Dr Maria B. Padua, PO Box 100294, Department of Obstetrics and Gynecology, University of Florida, Gainesville, FL 32610-0294, USA. E-mail: email@example.com
Citation Padua MB, Hansen PJ. Evolution and function of the uterine serpins (SERPINA14). Am J Reprod Immunol 2010
Uterine serpins (recently designated as SERPINA14) are hormonally induced proteins secreted in large quantities by the endometrial epithelium during pregnancy. The SERPINA14 proteins belong to the serine proteinase inhibitor (serpin) superfamily, but their apparent lack of inhibitory activity toward serine proteinases suggests that these proteins evolved a different function from the anti-proteinase activity typically found in most members of the serpin superfamily. The gene is present in a limited group of mammals in the Laurasiatheria superorder (ruminants, horses, pigs, dolphins and some carnivores) while being absent in primates, rodents, lagomorphs and marsupials. Thus, the gene is likely to have evolved by gene duplication after divergence of Laurasiatheria and to play an important role in pregnancy. That role may vary between species. In sheep, SERPINA14 probably serves an immunoregulatory role to prevent rejection of the fetal allograft. It is inhibitory to lymphocyte proliferation and natural killer cell function. In the pig, SERPINA14 is involved in iron transport to the fetus by binding to and stabilizing the iron-binding protein uteroferrin. It is possible that SERPINA14 has undergone divergence in function since the original emergence of the gene in a common ancestor of species possessing SERPINA14.
Pregnancy in mammals has evolved as a process that included the use of the existing genes for specific functions as well as the appearance of new genes formed by gene duplication that develop novel functions by sequence divergence from the parental gene.1 Among the proteins involved in pregnancy of a limited group of mammals are the uterine serpins (SERPINA14). During the course of evolution, SERPINA14 has been modified to exhibit high expression in the uterus under the regulation of progesterone. In addition, unlike most of the members of the serine proteinase inhibitor (serpin) superfamily, SERPINA14 has not retained the anti-proteolytic activity. Instead, SERPINA14 might have experienced continue divergence since its formation so that the function of the proteins may be species specific. Thus, SERPINA14 may regulate immune function in the sheep and placental iron transport in the pig.
Uterine serpins were first described as progesterone-induced proteins in the uterine fluid of the pregnant sheep (Ovis aries), pig (Sus scrofa) and cow (Bos taurus).2–4 The protein in sheep, discovered first, was called uterine milk protein (UTMP) because it was the major protein in uterine fluid (called uterine milk by Aristotle). The two uterine serpins found in the pig were originally known as uteroferrin-associated protein or uteroferrin-associated basic proteins (UfAP/UABP) because they form non-covalently heterodimers with uteroferrin.3,5 The homology between the sequence of the sheep UTMP and some members of the serpin superfamily was first found by Ing and Roberts.6 Later, Malathy et al.7 and Mathialagan and Hansen8 found that the pig and cow proteins were similar to the sheep protein and also related to the serpin family. Afterward, it was proposed that old designations be eliminated and proteins termed as uterine serpins (US).
Recently, serpin genes have been categorized according to their phylogenetic relationship into 16 clades or classes (A–P). The largest clades in the classification are Clade A and B which consists of extracellular or antitrypsin-like serpins and intracellular or ov-like serpins, respectively.9 All US that have been described so far possess a 25-amino acid signal peptide sequence and are secreted into the uterine lumen.8,10,11 Depending upon the analysis, phylogenetic studies have classified US as a highly divergent group of Clade A or as a separate clade.9,12,13 Upon discussion with James Whisstock and Gary Silverman, we asked the Human Genome Organization to reserve the subclade 14 designation for US and this was agreed to. Since that time, US have been referred to as SERPINA14 in print.11,14,15
Evolution and Phylogeny of SERPINA14
The SERPINA14 gene was first found in species with epitheliochorial placenta of the Ruminantia and Suidae orders of the Laurasiatheria superorder of eutherian mammals.2–4 It is now known that, while still somewhat limited in distribution among mammals, the SERPINA14 gene is present in a variety of other mammalian species (Fig. 1a) including those with epitheliochorial placenta [goat (Capra hircus) and water buffalo (Bubalus bubalis) and dolphin (Tursiops truncatus– based on genomic information) in Cetartiodactyla and horse (Equus caballus) in Perissodactyla] as well as one species of Carnivora with endotheliochorial placentation, the dog (Canis lupus familiaris).11 Based on sequence identity in genomic DNA, original data presented in this paper are indicative that another carnivore, the panda bear (Ailuropoda melanoleuca), also contains a SERPINA14 gene (Fig. 2).
There is also a non-functional SERPINA14 in a third carnivore, the cat (Felis catus).11 Thus, it may be that not every carnivore expresses a functional SERPINA14 gene. In addition, the SERPINA14 gene is not present in the Rodentia, Lagomorpha and Anthropoidea orders of the Euarchontoglires superorder nor in Marsupialia.11 These results suggest that the SERPINA14 gene evolved only within the Laurasiatheria superorder of mammals, probably by gene duplication from another serpin gene.
A phylogenetic tree with all the SERPINA14 proteins identified today, including the putative dolphin and panda bear SERPINA14, is shown in Fig. 1b. As would be expected from patterns of mammalian evolution, ruminant SERPINA14 cluster together, with bovine and water buffalo proteins forming a sister cluster and ovine and caprine forming another. Also, as expected, given the common ancestor between cetaceans and ruminants,24 the dolphin-like protein was in a separate branch, but closer to the ruminants than to other species. The porcine SERPINA14 is more closely related to ruminants than to other serpins and the equine, canine and panda bear-like SERPINA14 proteins cluster together, with the two carnivore species forming a sister clade. Note that while it was originally reported that there were two SERPINA14 genes in the pig,8 only one gene was identified in the porcine genome.11
Structural and Enzyme-inhibitory Properties of SERPINA14
The key region for serpins that inhibit proteinases is the reactive center loop (RCL), which is a flexible structure localized on the top of the serpin and contains a complementary sequence to the active site of the target proteinase. The RCL is usually formed by 20–25 amino acids and includes the hinge region at P15–P9 (with P1–P1′ representing the scissile bond).9,25,26 The hinge region provides mobility to the RCL, and there is a consensus pattern present in the sequence for inhibitory serpins. Glycine is usually present in the P15 position, threonine or serine is present at P14 position and acid residues with short side chains such as alanine, glycine or serine are abundant in positions P12–P9.9 Analysis of the hinge regions of all SERPINA14 proteins indicates that these serpins are not conserved with inhibitory serpins and are probably not functional proteinase inhibitors.11
In addition to the lack of conservation of key amino acid residues necessary for proteinase inhibitory activity, there is little biochemical evidence to indicate that the SERPINA14 proteins are non-functional proteinase inhibitors. Ovine SERPINA14 had non-inhibitory activity against trypsin, chymotrypsin, plasmin, thrombin, elastase, plasminogen activator, cathepsins B, D or E, or dipeptidyl proteinase IV.6,8,27 Similarly, porcine SERPINA14 did not inhibit trypsin or chymotrypsin.7
Mathialagan and Hansen8 demonstrated that freshly purified ovine SERPINA14 inhibited pepsin A and C activity at both pH 2.0 and 4.5, but an excess of 35- and 8-fold molar of the protein was required to achieve 50% inhibition of both pepsin A and C, respectively. Moreover, the complex formed by the sheep protein and pepsin could not be detected electrophoretically in the presence of sodium dodecyl sulfate8, whereas complexes formed by inhibitory serpins and their partner proteinases (at 1:1 ratio) are so stable that they can be resolved using the same approach.28 Furthermore, the binding of ovine SERPINA14 to pepsin seems to be electrostatic.29
Regulation of Expression and Secretion of SERPINA14
Two of the defining properties of SERPINA14 are that they are products of the uterine endometrium and are elevated in synthesis during pregnancy. This has been shown experimentally for sheep,2 pig,3 goat,10 horse,11 dog11 and cow.30 The expression and secretion of SERPINA14 in the endometrial epithelium is hormonal dependent, with progesterone being the main regulator of gene expression. A description of SERPINA14 secretion in two species, the sheep and cow illustrates the nature of synthesis and secretion of the protein.
In sheep, which is the species where SERPINA14 has been studied most extensively, SERPINA14 mRNA can be first detected in the sheep uterine endometrium around days 13–16 of the estrous cycle31 and pregnancy.31,32 There is then an increase in the steady-state amount of SERPINA14 mRNA in the glandular epithelium between days 20 and 80, and a decline at day 120 of pregnancy.31 SERPINA14 appears to be initially produced by epithelial cells of the uterine glands and then expression spreads to the luminal epithelium.2,33 Interestingly, mRNA has not been detected in luminal epithelial cells31; so it is possible that SERPINA14 in these cells was synthesized while cells were residing in the glands. SERPINA14 mRNA was only found in the stratum spongiosum of the glandular epithelium at post-gestational day 1 and was not detectable by day 7 postpartum.34
The main regulator of SERPINA14 expression in sheep is progesterone although other hormones modulate its activity. Treatment of ovariectomized ewes with progesterone induced SERPINA14 in endometrial epithelial cells after 6 days of treatment;32 a large-scale increase in secretion of the protein occurs after progesterone treatment for 14–60 days.30,32,35 Administration of progesterone and intrauterine injections of INF-τ combined with ovine placental lactogen and/or ovine growth hormone increased amounts of SERPINA14 mRNA in the glandular epithelium.36,37 The co-adminstration of estradiol with progesterone, where estradiol up-regulates the expression of progesterone receptors in the endometrium, decreased SERPINA14 mRNA in the glandular epithelium.37
In the cow, unlike the sheep, the SERPINA14 mRNA is expressed in the endometrium during estrus, with particular highly expression in the cranial area of the uterine horns.15,38 At this time, SERPINA14 mRNA is highly expressed in the superficial uterine glands and more weakly expressed in the deep glandular epithelium and some dispersed luminal epithelial cells of the endometrium.38 Moreover, immunoreactive SERPINA14 was also found in the glandular epithelium of endometrial tissues from early and late estrus.15 There is then a decline in SERPINA14 mRNA at day 3.5 and a subsequent, moderate increase during luteal phase (days 12–18).15 At gestational day 18, the expression of SERPINA14 mRNA was higher when compared with cyclic animals.15 However, no immunostaining was detected in either the glandular or luminal epithelium of endometrial tissues from cows on days 15, 17, 18, 19, 21 of pregnancy or days 17 and 19 of the estrous cycle.4,15 Like the sheep, therefore, abundant secretion of SERPINA14 occurs only later in pregnancy. SERPINA14 can be immunolocalized in the glandular epithelium and in both glandular and luminal epithelium of endometrial tissues from days 135 and 150 of gestation, respectively.4,15,30
Similar to the sheep, SERPINA14 secretion in the bovine uterus can be induced by progesterone.30 Bovine SERPINA14 expression is not limited to the uterus, however, can also be found in the ovarian follicles, corpus luteum, cumulus oocyte complex and placental cotyledon.15,39
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
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
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
The regulation of SERPINA14 by progesterone and their abundant secretion into the uterus during pregnancy make these proteins a distinctive group of the serpin superfamily. The evolution of the gene in a limited group of species of the Laurasiatheria superorder of eutherian mammals (ruminants, horses, pigs, dolphins and some carnivores) and the lack of anti-proteolytic activity against proteinases are indicative that SERPINA14 arose to serve some specific role during pregnancy in a subset of mammals. It is possible that formation of SERPINA14 was followed by continuing divergence and as a consequence it may play species-specific roles during pregnancy so that SERPINA14 functions as an immunomodulator of the maternal immune system against the fetus in the sheep and as an iron-transport protein for the fetus in the pig.