocytes. In contrast, the 8B1 mab only reacted with lymphocytes and monocytes. 8B1 expression was found on almost all blood αβ T cells, while a γδ T cell subset and virtually all B cells lacked 8B1 reactivity. The fraction of 8B1^- αβ and γδ cells was larger in splenocytes as compared to PBL and there was also a population of 8B1⁺ splenic B cells. CD3 stimulation of splenic T cells resulted in upregulation of the 8B1 antigen on all T cells. Three-color immunofluorescence revealed differences in CD28 expression between the 8B1⁺ and 8B1¯ γδ T cell subsets with a higher CD28 expression level on 8B1¯ cells. The CD28 antigen was upregulated upon stimulation of the cells with IL-2 and IL-12. This novel mab will be a useful tool to further analyze chicken γδ T cells in more detail.     

A novel HLA-G allele, HLA-G*010111, in the Brazilian population

The human leukocyte antigen-G (HLA-G) gene plays an important role in pregnancy and is related to negative signals for natural killer cells and T lymphocytes. Herein a new HLA-G allele (HLA-G*010111) is described in the Brazilian population--one of the most heterogeneous populations in the world. The new allele is associated with the 14-bp deletion at exon 8 and is similar to the HLA-G*01010105 allele, except for a C to G transversion at codon 117 in exon 3.     

Expression of soluble isoforms of rat CD45. Analysis by electron microscopy and use in epitope mapping of anti-CD45R monoclonal antibodies.

The CD45 or leucocyte-common antigens are encoded by a single gene but can be found in various forms due to alternative splicing of three exons near the 5' end of the gene. The CD45 antigens are major glycoproteins of all types of leucocytes. Monoclonal antibodies recognizing restricted epitopes of CD45 have been used to distinguish phenotypic and functional subsets of lymphocytes. To facilitate epitope mapping and biochemical studies, we have expressed the extracellular portions for four different isoforms of rat CD45 in Chinese hamster ovary cells. Constructs were prepared to give four soluble CD45 isoforms, with sequence incorporating either all three alternative exons (sCD45.ABC), the B exon (sCD45.B), the C exon (sCD45.C), or no alternative exons (sCD45.O). These were expressed at approximately 5 mg/l of spent tissue culture supernatant and were antigenically active with monoclonal antibodies (mAb) that recognize all CD45 isoforms. The MRC OX22 and OX32 mAb have been used to split rat CD4+ T cells into functionally distinct subpopulations and the epitopes for these were mapped to the product of exon C. The epitope for MRC OX33, a marker for B cells, requires expression of either the A exon or the A/B exon junction. Electron microscopy showed that the extra segments contributed to an extended structure as has been predicted from the sequence. The shape of the molecule is discussed with regard to other molecules at the leucocyte cell surface.