Plasmin generates vasoinhibin-like peptides by cleaving prolactin and placental lactogen.

Vasoinhibin is an antiangiogenic, profibrinolytic peptide generated by the proteolytic cleavage of the pituitary hormone prolactin by cathepsin D, matrix metalloproteinases, and bone morphogenetic protein-1. Vasoinhibin can also be generated when placental lactogen or growth hormone are enzymatically cleaved. Here, it is investigated whether plasmin cleaves human prolactin and placental lactogen to generate vasoinhibin-like peptides. Co-incubation of prolactin and placental lactogen with plasmin was performed and analyzed by gel electrophoresis and Western blotting. Mass spectrometric analyses were carried out for sequence validation and precise cleavage site identification. The cleavage sites responsible for the generation of the vasoinhibin-like peptides were located at K170-E171 in prolactin and R160-T161 in placental lactogen. Various genetic variants of the human prolactin and placental lactogen genes are projected to affect proteolytic generation of the vasoinhibin-like peptides. The endogenous counterparts of the vasoinhibin-like peptides generated by plasmin may represent vasoinhibin-isoforms with inhibitory effects on vasculature and coagulation.

Tobacco as biofactory for biologically active hPL production: a human hormone with potential applications in type-1 diabetes.

Human placental lactogen (hPL) is a peptidic hormone that belongs to the short list of growth factors that could treat type-1 diabetes through pancreatic islet transplantation. Placental lactogen has the capacity to improve islet survival and function before or after transplantation. In this study, transgenic tobacco plants were used as a novel expression system for the production of recombinant hPL protein (rhPL). The expression vector pNEKhPL2 containing hPL cDNA was introduced into tobacco plants; the transcriptional activity was confirmed by real-time PCR, and the rhPL levels reached 1% of the total soluble protein (TSP) content in plants cultivated in the greenhouse. In vitro bioassays using the rat insulinoma (INS-1) cell line showed that recombinant protein was able to induce cell proliferation and activate the JAK-2/STAT-5 signal transduction pathway, demonstrating that plant cells can produce the biologically active hPL protein. To further characterize the plant expression system for hPL production, we analyzed the stability of the protein during the life cycle of tobacco plants as well as the transmission of the transgenic trait to the progeny. The recombinant protein was stably accumulated in young leaves, reaching the maximum level in the first month (6.51 µg/g of fresh weight), but showing a decreasing trend of 26% from the initial sampling time until the end of plant's life cycle. The progeny of the selected pNEKhPL2 plant showed in vitro expression levels of up to 1.1% of TSP. Our results therefore indicate that transgenic plants are a suitable expression system for hPL production.

Obligate ordered binding of human lactogenic cytokines.

Class 1 cytokines bind two receptors to create an active heterotrimeric complex. It has been argued that ligand binding to their receptors is an ordered process, but a structural mechanism describing this process has not been determined. We have previously described an obligate ordered binding mechanism for the human prolactin/prolactin receptor heterotrimeric complex. In this work we expand this conceptual understanding of ordered binding to include three human lactogenic hormones: prolactin, growth hormone, and placental lactogen. We independently blocked either of the two receptor binding sites of each hormone and used surface plasmon resonance to measure human prolactin receptor binding kinetics and stoichiometries to the remaining binding surface. When site 1 of any of the three hormones was blocked, site 2 could not bind the receptor. But blocking site 2 did not affect receptor binding at site 1, indicating a requirement for receptor binding to site 1 before site 2 binding. In addition we noted variable responses to the presence of zinc in hormone-receptor interaction. Finally, we performed Förster resonance energy transfer (FRET) analyses where receptor binding at subsaturating stoichiometries induced changes in FRET signaling, indicative of binding-induced changes in hormone conformation, whereas at receptor:hormone ratios in excess of 2:1 no additional changes in FRET signaling were observed. These results strongly support a conformationally mediated obligate-ordered receptor binding for each of the three lactogenic hormones.

Crystal structure of an affinity-matured prolactin complexed to its dimerized receptor reveals the topology of hormone binding site 2.

We report the first crystal structure of a 1:2 hormone.receptor complex that involves prolactin (PRL) as the ligand, at 3.8-A resolution. Stable ternary complexes were obtained by generating affinity-matured PRL variants harboring an N-terminal tail from ovine placental lactogen, a closely related PRL receptor (PRLR) ligand. This structure allows one to draw up an exhaustive inventory of the residues involved at the PRL.PRLR site 2 interface, consistent with all previously reported site-directed mutagenesis data. We propose, with this description, an interaction model involving three structural components of PRL site 2 ("three-pin plug"): the conserved glycine 129 of helix alpha3, the hydrogen bond network involving surrounding residues (glycine cavity), and the N terminus. The model provides a molecular basis for the properties of the different PRL analogs designed to date, including PRLR antagonists. Finally, comparison of our 1:2 PRL.PRLR(2) structure with those of free PRL and its 1:1 complex indicates that the structure of PRL undergoes significant changes when binding the first, but not the second receptor. This suggests that the second PRLR moiety adapts to the 1:1 complex rather than the opposite. In conclusion, this structure will be a useful guiding tool for further investigations of the molecular mechanisms involved in PRLR dimerization and activation, as well as for the optimization of PRLR antagonists, an emerging class of compounds with high therapeutic potential against breast and prostate cancer.

Native and recombinant bovine placental lactogens.

The bovine placenta produces a wide variety of proteins that are structurally and functionally similar to the pituitary proteins from the GH/PRL gene family. Bovine placental lactogen (bPL) is a 200-amino acid long glycoprotein hormone that exhibits both lactogenic and somatogenic properties. The apparent molecular masses of purified native (n) bPL molecules (31-33 kDa) exceed 23 041 Da, which is the theoretical molecular mass of the protein core. At least six isoelectric variants (pI: 4.85-6.3) of bPL were described in cotyledonary extracts and three different bPL isoforms (pI: 4.85-5.25) were found in fetal sera. The bPL molecules that are detected in higher concentrations in peripheral circulation exhibit a more acidic pI than those present in placental homogenates. This may reflect an important glycosylation process occurring just prior to the bPL secretion. The bPL mRNA is transcribed in trophectoderm binucleate cells starting from Day 30 of pregnancy until the end of gestation. In mothers, bPL is involved in the regulation of ovarian function, mammogenesis, lactogenesis, and pregnancy stage-dependent adaptation of nutrient supplies to the fetus. Due to the higher fetal, compared to maternal concentrations of circulating hormone, it has been suggested that bPL primarily targets fetal tissues.

P57kip2 immunohistochemical expression and ultrastructural findings of gestational trophoblastic disease and related disorders.

Gestational trophoblastic disease (GTD) is a unique spectrum of diseases ranging from complete hydatidiform mole (CHM), partial hydatidiform mole (PHM), and invasive mole (IM) to choriocarcinoma (CC). Placental site trophoblastic tumor (PSTT) and epithelioid trophoblastic tumor (ETT) have been classified as related disorders. Mesenchymal dysplasia (MD) may be misdiagnosed as PHM; however, it is said to have a quite different histogenesis from PHM. P57kip2 is the protein product of a paternally imprinted or maternal gene that inhibits cyclin-dependent kinases (CDK), thus serving to inhibit cell proliferation and to suppress tumor growth. Its lack of expression in trophoblastic disease plays a role in its abnormal proliferation and differentiation. In this study, P57kip2 immunostaining was absent in the trophoblastic layers of CHM and was positive in the trophoblast layer of nonmolar villi and MD. Ultrastructure of complete molar cystic villi showed tree-like branching of microvillous processes and intracytoplasmic lacunae without capillaries in the stroma, whereas MD contained many newly formed blood vessels and collagen. Also, large lacunae with microvilli and polymorphic nuclei of syncytiotrophoblast cells with well-developed organelles were observed in IM. Lung ETT following CHM and normal deliveries showed two types of large mononuclear cells and binuclear cells with abundant organelles and bundles of intermediate-type filaments in the stroma.

Vasoinhibins: endogenous regulators of angiogenesis and vascular function.

Vasoinhibins are a family of peptides derived from prolactin, growth hormone and placental lactogen that act on endothelial cells to suppress vasodilation and angiogenesis and to promote apoptosis-mediated vascular regression. Some of the pathways by which vasoinhibins act have now been defined, and recent developments indicate that endogenous vasoinhibins exert tonic and essential actions on blood vessel growth, dilation and regression in vivo. By studying the pathways that can generate vasoinhibins, and the nature of their receptors and key biological mediators, it should be possible to clarify the role of vasoinhibins in controlling vascular function in health and disease.

Crystal structure and site 1 binding energetics of human placental lactogen.

In primates, placental lactogen (PL) is a pituitary hormone with fundamental roles during pregnancy involving fetal growth, metabolism, and stimulating lactation in the mother. Human placental lactogen (hPL) is highly conserved with human growth hormone (hGH) and both hormones bind to the hPRLR extracellular domain (ECD), the first step in receptor homodimerization, in a Zn2+-dependent manner. A modified surface plasmon resonance method was developed to measure the kinetics for hPL and hGH binding to the hPRLR ECD, with and without Zn2+ and showed that hPL has about a tenfold higher affinity for the hPRLR ECD1 than hGH. The crystal structure of the free state of hPL has been determined to 2.0 A resolution showing the molecule possesses an overall structure similar to other long chain four-helix bundle cytokines. Comparison of the free hPL structure with the 1:1 complex structure of hGH bound to the hPRLR ECD1 suggests that two surface loops undergo conformational changes >10 A upon binding. An 18 residue Ala-scan was used to characterize the binding energy epitope for the site 1 interface of hPL. Individual alanine substitutions at five positions reduced binding affinity by a DeltaDeltaG > or = 3 kcal mol(-1). A comparison of the hPL site 1 epitope with that previously determined for hGH indicates contributions of individual residues track reasonably well between hPL and hGH. In particular, residues involved in the zinc-binding site and Lys172 constitute the principal binding determinants for both hormones. However, several residues that are identical between hPL and hGH contribute quite differently to the binding of the hPRLR ECD1. Additionally, the overall magnitudes of the DeltaDeltaG changes observed from the Ala-scan of hPL were markedly larger than those determined in the comparative scan of hGH to the hPRLR ECD1. The structural and biophysical data presented here show that subtle changes in the structural context of an interaction can lead to significantly different effects at the individual residue level.

Bacterial expression of prolactin family proteins.

Complementary DNAs of three recombinant proteins related to the prolactin family: ovine placental lactogen (oPL), ovine prolactin (oPRL), and rabbit soluble extracellular domain of prolactin receptor (rbPRLR-ECD) were subcloned by different methods and inserted into prokaryotic expression plasmids. Escherichia coli cells transformed with those plasmids overexpressed the respective proteins either by induction or constitutively, resulting in accumulation of the recombinant proteins in insoluble inclusion bodies, which were subsequently purified, used for refolding and purifying of the proteins by one-step chromatography. The isolated oPL, oPRL, and rbPRLR-ECD were biologically active over >95% pure monomers. Ten-liter bacterial culture yielded hundreds of milligrams or more than gram quantities of recombinant proteins. The methodology described in the present chapter allows large-scale preparation of pure, monomeric, biologically active oPL, oPRL, and rbPRLR-ECD suitable for performing in vitro and in vivo experiments.

Solution structure of human prolactin.

We report the solution structure of human prolactin determined by NMR spectroscopy. Our result is a significant improvement over a previous structure in terms of number and distribution of distance restraints, regularity of secondary structure, and potential energy. More significantly, the structure is sufficiently different that it leads to different conclusions regarding the mechanism of receptor activation and initiation of signal transduction. Here, we compare the structure of unbound prolactin to structures of both the homologue ovine placental lactogen and growth hormone. The structures of unbound and receptor bound prolactin/placental lactogen are similar and no noteworthy structural changes occur upon receptor binding. The observation of enhanced binding at the second receptor site when the first site is occupied has been widely interpreted to indicate conformational change induced by binding the first receptor. However, our results indicate that this enhanced binding at the second site could be due to receptor-receptor interactions or some other free energy sources rather than conformational change in the hormone. Titration of human prolactin with the extracellular domain of the human prolactin receptor was followed by NMR, gel filtration and electrophoresis. Both binary and ternary hormone-receptor complexes are clearly detectable by gel filtration and electrophoresis. The binary complex is not observable by NMR, possibly due to a dynamic equilibrium in intermediate exchange within the complex. The ternary complex of one hormone molecule bound to two receptor molecules is on the contrary readily detectable by NMR. This is in stark contrast to the widely held view that the ternary prolactin-receptor complex is only transiently formed. Thus, our results lead to improved understanding of the prolactin-prolactin receptor interaction.

Colloidal effect of albumin on the placental lactogen and chorionic gonadotrophin releases from human term placental explants.

Albumin has been reported to stimulate the release of placental lactogen and chorionic gonadotrophin from human term placental explants within physiological concentrations. This study aimed at characterizing further its effect on the placental hormonal secretion. The placental lactogen and chorionic gonadotrophin secretory response of incubated explants to 5% albumin was reproduced by colloidal agents, i.e., dextran (4.5%) and polygelin (4%), indicating that a rise in colloidal osmotic pressure can elicit hormonal release from the syncytiotrophoblast. Their secretory effects were not modified by the absence of extracellular calcium or the presence of verapamil in the medium. The three agents also provoked a marked increase in (45)calcium outflow from preloaded and perifused explants that persisted in absence of extracellular calcium. These data indicate that the triggering effect of albumin on placental lactogen and chorionic gonadotrophin release can be partly reproduced by colloidal agents and is independent of extracellular calcium.

The mouse prolactin gene family locus.

In the mouse, there is a large family of paralogous genes closely related to PRL. The objective of this report was to investigate the organization of the mouse PRL gene family locus. PRL family genes reside on chromosome 13 of the mouse genome. The PRL gene family members were localized to a series of overlapping bacterial artificial chromosome clones and arranged based on structural relationships. Additionally, several new members of the PRL gene family were identified. Placental lactogen I (PL-I) was found to be encoded by three closely related (>98% exon sequence identity) contiguous genes (termed: PL-Ialpha, PL-Ibeta, and PL-Igamma). Two previously unidentified mouse orthologs for members of the rat PRL family, PRL-like protein-I (PLP-I) and PLP-K were discovered, as were two new members of the PLP-C subfamily, PLP-Cgamma and PLP-Cdelta, and two new entirely unique members of the PRL family, PLP-N and PLP-O. Amino acid sequences predicted from the latter two genes most closely resembled proliferin-related protein. Each of the nine newly discovered genes is expressed in trophoblast cells of the mouse placenta in a gestationally specific pattern. In summary, elucidation of the mouse PRL gene family locus provides new insights into the expansion of the mouse PRL family and new tools for studying the genetics and biology of its members.

Mechanism of ruminant placental lactogen action: molecular and in vivo studies.

Ruminant placental lactogens (PLs) are structurally related to prolactins (PRLs) and growth hormones (GHs) and are secreted by placentae. Ruminant PLs are unusual in their capacity to bind and activate PRL and GH receptors (Rs) from other species. The present minireview summarizes several works showing that unlike in heterologous species (rat, rabbit, human), in homologous (ruminant) species, PLs act by activating PRLRs or by heterodimerizing GHRs and PRLRs, and suggests that this may be the main mechanism of PL action in vivo. Mutations impairing the ability of ovine (o)PL or bovine (b)PL to form complexes with PRLRs (but not with GHRs) do not cause loss of biological activity, because the transient existence of the homodimeric complex is still sufficient to initiate the signal transduction; however, mutants do lose their ability to activate homologous PRLRs. To explain this difference, we proposed a novel term-minimal time of homodimer persistence-which assumes that to initiate the signal transduction, a "minimal time" of homodimer existence is required for transphosphorylation of associated JAK2s. In interactions between ruminant PLs and homologous PRLRs, this minimal time is met through the interaction with homologous PRLRs, which has a shorter half-life than with heterologous PRLRs. Thus oPL or bPL are active in cells possessing both homologous and heterologous PRLRs. Mutations of PLs decrease the affinity, shortening the "time of homodimer persistence." In heterologous interactions, the minimal time is still sufficient to initiate the biological activity, whereas in homologous interactions, which in any case are weaker, further destabilization of the complex shortens its persistence below the minimal time, causing loss of biological activity.

An unusual member of the human growth hormone/placental lactogen (GH/PL) family, the testicular alternative splicing variant hPL-A2: recombinant expression revealed a membrane-associated growth factor molecule.

The human growth hormone/placental lactogen (GH/PL) gene cluster consists of five highly-related genes (GH-N, GH-V, PL-L, PL-A, PL-B). This evolutionarily young gene cluster codes for an array of mRNAs and proteins, such as the major 22 k forms (hGH-N/V, identical PL-A and B), 20 k and 17.5 k hGH-N and the recently described 25 k hGH-Delta4, a presumably chimeric molecule. In addition, two longer alternatively spliced, (intron D retaining) mRNAs isoforms, termed PL-A2 and GH-V2, have been described in placenta and testis. To elucidate the role of hPL-A2 in male reproduction and pregnancy, testicular PL-A2 cDNA was cloned in a complementary overlapping 2-way RT-PCR approach to analyze translation, localization and structure/function of this unusual member of the GH/PL growth factor family. Analysis of insect mRNA revealed that intron D-retaining PL-A2 cDNA was expressed without splicing in the baculovirus expression system. Thus, PL-A2 mRNA does not represent a nuclear intermediate splicing product simply co-isolated with the mature RNA, but is a stable mRNA isoform generated by placental/testis-specific splicing factors. Recombinant protein was present in whole cell extracts, and no secreted protein was detected in the supernatant. Immunologically, the N-terminus of the 230 amino acid protein is similar to 22 k hPL-A/B, as determined by hPL-specific monoclonal antibodies. In contrast, the C-terminus shares a hydrophobic region presumably responsible for membrane insertion. By the use of confocal microscopy recombinant hPL-A2 was localized in the cell membrane. Thus, hPL-A2 might exert its function by modulating GH/PL actions or act as an independent growth-regulatory molecule itself and its functions in male reproduction and embryonic development remain to be investigated.

Ternary complex between placental lactogen and the extracellular domain of the prolactin receptor.

The structure of the ternary complex between ovine placental lactogen (oPL) and the extracellular domain (ECD) of the rat prolactin receptor (rPRLR) reveals that two rPRLR ECDs bind to opposite sides of oPL with pseudo two-fold symmetry. The two oPL receptor binding sites differ significantly in their topography and electrostatic character. These binding interfaces also involve different hydrogen bonding and hydrophobic packing patterns compared to the structurally related human growth hormone (hGH)-receptor complexes. Additionally, the receptor-receptor interactions are different from those of the hGH-receptor complex. The conformational adaptability of prolactin and growth hormone receptors is evidenced by the changes in local conformations of the receptor binding loops and more global changes induced by shifts in the angular relationships between the N- and C-terminal domains, which allow the receptor to bind to the two topographically distinct sites of oPL.

Ovine placental lactogen and ovine prolactin: partial proteolysis and conformational stability.

The high-resolution structure of ovine placental lactogen (oPL) and ovine prolactin (oPRL), not yet established in detail, was probed by limited proteolysis with the Glu-specific protease from Staphylococcus aureus V8. While in hGH there were no cleavage sites inside of any of the four alpha-helices, the analysis of the fragments obtained after partial proteolysis of oPL showed a site of cleavage at the putative third helix, suggesting that this helix is partially unwound at this point. The partial proteolysis of the rest of the molecule was compatible with a similar folding pattern for oPL, hGH and pGH, on the basis of the crystal structure of these last hormones. In the case of oPRL, proteolytic cleavage occurred at Glu residues which would be located at the end of the first helix and the beginning of the second in the hGH folding model, suggesting that these helices are shorter in oPRL than in hGH. In order to gain further insight on the folding of these molecules, circular dichroism and intrinsic fluorescence measurements were used to examine the effect of denaturing conditions on oPL and oPRL. After exposure to 6 M guanidine the unfolding of both proteins was completely reversed upon elimination of the denaturing agent. In contrast, exposure to pH 3.0 caused an irreversible decrease in the alpha-helical content in both hormones, most striking for oPL, indicating that this hormone is less stable than oPRL or hGH.

Functional domains of human growth hormone necessary for the adipogenic activity of hGH/hPL chimeric molecules.

Genetic analysis through construction of chimeric genes and their transfection in mammalian cells could provide a better understanding of biological functions of native or modified proteins, and would allow the design of new gene constructs encoding peptides that mimic or block ligand interaction with target tissues. To identify the hGH domains responsible for induction of adipose differentiation we constructed hGH/hPL chimeric molecules using homologous DNA mutagenesis, since hGH, but not human placental lactogen (hPL), promotes adipose differentiation in mouse 3T3-F442A cells. We assayed their adipogenic activity in an autocrine/paracrine biological model consisting of transiently transfected 3T3-F442A cells with the chimeric constructs. Plasmid DNAs carrying these constructs were transfected into growing 3T3-F442A cells, and cultures were further maintained for 7 days to differentiate into adipocytes. Secretion of transfected hGH/hPL chimeric proteins into the medium was in the range of 5-25 ng/ml. Adipogenic activity was a property only of those chimeric proteins that contained hGH exon III together with either hGH exon II or hGH IV. Our results also suggest that hGH binding site-2 is composed of two structural subdomains: subsite 2A encoded by exon II of hGH and subsite-2B encoded by exon IV. We also suggest that full adipogenic activity requires the presence of binding site-1 and any of the subsites of binding site-2. This simple autocrine/paracrine biological model of gene transfection allows the analysis of specific biological activity of products encoded by modified genes.

Opposing actions of intact and N-terminal fragments of the human prolactin/growth hormone family members on angiogenesis: an efficient mechanism for the regulation of angiogenesis.

Angiogenesis, the process of development of a new microvasculature, is regulated by a balance of positive and negative factors. We show both in vivo and in vitro that the members of the human prolactin/growth hormone family, i.e., human prolactin, human growth hormone, human placental lactogen, and human growth hormone variant are angiogenic whereas their respective 16-kDa N-terminal fragments are antiangiogenic. The opposite actions are regulated in part via activation or inhibition of mitogen-activated protein kinase signaling pathway. In addition, the N-terminal fragments stimulate expression of type 1 plasminogen activator inhibitor whereas the intact molecules have no effect, an observation consistent with the fragments acting via separate receptors. The concept that a single molecule encodes both angiogenic and antiangiogenic peptides represents an efficient model for regulating the balance of positive and negative factors controlling angiogenesis. This hypothesis has potential physiological importance for the control of the vascular connection between the fetal and maternal circulations in the placenta, where human prolactin, human placental lactogen, and human growth hormone variant are expressed.

Endocrinology of pregnancy: chorionic somatomammotropins and pregnancy-associated glycoproteins: review.

The two main groups of placental proteins of ruminants are discussed in this paper: chorionic somatomammotropins (placental lactogens) and pregnancy-specific (-associated) proteins. Placental lactogens belong to the prolactin and growth hormone family. They stimulate mammogenesis, fetal growth and maternal metabolism. Pregnancy-specific proteins and pregnancy-associated glycoproteins belong to the aspartic proteinase family like pepsin, cathepsin D and E. These two groups of proteins are secreted in the maternal circulation by the binucleate cells after their migration to and fusion with the uterine cells. Their profiles were determined by radioimmunoassay (RIA). Further investigations are in progress to relate secretory profiles with alterations of the trophoblastic function such as those occurring in embryonic mortality, abortion, and fetal distress. The endocrine function of the primate and equine placenta is also discussed.