[Aves Labs will periodically post research highlights in this space focusing on published articles from clients who have used our custom antibody production services. These highlights have been submitted for review to the principal investigators to ensure accuracy.]
Progesterone (P4) – the steroid hormone involved in the menstrual cycle and pregnancy – was classically thought to act via receptor proteins localized within the nucleus of its target cells (Evans, 1988). These nuclear P4 receptor proteins were believed to bind to P4-specific promoter regions in various genes to effect changes in their expression. One such target of P4 are the follicular granulosa cells of the female ovary, in which this hormone acts to inhibit apoptosis, allowing these cells to continue supporting egg maturation. Accumulating levels of P4 within the nucleus are also involved in follicular ovulation, among other effects (Robker et al., 2000). Interestingly, P4 is also synthesized by follicular granulosa cells, consistent with a self-stimulating mechanism (Robker et al., 2000).
One quandary, though, had to do with the timing of this ‘self-stimulating’ effect. It turns out that the known nuclear receptor proteins for P4 [PR-A and PR-B – differentially processed products of a common gene (Conneely et al., 2001)] aren’t yet present at early stages of follicular growth, and yet P4 suppresses apoptosis of granulosa cells at these stages (Peluso et al., 2005). The presence of the PR-A and PR-B gene products seemed only to occur after the gonadotropin surge, but P4-suppression of apoptosis occurred prior to the gonadotropin surge. This suggested that the P4 hormone was acting via receptors unrelated to PR-A or PR-B.
In earlier work, Dr. John J. Peluso (University of Connecticut Health Center, Farmington, CT) found reasons to believe that P4 may have been acting at the plasma membrane of early-stage granulosa cells. He found, for example, that there was direct binding of both fluorescein-labeled P4, as well as binding of the nuclear P4 receptor antibody C-262 , at the cell surface (Peluso et al., 2005). Using the C-262 antibody bound to an affinity column, he then isolated a 60 kDa protein from membrane preparations (Peluso et al., 2004). Using limited proteolysis and mass spectrometric analysis, this novel protein was sequenced and named “RDA288” (NCBI accession number XM_216160). [Note that this gene product was later renamed “PAIRBP1 (Plasminogen Activator Inhibiting RNA Binding Protein),” consistent with its other function – inhibiting the proteinase plasminogen activator.]
At this point, Dr. Peluso approached Aves Labs with his desire to produce antibodies specific to RDA288/PAIRBP1. Using our proprietary Immunogenicity Algorithm®, we identified five peptide sequences within the gene product that fit our criteria for immunogenicity (see Figure 1, below). Dr. Peluso chose the one closest to the N-terminus of the protein. After consultation with Dr. Peluso, we added a cysteine and a synthetic spacer amino acid to the N-terminus of this sequence, and synthesized the 13-mer peptide [CZ KQL RKE SQK DRK N (residues #68-80), where “Z” stands for aminocaproic acid – a spacer amino acid that offers some technical advantages for increased affinity antibody yields].
A keyhole limpet hemocyanin (KLH)-conjugate of this peptide was injected over a 7-week period into the breast muscles of two laying hens, and then eggs were collected over a 3-week period after the 4th injection. From 12 of these eggs, we purified the IgY fraction using our proprietary methods, and from this preparation, we further isolated affinity purified antibody. Since the hens were unharmed during the egg collection phase, of course, we also had the option of doing additional injections and collecting additional eggs for greater affinity purified antibody yields.
Dr. Peluso used this antibody in various applications to better understand this novel membrane-localized, P4-associated signaling pathway. First, in Peluso et al. (2004), he showed that the antibody recognized a single band at the predicted molecular weight using western blot analysis [Fig.2 (from Peluso et al., 2004; see also Peluso et al., 2006)], and using immunocytochemistry, was found at the predicted subcellular locale – the plasma membrane [Fig.3 (from Engmann et al., 2006)]. Interestingly, he found that the antibody Aves Labs produced for him actually was function-blocking. When added to culture medium, these chicken IgY antibodies against RDA288/PAIRBP1 blocked the ability of P4 to inhibit apoptosis (Fig.4 from Engmann et al., 2006). This suggests that antibody binding to this epitope somehow blocked the ability of P4 to effect downstream events involved in apoptosis. Most recently, Dr. Peluso found that RDA288/PAIRBP1 wasn’t the primary receptor for P4, but that it interacted with another protein – Progesterone Receptor Membrane Component 1 (PGRMC1) – and that this protein-protein interaction was necessary for P4 to inhibit apoptosis in granulosa cells (Peluso et al., 2013).
Finally, in my email conversations with Dr. Peluso during my writing of this “Custom Antibody Highlight,” he allowed me to quote him saying “this antibody was really useful in that it worked for rats, mice and human and could be used for immunocytochemistry, immunohistochemistry on fixed paraffin sections, Western blots and IP protocol. It was also useful as a blocking antibody.”
Conneely, O.M., Mulac-Jericevic, B., Lydon, J.P., De Mayo, F.J. (2001). Reproductive functions of the progesterone receptor isoforms: Lessons from knock-out mice. Molec. Cell. Endocrin. 179: 97-103.
Engmann, L., Losel, R., Wehling, M., Peluso, J.J. (2006). Progesterone regulation of human granulosa/luteal cell viability by an RU486-independent mechanism. J. Clin. Endocrin. Metab. 91: 4962-4968.
Evans, R.M. (1988). The steroid and thyroid hormone receptor superfamily. Science 240 (4854): 889-895.
Peluso, J.J., Pappalardo, A. (1999). Progesterone maintains large rat granulosa cell viability indrectly by stimulating small granulosa cells to synthesize basic fibroblast growth factor. Biol. Reprod. 60: 290-296.
Peluso, J.J., Fernandez, G., Pappalardo, A., White, B. (2002). Membrane-initiated events account for progesterone’s ability to regulate intracellular free calcium and inhibit rat granulosa cell mitosis. Biol. Reprod. 67: 379-385.
Peluso, J.J., Pappalardo, A., Fernandez, G., Wu, C.A. (2004). Involvement of an unnamed protein, RDA288, in the mechanism through which progesterone mediates its antiapoptotic action in spontaneously immortalized granulosa cells. Endocrin. 145 (6): 3014-3022.
Peluso, J.J., Pappalardo, A., Losel, R., Wehling, M. (2005). Expression and function of PAIRBP1 within gonadotropin-primed immature rat ovaries: PAIRBP1 regulation of granulosa and luteal cell viability. Biol. Reproduc. 73: 261-270.
Peluso, J.J., Pappalardo, A., Losel, R., Wehling, M. (2006). Progesterone membrane receptor component 1 expression in the immature rat ovary and its role in medicating progesterone’s antiapoptotic action. Endocrinol. 147 (6): 3133-3140.
Peluso, J.J., Yuan, A., Liu, X., Lodde, V. (2013). Plasminogen activator inhibitor 1 RNA-binding protein interacts with progesterone receptor membrane component 1 to regulate progesterone’s ability to maintain the viability of spontaneously immortalized granulosa cells and rat granulosa cells. Biol. Reproduc. 88 (1): 1-10.Robker, R.L., Russell, D.L., Yoshioka, S., Sharma, S.C., Lydon, J.P., O’Malley, B.W., Espey, L.L., Richards, J.S. (2000). Ovulation: A multi-gene, multi-step process. Steroids 65: 559-570.
Figure 1. Diagram of the RDA288/PAIRBP1 gene product, showing the peptide sequence recognized by the antibody Aves Labs produced (yellow box at the left). The location of the mRNA binding site is designated by the pink box. Sites of putative serine phosphorylations is designated by the single asterisks; sites of a lysine acetylation is designated by the double asterisk.
Figure 2 [from Peluso et al., 2004]. Western blot analysis, showing a single band at the predicted molecular weight of RDA288/PAIRBP1 in rat ovaries, cultured granulosa cells ("Rat GC"), and spontaneously immortalized granulosa cells ("SIGC"), but not in the negative control lane.
Figure 3 [from Engmann et al., 2006]. Immunocytochemical staining of 3 day old cultures of rat granulosa cells, showing a plasma membrane localization of the RDA288/PAIRBP1 gene product. On the left is immunostaining with the immune antibody; on the right is a negative control (immunostaining with a pre-immune IgY fraction).
Figure 4 [from Peluso et al., 2005]. Antibodies against RDA288/PAIRBP1 neutralize the effects of P4 on apoptosis in cultured rat granulosa cells. Note that about 100 nM P4 hormone reduced the percentage of apoptotic nuclei to about 30% of the original (compare the white bar on the right with the white bar at the left), but that that percentage reduction was only less than 50% of the original when RDA288/PAIRBP1 antibody was included (compare the black bar on the right with the black bar at the left).