It has been difficult to stablish a function of the SERPINA14 with certainty because of the inability to perform knockout or knockdown experiments in the species where the gene is present. It may also be that evolutionary divergence has resulted in species-specific roles for the SERPINA14. However, examination of the characteristics of the SERPINA14 reveals some important clues. The fact that the proteins are produced in the uterus, especially during pregnancy means that they probably play an important role to maintain the developing conceptus. Because the gene is present only in a limited number of mammals, the role of SERPINA14 in pregnancy is one that is either not important for other species or is served by other genes. Finally, the structural and biochemical features of the proteins are such that the SERPINA14 is probably not functioning as a proteinase inhibitor.
While the specific role of SERPINA14 is still open to question, it is clear that it is an important molecule. Khatib et al.39 showed that a single nucleotide polymorphism (A/G) at position 1269 of the bovine SERPINA14 was associated with productive life in cattle populations. Productive life is determined in large part by culling rate, and major causes of culling are problems with reproductive health or infectious diseases. There is evidence for a variety of functions for SERPINA14 as follows.
SERPINA14 as Immune Regulator
It has long been known that progesterone can inhibit uterine immune function, and this can been seen in sheep as delayed rejection of allografts placed into the uterus of ovariectomized ewes treated with progesterone for 60 days35,40 (Fig. 3). Given that prolonged treatment with progesterone is required to affect skin graft survival, and that the amounts of progesterone administrated are too low to directly affect lymphocyte function, it is likely that progesterone regulates uterine immune function indirectly by inducing synthesis of an immunoregulatory molecule. A plausible function for SERPINA14, at least in the sheep, is to be the mediator of progesterone’s immunosuppressive actions on the uterus. Through this role, SERPINA14 may provide immunological protection to the allogenetically distinct conceptus.41
Figure 3. Effect of progesterone on skin graft survival in the uterus of ovariectomized ewes. (a) Loss of an allograft in an ovariectomized ewe treated with vehicle (corn oil) for 60 days. The arrow shows remains of wool from the skin graft. Note, however, that the autograft is still present. (b) Presence of an allograft in an ovariectomized ewe treated with progesterone for 60 days (30 days before and 30 days after grafting). The figure was modified from Padua et al.35 with permission of Molecular Reproduction and Development.
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Purified ovine SERPINA14 inhibited lymphocyte proliferation induced by mixed lymphocyte reactions and mitogens such as concanavalin A (Con A), phytohemagglutinin (PHA) and Candida albicans antigen.42–47 The protein did not cause any inhibitory activity against pokeweed-activated lymphocytes,44 which activates T and B cells. In addition, ovine SERPINA14 reduced the antibody titer in ewes immunized against the T-cell dependent antigen ovalbumin.46
Ovine SERPINA14 also inhibits NK cell activity. SERPINA14 blocked abortion in pregnant mice induced by poly(I)•poly(C) (Fig. 4) and reduced basal splenocyte NK cell activity.48 Additional experiments demonstrated that the protein inhibited NK-like activity in sheep lymphocytes and mouse splenocytes.48,49
Figure 4. Effect of ovine SERPINA14 on poly(I)•poly(C)-induced abortion in pregnant mice. Note that injection of poly(I)•poly(C) on Days 3 or 4 of pregnancy increased the number of degenerated fetuses in mice treated with vehicle or a control protein (ovine serum albumin; OSA). However, SERPINA14 greatly reduced the percentage of degenerated conceptuses in mice treated with poly(I)•poly(C). The figure was adapted from Liu and Hansen.48
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In addition to immune cells, ovine SERPINA14 inhibited the proliferation of other cell types such as human prostatic adenocarcinoma (PC-3), mouse lymphoma P388D1 and canine primary osteogenic sarcoma (D-17) cell lines, and bovine pre-implantation embryos.50,51 However, the protein did not affect proliferation of γδT+ cells, an endometrial fibroblast cell line or kidney epithelial bovine cells.46,50,51
Ovine SERPINA14 does not inhibit proliferation by being cytotoxic or inducing apoptosis.44,45,47,50 The protein can bind to lymphocytes in a concentration-dependent and saturable manner.52 High concentrations (0.1–1 mg/mL) of protein are required for inhibition of cell proliferation, but these high concentrations are physiological because, in the pregnant ewe, SERPINA14 is present in multiple milligrams per milliliter concentrations in uterine fluid.2 The fact that high concentrations of SERPINA14 are required for anti-proliferative effects may imply that, rather than binding to a specific, high-affinity cell surface receptor, SERPINA14 may act on the cell by activating or inhibiting receptors for other ligands.
Ovine SERPINA14 did not cause inhibition of lymphocyte proliferation stimulated by PHA through the protein kinase (PK) A pathway as was shown by experiments using a selective inhibitor of cAMP-dependent type-I PKA (Rp-8-Cl-cAMPS).50 Instead, ovine SERPINA14 reduced proliferation of phorbol myristol acetate-activated lymphocytes, suggesting that the protein inhibits some PKC-mediated events.53 In the same study, the protein-blocked IL-2 induced proliferation and reduced expression of CD25 (IL-2Rα chain), but it did not affect the steady-state amounts of IL-2 mRNA caused by Con A.53
It was recently determined that ovine SERPINA14 inhibited proliferation of PHA-stimulated lymphocytes and PC-3 cells by blocking cell cycle progression at specific stages. Ovine SERPINA14 increased the proportion of activated lymphocytes at the G0/G1 phase and decreased the proportion of these cells at the S phase of the cell cycle.47 Moreover, the protein blocked the progression of the PC-3 cells through the cell cycle at the G2/M and G0/G1 phases when cells were incubated with the protein for 12 and 24 hr, respectively.47 Additional experiments indicated that, in PC-3 cells, ovine SERPINA14 increased the expression of genes involved in checkpoints and arrest of the cell cycle such as CDKN1A (p21cip1) CCNG2 (cyclin G2) and CDKN2B (p15ink) and decreased the expression of genes required for DNA synthesis and progression at the S and M phase, respectively.14
SERPINA14 as a Carrier Protein
Perhaps not surprising for a protein with a basic isoelectric point, SERPINA14 has the propensity to bind other proteins. Ovine SERPINA14 can cross the placenta, as has shown in the sheep54,55 and pigs.3,56 It may, therefore, function to stabilize proteins in the uterus or placental fluids or facilitate transplacental transport.
Examples of binding partners for ovine SERPINA14 are the pregnancy-associated glycoproteins,8 which are a large family of inactive aspartic proteinases secreted by the ungulate placenta,57,58 activin,55 IgM and IgA.59 Binding to immunoglobulins was inhibited by the presence of high salt concentrations, indicating the ionic nature of the binding.59
Porcine SERPINA14 has been implicated as an important component of the iron-transport system to the conceptus through its interaction with another endometrial protein called uteroferrin. Porcine uteroferrin is an iron-binding alkaline phosphatase of purple-colored whose secretion by the glandular epithelium is also stimulated by progesterone.3,56,60,61 Uteroferrin is transported by the areolae of the placenta into the allantoic fluid61–63 (Fig. 5). Porcine SERPINA14 binds non-covalently to uteroferrin to create a heterodimer of pink color that is stable for long periods of time in the presence of oxygen whereas purple uteroferrin is not.3,5 Also, the association between porcine SERPINA14 and uteroferrin promotes the latter’s enzymatic activity in the heterodimer conformation.3
Figure 5. Proposed model for the path (showed by arrows) for the transport of iron to the pig fetus by the uteroferrin-SERPINA14 complex. The purple circles represent uteroferrin whereas the gray circles represent porcine SERPINA14. Uteroferrin and porcine SERPINA14 are secreted by the endometrial epithelium, bind non-covalently to form a heterodimer, are taken up by the areolae of the placenta (specialized structures that form adjacent to endometrial glands) and move into the umbilical vein, heart, arterial system and fetal liver. Some uteroferrin is also cleared by the kidney where it ends up in the allantoic fluid. The figure was adapted from Renegar et al.63 and is reproduced with permission of Biology of Reproduction.
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