Federica Andorno e Rosa Nevola
Folate receptors bind folate and reduced folic acid derivatives and mediates delivery of tetrahydrofolate to the interior of cells.
Folic acid or folate is a B vitamin. Folic acid or folate, is converted by humans to dihydrofolate (dihydrofolic acid) (DHF) (FH2) by dihydrofolate reductase. DHF is then reduced to THF (tetrahydrofolate), which have various biological activities.
Three folate receptors (FRs) are expressed at the cell surface, anchored in the cell membrane by a glycosylphosphoinositol (GPI) domain.
Folate receptor alpha is a protein that in humans is encoded by the FOLR1 gene. FRα is expressed on the membrane of epithelial tissues, in particular the placenta, the apical brush-border membrane of proximal renal tubular cells, retinal pigment epithelium, and the choroid plexus.
Folate receptor beta is a protein that in humans is encoded by theFOLR2 gene. This protein has a 68% and 79% sequence homology with the FOLR1 and FOLR3 proteins, respectively. The FOLR2 protein was originally thought to exist only in placenta, but is also detected in spleen, bone marrow, and thymus. FRβ is expressed in hematopoietic tissues (e.g., spleen, thymus, and CD34+ monocytes).
The Folate receptor gamma is a protein, one of the isoforms of folate receptor, encoded by the gene FOLR3.
Juno(protein), also known as folate receptor 4 or folate receptor delta, is a protein that in humans is encoded by the FOLR4gene on chromosome 11.
Based on a sequence homology search for genes relate to the folate receptor, the gene for folate receptor 4 was first identified in mice and humans in 2000 at the University of Nebraska.
In 2014, the function of folate receptor 4 was discovered by the researchers of the Wellcome Trust Sanger Institute. Juno was initially found in murine oocytes, but its interaction with Izumo was subsequently found in other mammalian species, including humans. Because of its role in fertilization and inability to bind folate, the protein has renamed ‘Juno’, the Roman goddess of fertility and marriage.
Mechanisms of Membrane Transport of Folates into Cells and Across Epithelia, 2011
APPROVED NAME: folate receptor 4, delta (putative)
SYNONYMS: Folbp3, JUNO
OFFICIAL SYMBOL: FOLR4
LOCUS TYPE: gene with protein product
CHROMOSOMAL LOCATION: 11q14
Protein amino acids percentage
Folr4 is one of three folate receptor paralogues in mouse whose main role is thought to involve folate uptake. Using recombinant proteins, researchers showed that, unlike Folr1 and Folr2, Folr4 was unable to bind to folate which was expected given differences in amino acids knownto be critical for folate binding.
In picture “a” : Soluble biotinylated recombinant proteins corresponding to the entire ectodomains of mouse Folr1, Folr2 and Juno or human Juno were captured on a streptavidin-coated plate and washed. Folic acid binding was quantified relative to a CD200R negative control by adding a folic acid–HRP conjugate followed by an HRP substrate. Folic acid bound Folr1 and Folr2, but not mouse or human Juno.
In picture “b”: A multiple sequence alignment of human and mouse Folr1, Folr2 and Juno highlighting residues which are critical for folic acid binding. Sequences were aligned and structural features annotated: the signal peptide is in green, the GPI-anchor cleavage sites are underlined in bold for Folr1 and Folr2, and the best-scoring prediction is underlined pink for Juno. Residues located in the folic acid binding pocket and which affect folic acid binding are marked in red and differences highlighted in blue. Only 9 out of 15 residues involved in folic acid binding are conserved in human Juno relative to human Folr1, and 6 out of 15 are conserved in mouse Juno.
Fertilization is the culminating event in sexual reproduction and requires the fusion of haploid sperm and egg to create a new, genetically distinct, diploid organism.
Sperm acquire the ability to fertilize the egg within the female reproductive tract. Capacitation is the first step to render sperm capable of interaction with the egg. It is basically a functional maturation of the sperm, involving an increase in membrane fluidity due to cholesterol efflux, changes in sperm membrane potential, increased tyrosine phosphorylation and induction of hyperactivation. It is followed by the acrosome reaction, fusion of the plasma and outer acrosome membranes, exposing the inner acrosome membrane and releasing the acrosomal content.
The fusion site is specific on both gametes, which leads us to believe that there are topologically unique protein populations or lipid organization sites with the distinct membrane morphology required for fusion. The sperm membrane overlying the acrosome, which does not take part in the acrosome reaction, is called the equatorial region, and the sperm-egg fusion is long believed to be initiated in this region. The surface of egg plasma membrane can be divided into two parts: the microvillar-free smooth region, which overlays the meiotic spindle, and the microvillar protrusions-rich region, covering the rest of the egg, forming a dome shaped structure antipodal to eccentric nucleus. Gamete fusion occurs predominantly or exclusively in the microvillar-rich region.
Several receptor proteins have been implicated in the recognition and/or fusion process3, but just two significantly affect fertility in vivo: Izumo1 on sperm4, and on eggs CD9 (an important member of the tetraspanin family).
IZUMO was found to belong to an immunoglobulin superfamily of type I membrane proteins with one extracellular immunoglobulin (Ig) domain and one N-terminal domain. The superfamily consists of four proteins, coded with numbers 1 to 4, showing a significant homology in the N-terminal domain, hence known as “IZUMO domain”. IZUMO1 (originally described by Inoue’s group), 2 and 3 are transmembrane proteins expressed only in the testis, whereas IZUMO4 is soluble and expressed in the testis and other tissues.
Recombinant Izumo1 binds both wild-type and CD9- deficient eggs, suggesting that Izumo1 interacts with an egg receptor other than CD99. Glycophosphatidylinositol (GPI)-anchored receptors on the egg are essential for fertilization because removing them either enzymatically10 or genetically11 renders eggs infertile.
Sperm-egg fusion: a molecular enigma of mammalian reproduction, 2014
Juno is the binding partner for IZUMO1.
In Figure A, an avid recombinant Izumo1 protein, but not a control, binds the oolemma; in Figure B, Izumo1 binds HEK293 cells transfected with a Juno cDNA (clone B2), but not untransfected controls.
Using an anti-Juno monoclonal antibody, we can see that Juno expression matched the binding pattern of the recombinant Izumo probe on ovulated oocytes (Figure C). Preincubating oocyteswith the anti-Juno antibody prevented all detectable binding of the Izumo1 probe, demonstrating that Juno is the sole Izumo1 receptor on oocytes (Figure D). The protein sequence of Juno suggested the presence of a carboxy-terminalGPI-anchor site. A large fraction of cell-surface Juno staining was lost after phosphoinositide phospholipaseC (PIPLC) treatment of eitherHEK293 cells transfected with the JunocDNA or oocytes (Figure F), demonstrating that Juno was GPI-anchored.
The Izumo1–Juno interaction is direct, transient and conserved across mammals.
Clearly identifiable Izumo1 and Juno orthologues exist in all sequenced mammalian genomes, including marsupials. To determine whether the interaction was conserved within mammals, researchers expressed the entire ectodomains of both Izumo1 and Juno orthologues from humans, pig (Sus scrofa) and opossum (Monodelphis domestica) and assessed binding using the AVEXIS assay. Clear binding between the orthologues was observed, demonstrating that the interaction is conserved within Mammals.
To determine whether Juno was essential for fertilization, is made created Juno-deficient mice using embryonic stem cells disrupted at the Folr4 Locus.
Both Juno2/2 male and female mice developed indistinguishably from wild-type controls, showing normal rates of growth and morphology and were overtly healthy. Whereas Juno2/2 male and Juno1/2 female mice were fertile, by contrast, Juno2/2 females failed to produce any litters during three months of continuous mating with wild-type males of proven fertility . Female Juno2/2 mice exhibited natural mating behaviours, as assessed by vaginal plug formation and the presence of motile spermin the reproductive tract when paired with fertile wild-type males. Juno2/2 females responded to hormone treatment by ovulating morphologically normal eggs at numbers that did not significantly differ from wild-type.
Researchers suggest that the Izumo1–Juno interaction performs a necessary adhesion step rather than acting as a membrane ‘fusogen’.
The Izumo1–Juno interaction is not sufficient for cell fusion but is required for efficient sperm–egg adhesion.
In picture “a”: HEK293T cells transfected with plasmids encoding either Juno or full-length, GFP-tagged Izumo1 were mixed and cultured for 24 h before analysis by confocalmicroscopy. Izumo1 was detected by GFP fluorescence (green) and Juno using an anti-Juno antibody (red) and nuclei using DAPI (blue). No fused cells were observed, but both Izumo1 and Juno were enriched at sites of cellular contact. Scale bar, 10mm.
In picture “b”: Sperm were collected from acrosome reporter mice and capacitated before mixing with zona-free eggs at a 1:70 egg:sperm ratio. Acrosome-reacted (GFP-negative) sperm bound less efficiently to Juno-deficient (2/2) than wild-type (1/1) control eggs. Bars represent mean 6s.e.m., the number of eggs is indicated in parentheses.
After fertilization, oocytes become largely refractory to further sperm fusion events to prevent the creation of nonviable polyploid embryos due to polyspermy. This is achieved through both a relatively slowacting(1 h) hardening of the zona pellucida caused by the action of enzymes released fromcortical granules after egg activation, and a fasteracting block involving biochemical changes in the oolemmalmembrane.
In mammals, the membrane block occurs over a longer timeframe (30–45 min), and despite being first described 60 years ago, its mechanistic basis remains a mystery.
We can see that cell-surface Juno rapidly becomes undetectable after fertilization. Juno (green) is expressed on ovulated metaphase II eggs, but is barely evident at telophase II and undetectable on pronuclear-stage zygotes. Arrow and asterisk indicate sites of first and second polar body extrusion, respectively; chromosomes are not within the plane of focus. Using immunogold electron microscopy we can determine the subcellular localization of Junofollowing fertilization. Surprisingly,we observed that Juno was not internalised after fertilization but present in extracellular vesicles, presumably derived fromthemicrovillus-rich oolemma that undergoes significant architectural changes upon fertilization.
Juno is the egg Izumo receptor and is essential for mammalian fertilization, 2014
Izumo meets Juno: preventing polyspermy in fertilization, 2014
While these findings may eventually be used to improve assisted fertility treat-ments, the essential requirement for the Izumo1–Juno interaction could provide novel opportunities for the development of non-hormonal and/or more flexible contraceptives, something that may be important given the rapid expansion of the human population on a planet of finite resources.