| The vitamin B9 occurs physiologically in its reduced forms as small hydrophilic molecules (Mr～500), functioning as co-factors in one-carbon transfer reactions including de novo biosynthesis of nucleotides and plays a key role in metabolic processes involved in DNA and RNA synthesis, epigenetic processes, cellular proliferation and survival . The major circulating form of the vitamin, 5-methyltetrahydrofolate occurs at a serum concentration ranging from 5 to 30 nM. Numerous in vitro and in vivo studies have established profound effects of folate deficiency on DNA replication and repair and on gene expression and its causal role in several pathological conditions such as fetal nerve development disorders and cardiovascular disease .The hydrophilic and anionic nature of folates at physiologic pH precludes their passive diffusion through the plasma membrane. Cellular folate uptake is mediated by at least three distinct transporters. Among these, the reduced folate carrier (RFC), so named because of its inability to bind folic acid, the unphysiologic oxidized form of the vitamin, is the predominant folate transporter that is ubiquitously expressed throughout development and in normal adult tissues, despite significant variability in its expression levels among tissue types . The RFC is a transmembrane anion exchanger that has a dissociation constant for 5-methyltetrahydrofolate in the micromolar range and although it operates suboptimally in the physiologic concentration range of folate, the transport is rapid and generally adequate to supply the cellular stores of the co-enzyme. A proton- coupled high-affinity folate transporter has recently been reported; this protein has been shown to be the major transporter of folate in low pH environments and appears to be the principal transporter in the intestinal absorption of folate.A third folate transporter is known as the folate receptor (FR), often referred to as the high affinity folate binding protein. FR is represented by a family of glycopolypeptides of apparent Mr in the range of 38,000–45,000 . The receptor binds folic acid, reduced folate, many antifolates and folate conjugates with a 1:1 stoichiometry and a high affinity (Kd～0.1–1 nM). One of the FR isoforms has been shown to be essential for normal nerve tube development and growth of mouse embryos under conditions of limiting dietary folate. However, the physiologic function of FR in adult tissues has largely remained a mystery since, in addition to its narrow tissue distribution, FR is predominantly expressed on the apical (luminal) surface of polarized epithelial cells where it is not in contact with circulating folate. Further, FR expressed in mature hematopoietic cells is unable to bind folate.A homology search of the human genome reveals four distinct FR genes termed FRs-α,-β, -γand -δin addition to several pseudogenes. A polymorphism in the FR-γgene is caused by a mutation that results in a carboxyl-terminal truncation of the protein; the mutated protein is referred to as FR-γ′. The genes encoding FRs-α, -βand -γare located on chromosome 11 (q11.3–q13.5) . The gene encoding FR-δoccurs on chromosome 11q14 and is predicted to encode a 27.7 KDa protein. FRs-α, -βare membrane associated protein while FR-γ,-γ′are predominantly secreted due to the lack of GPI anchor signal.FR-αis expressed at the luminal surface of polarized epithelial cells of normal adult tissues including proximal kidney tubules, type I and II pneumocytes in the lungs, choroids plexus, ovary, fallopian tube, uterus, epididymis, submandibular salivary and bronchial glands and trophoblasts in placenta, as well as the basolateral membrane of retinal pigment epithelial cells. FR-βis expressed in later stages of normal myelopoiesisand in placenta, spleen and thymus, and in leukemic blasts in chronic (CML) and acute (AML) myelogenous leukemia. FR-γexpression was detected in normal and malignant hematopoietic cells present in the spleen, bone marrow and thymus, as well as ovarian, cervical and uterine carcinomaThe murine FR isoforms referred to in the literature as FR1, 2 and 3 are frequently assumed to correspond to the human FRs-α,-βand -γ, respectively. A third mouse FR, FR4 was discovered from genome database mining, and is expressed in spleen and thymus. Further study suggested that splenic lymphocytes, T cells (especially), and mature thymocytes expressed mRNA and protein for FR4 but not for FR1 and FR2, and moreover, it also found that natural Treg cells, constitutively expressed high amounts of FR4. However, whether it is secreted or anchored to the membrane remains unclear. Here, we isolated a novel full-length cDNA of FR4 gene from murine splenocytes by gene cloning, termed FR4v. FR4v was 843bp in size and contained an 108bp complete intron. Western blot analysis identified FR4v as a 31-38 kDa larger protein distinct from FR4. We firstly demonstrated that FR4 variants are mainly membrane -associated forms through GPI anchor by PI-PLC treatment of splenocytes prior to Western blot and FACS analysis. Importantly, Western blot showed FR4v was predominantly expressed in CD4+CD25+ regulatory T cells(Treg).In summary, we have identified a Treg constitutively expressed, alternatively spliced mouse FR4 variant containing a retained and encoding a new GPI-anchored FR4 isoform, FR4v, which may be involved in the anergy state of Tregs. |
PDF Full Text Request