Multiple forms of Pregnancy-Associated Glycoproteins released in vitro by porcine chorion or placentomal and interplacentomal explants of wild and domestic ruminants

Characterization of the Pregnancy-Associated Glycoproteins (PAG) is important for studies of reproduction of various eutherian domestic, wild and endangered mammals. Distinct chorionic PAG genes are expressed in embryo-origin cells: pre-placental trophoblast (TR) and in placental trophectoderm (TRD) of various entherians. This study demonstrates in vitro production of the PAG proteins during long-term cultures of various chorionic explants: porcine TR or TRD, cotyledonary (CT) of European bison (Eb), and CT or intercotyledonary (intCT)-TRD of the cattle. Chorionic proteins isolated from media were analyzed by homologous or heterologous Western immunoblotting with anti-PAG sera, raised against cellular bovine or secretory porcine antigens. Used anti-PAG sera identified diverse molecular forms of released PAG proteins: 43-69 kDa for EbPAG proteins, 40-85 kDa for bovine PAG (bPAG), and 43-73 kDa for porcine PAG (pPAG). Immunoblotting revealed also that both CT and intCT-TRD explants secreted equivalent amounts of bPAG proteins. This useful system of in vitro protein production can provide native chorionic PAG proteins with placental unique carbohydrate chains. The PAG proteins are required as standard markers for diagnostic tests of pregnancy in domestic and wild mammals, in which seasonal reproductive processes are relatively difficult to control.     

Pregnancy-associated glycoprotein family (PAG)--as chorionic signaling ligands for gonadotropin receptors of cyclic animals

The chorionic pregnancy-associated glycoprotein (PAG) family was identified in pigs, cattle and other eutherian mammals. The objective of this study was to examine whether secretory chorionic proteins (including PAGs), produced in vitro by explants of porcine and bovine placental membranes, may interact with other proteins, i.e. gonadal and extragonadal binding sites. Trophoblast (TRF) and trophectoderm (TRD) explants of pigs (n=38; 14-61 dpc-day post coitum) or cotyledons (CT) of cows (n=5; 40-110 dpc) were long-term cultured. Released chorionic proteins were ultra-fractionated from media (>10 kDa) or precipitated [20-75% of (NH(4))(2)SO(4)]. The PAGs were monitored by Western/PAGE (30-73 kDa). Secretory TRF/TRD/CT (+PAG) proteins (0.78-25 microg/ligand) were examined by radioreceptor assay (RRA) with iodinated hCG ((125)I-hCG) for binding-effectiveness by gonadotropin receptors of cyclic pigs and cows (cRc). Gonadal and extragonadal cRc isolated from luteal-phase corpora lutea and uteri (cCLRc, cMYORc and cENDRc) were tested with positive control ligands: porcine LH and hCG (0.39-50 ng/ml). Control proteins produced in vitro by endometrial (END) explants of cyclic (cEND), pseudopregnant (PsEND) and pregnant (pEND) gilts were utilised as negative ligands (0.78-25 microg/ligand). Positive control ligands competed with (125)I-hCG for binding by cCLRc, cMYORc and cENDRc (18-61%/B(0) for hCG and 27-57%/B(0) for LH). Negative ligands (cEND, PsEND and pEND) did not show cRc bindings. This is the first RRA report indicating that in vitro produced porcine TRF/TRD proteins (+PAG) competed (P< or =0.05) with (125)I-hCG for binding by cCLRc, cMYORc and cENDRc in a concentration- and pregnancy stage-dependent manner. The highest competition with (125)I-hCG (up to P< or =0.001) was found for ultra-fractionated TRF/TRD proteins (>10 kDa) during early pregnancy (<22 dpc). The greatest competition (P< or =0.05) of precipitated porcine TRD proteins (>30 dpc) was detected for fractions obtained by saturation with use of 20% of (NH(4))(2)SO(4). Bovine CT proteins revealed lower competition of (125)I-hCG for bovine cCLRc (during 45 dpc only) that was more efficient with CT (up to 71%) than with non-labelled hCG (82%). The PAG proteins may play a role as potential "signal molecules", because they were able to interact with gonadotropin receptors of luteal-phase animals. It seems that the pPAG proteins may be luteoprotective chorionic-origin signals during implantation and placentation, according to binding-effectiveness of the chorionic ligands that was comparable to LH/hCG ligands with gonadal and extragonadal receptors of cyclic animals.     

A cloning and expression analysis of pregnancy-associated glycoproteins expressed in trophoblasts of the white-tail deer placenta

The pregnancy-associated glycoproteins (PAGs) are placental proteins that have been cloned from swine, sheep, goats, and cattle, but never from animals within the Cervidae family. The goal of this work was to characterize PAGs in white-tailed deer. Placenta and uterine tissues were collected from pregnant does at days 85 and 90 of pregnancy. RNA from cotyledons was used to amplify deer PAGs by RT-PCR. Ten distinct cDNAs were cloned and sequenced. Some normally conserved amino acids comprising the catalytic site were found to be altered in deer PAGs 4, 5, and 8; another PAG, (PAG-9) was a splice variant that lacked exon 7. In each case, these mutations would likely preclude proteolytic activity for these proteins. A phylogenetic analysis revealed that most of the deer PAGs fell within the ancient PAG grouping. The remainder fell within the more modern (BNC-specific) PAG group. Western blotting was performed with anti-PAG antibodies and this analysis revealed that deer PAGs comprise a heterogeneous group based on different antigenicities and electrophoretic mobilities. Immunohistochemistry and in situ hybridization revealed some unique localization patterns of PAGs in the deer placentome compared to those in other ruminants. Most notably, deer PAGs 4 and 5, which according to the phylogeny, are "ancient PAGs," were expected to be present in all trophoblasts; instead, they were localized to the BNC. Although many of the PAGs identified here are very similar to those in Bovidae, some are clearly distinct in their expression pattern and probably possess functional roles unique to cervid reproduction.     

In bovine binucleate trophoblast giant cells,pregnancy-associated glycoproteins and placental prolactin-related protein-I are conjugated to asparagine-linked<Emphasis Type="Italic"> N</Emphasis>-acetylgalactosaminyl glycans

Bovine binucleate trophoblast giant cells (BNCs) produce large amounts of PAS-positive cytoplasmic granules. After fusion of BNCs with uterine epithelial cells, the contents of these granules are released into the maternal stroma which underlies the uterine epithelium. Histochemically, the granules can be labeled with N-acetylgalactosamine-specific lectins ( Dolichos biflorus, Vicia villosa, and Wisteria floribunda agglutinins) and with Phaseolus vulgaris leucoagglutinin. In this study, we used lectin western blot analysis of proteins from fetal cotyledons to characterize the lectin binding glycoproteins. Lectin western blots showed several bands. A main band of approximately 65 kDa was identified as pregnancy-associated glycoproteins (PAGs) and a double band at 34-35 kDa as prolactin-related protein-I (PRP-I) by their crossreactivity with specific antisera. Enzymatic cleavage of N-linked glycans with peptide- N-glycanase F abolished the lectin binding to PRP and PAGs in western blots, revealing that the lectins bound to asparagine-linked glycans. The high specificity of the lectins was used for the enrichment of PRP-I and PAGs from placental cotyledons with Vicia villosa lectin affinity chromatography. The occurrence of the relatively uncommon asparagine-linked N-acetylgalactosaminyl glycans on secretory proteins of the BNCs suggests a functional role of this specific glycosylation pattern.     

N-glycodiversity of the Pregnancy-Associated Glycoprotein family (PAG) produced in vitro by trophoblast and trophectoderm explants during implantation, placentation and advanced pregnancy in the pig

The PAG family is encoded by distinct genes expressed in extra-embryonic chorionic membranes (TR--trophoblast, TRD--trophectoderm) of various pregnant mammals. The objective of our study was to determine N-glycodiversity of porcine PAG protein family (pPAG) produced in vitro by TR or TRD explants of gilts (n=26) throughout pregnancy (16-77 dpc). Explants were cultured for over 1200 h (TR, 16 dpc) or for 8 h (TRD, 17-77 dpc). Released proteins were isolated from media by separating ultra-filtration (>10 kDa). A deglycosylation (removal of N-linked carbohydrate side chains) of proteins was performed by glycopeptidase F, and compared to non-deglycosylated forms by PAGE-Western blotting with anti-pPAG sera and additionally to polypeptide pPAG precursors, coded by ORF of their cloned cDNAs. We demonstrated gestation-stage dependent diversity of deglycosylated/glycosylated forms of the pPAG proteins produced in vitro in the pig. TR explants harvested on 16 dpc during long term culture released 43 kDa pPAG proteins. These proteins were deglycosylated to approximately 36.9 and approximately 39.6 kDa (16 dpc). Tissue harvested on 17 dpc in vitro secreted 65-68 kDa pPAG proteins which were reduced to three forms, 50.6, 58.7 and 63.5 kDa. In addition, approximately 73.3 kDa major pPAG proteins (77 dpc) were reduced to at least three forms: approximately 39.6, approximately 36.9 and approximately 33.4 kDa. Such N-deglycosylation was not detected on days 25-61. N-deglycosylation of native pPAG proteins clearly corresponded to three N-glycosylation sites of asparagines (N-x-S/T) found in ORF of the pPAG2-like precursors, identified by their in silico translated cDNAs. Thus, the pregnancy-stage dependent N-glycodiversity of the pPAG protein family, containing an average 9.66% of N-linked oligosaccharides, may play some role(s) in porcine conceptus attachment, successful implantation and during advanced pregnancy.     

Pregnancy associated glycoprotein-1, -6, -7, and -17 are major products of bovine binucleate trophoblast giant cells at midpregnancy

Pregnancy associated glycoproteins (PAGs) are extensively glycosylated secretory proteins of ruminant trophoblast cells. In cattle placenta several PAG cDNAs are expressed, but the variety of correspondent proteins and their degree of glycosylation are not well characterized. Thus, we purified PAGs by using a protocol which included a lectin (Vicia villosa agglutinin) affinity chromatography. Due to their specific glycosylation pattern, PAGs derived from binucleate trophoblast giant cells were highly enriched by this protocol. PAGs were purified from cotyledons of 2 day 100 placentas and from a single placenta at day 155 and 180. In all samples three major bands (75; 66; 56 kDa) were detected by one-dimensional SDS-PAGE. Mass-spectrometric analysis identified the 75 kDa band as a mixture of PAG-7 and PAG-6, the 66 kDa band as PAG-1 and the 56 kDa band as PAG-17. N-terminal sequencing of the day 100 sample confirmed the mass spectrometric identifications. Enzymatic release of N-glycans with peptide-N-glycanase-F from PAGs reduced the molecular weight to approximately 37 kDa which corresponds to the theoretical molecular mass of PAGs. Limited peptide-N-glycanase-F treatment revealed that all four N-glycosylation sites are quantitatively occupied in PAG-1. Compared to PAG-1 the number of potential N-glycosylation sites is lower in PAG-17 (three sites) and higher in PAG-6 and -7 (five and six sites, respectively). This suggests that the number of attached N-glycans is the main determinant of molecular mass of bovine PAGs. The degree of glycosylation may be a major factor regulating the plasma half life of PAGs.     

Mapping of surface glycoproteins of Trypanosoma cruzi by two-dimensional electrophoresis. A correlation with the cell invasion capacity

The cell-surface iodinatable proteins of Trypanosoma cruzi have been analyzed by two-dimensional polyacrylamide gel electrophoresis under equilibrium conditions. Antigenic polypeptides were characterized after immunoprecipitation and glycoproteins were identified by means of lectin-affinity chromatography. Two glycoproteins, with affinity for concanavalin A, were found to be common to both infective (trypomastigote) and non-infective (epimastigote) forms: protein 1 (90 kDa, pI 5.5-6.5) and protein 2 (80 kDa, pI 5.3-6.3). In epimastigotes a specific concanavalin-A-binding surface glycoprotein (70 kDa, pI 5.5) was identified. Trypomastigote forms, on the other hand, presented several specific iodinatable surface components: glycoproteins 3(85 kDa, pI 5.5), 4 (85 kDa, pI 5.0), 6 (100 kDa, pI 6.5), 7 (120 kDa, pI 6.3), 8 (68 kDa, pI 6.7) and several minor high-molecular-mass acid proteins, all containing glucose and/or mannose, and glycoprotein 5 (85 kDa, pI 6.3-7.5), containing N-acetyl-D-glucosamine (Tc-85). Proteins 1, 2 and 5 were the only ones which gave clear evidence of charge heterogeneity. Most of the surface proteins of trypomastigote forms, the exception being proteins 3, 4 and 8, were removed by treatment with trypsin. This proteolytic treatment results in 90% inhibition of the in vitro vertebrate-cell-invasion capacity of the parasites. Upon reincubation in culture medium for 4 h, the trypsin-removed glycoproteins are again detected, an observation that correlates well with the recovery of the cell-penetration capacity observed in the same period.     

Double radial immunodiffusion as a tool to identify pregnancy-associated glycoproteins in ruminant and nonruminant placentae

Pregnancy-associated glycoproteins (PAGs) are antigens synthesized in the superficial layers of the ruminant trophoblast. Initially, they were identified either as proteins released into the maternal bloodstream (where they have applications in pregnancy diagnosis) (PAG1) or as molecules binding to the LH receptor (PAG2). In this study, double radial immunodiffusion was used to test the ability of antisera raised against different PAG molecules (bovine, ovine and caprine) to react with placental extracts from nonruminants (rabbit, cat, mouse, pig, and wild pig) and ruminants (cow, ewe, and goat). Placental extracts from all nonruminants tested except rabbit reacted with anti bovine PAG2 (anti-boPAG2). Extracts of ruminant placentas reacted with different antisera, confirming the expression of various PAG molecules. According to the time at which the placentas were collected (early or middle pregnancy), the reaction differed as regards the thickness, position, and number of precipitation lines, suggesting that PAG expression varies as pregnancy progresses. Bos indicus and Bos taurus placental extracts exhibited different reactions with anti-boPAG2: a single precipitation line in the former case and two lines in the latter. This suggests differential expression of boPAG2 related glycoproteins in these two subspecies.     

Radiocompetition of secretory pregnancy-associated glycoproteins as chorionic ligands with luteal and uterine gonadotrophin receptors of pregnant pigs

Porcine pregnancy-associated glycoprotein (pPAG) family is very promiscuous and its role(s) remains unknown. The objective of this study was to identify whether secretory placental proteins (including pPAGs), produced in vitro by porcine chorionic explants, may interact with other proteins/targets, i.e. luteal and uterine binding sites of pregnant pigs. Trophoblast (TRF) and trophectoderm (TRD) were harvested during peri-implantation and placentation periods (14-61 dpc-day post coitum). In vitro-produced TRF/TRD proteins were isolated from media by ultrafractionation (>10 kDa MWCO) or precipitation with 20-75% saturation of (NH(4))(2)SO(4) and pPAG proteins were monitored by Western blotting. Secretory TRF/TRD ligands (including PAGs) were serially diluted (0.78-25 microg/ligand) and examined by radioreceptor assay (RRA). Luteal and uterine membrane receptors of pregnant pigs (pRc) were isolated from corpora lutea (pCLRc), myometrium (pMYORc) and endometrium (pENDRc). The three pRc types were harvested during three periods of pregnancy: 14 dpc (14 Rc), 21-26 dpc (21-26 Rc) and 31 dpc (31 Rc). The RRA competitions of individual TRF or TRD ligands were performed with (125)I-hCG as tracer and different pRc types. The RRA results of TRF/TRD were compared to hCG/pLH ligands--as positive controls (0.39-50 ng/ml), and endometrial (END) proteins (0.78-25 microg/ml) produced in vitro by END explants of cyclic, pseudopregnant and pregnant gilts (cEND, PsEND and pEND, respectively)--as negative control ligands. Results indicated that secretory TRF/TRD proteins (+pPAGs) were able to compete with (125)I-hCG for binding with other proteins/targets, i.e. luteal and uterine receptors of pregnant pigs (pCLRc, pMYORc and pENDRc) in a concentration- and pregnancy stage-dependent manner. This study indicated that porcine secretory 14-15 dpc TRF (pPAG; 30-73 kDa) ligands, effectively displaced (125)I-hCG tracer from pCL14Rc (up to P< or =0.01), corresponding to displacement by hCG and porcine LH. During the early stage of pregnancy, some competition tendency (P< or =0.01) was also detected for TRF ligands (14-15 dpc) with pEND14Rc. As pregnancy advanced, significant (125)I-hCG competition (at least P< or =0.05) with secretory semi-purified TRD ligands (30-42 dpc) was determined for all types of examined receptors pCL31Rc, pMYO31Rc and pEND31Rc, mainly with TRD fractions precipitated by 20% saturation of (NH(4))(2)SO(4). It seems that chorionic pPAG family can be involved in luteoprotective mechanism during implantation and placentation, according to the binding-interaction with luteal and uterine gonadotropin receptors of pregnant pigs.     

Isolation and partial characterization of three pregnancy-associated glycoproteins from the ewe placenta

Pregnancy-associated glycoproteins (PAGs) are synthesized in the outer epithelial layer of the placenta in artiodactyls. In this work, three novel ovine PAGs were isolated from late-pregnancy fetal cotyledons and characterized biochemically. The isolation procedure included acid and ammonium sulfate precipitations and anion and cation exchange chromatographies. The isolated PAGs have different NH(2)-terminal amino acid sequences (RGSXLTILPLRNMRDIVY, ISRVSXLTIHPLRNIMDML, and RGSNLTIHPLRNIRD) and apparent molecular masses (55, 57, and 59 kDa). Each shows several isoforms with different pI values. The three proteins share high sequence identity with each other and with other ovine, bovine, and caprine PAGs. They have not been described previously. The ovPAG-59 sequence differs from the previously identified ovPAG-4 sequence (determined by DNA cloning and sequencing) at only one position among the 15 N-terminal residues. The newly characterized ovPAGs and the procedure used to isolate them will be helpful in producing new antisera for investigating PAG secretion in pregnant ewes.     

Ultrasound fetal measurements and pregnancy associated glycoprotein secretion in early pregnancy in cattle recipients carrying somatic clones

Somatic cloning in the bovine species leads to high levels of fetal losses which occur throughout pregnancy. These losses are most often associated with fetal overgrowth, a syndrome known as large offspring syndrome (LOS), and excessive maternal plasma pregnancy serum protein 60 (PSP60), a protein similar to a pregnancy-associated glycoprotein of 67 kDa (PAG I67) produced by the bovine placenta. Predicting the outcome of pregnancies initiated from cloned embryos has become an important issue both to prevent potential harm to the mother because of excessive fetal size at birth and also to get a better understanding of the relationships between growth, differentiation and placental functions in developing cloned fetuses. Here, we report on a systematic analysis of fetal and placental development in the first trimester of pregnancy performed by ultrasonographic imaging and by measurement of the maternal concentrations of pregnancy associated glycoproteins (PAGS), using four different radioimmunoassays (RIA) (two homologous RIA systems with PSP60 and PAG I67; two heterologous RIA systems with PAG I67 as standard and tracer, and antisera anti-caprine PAGs). We showed that crown-rump length (CRL) in clones appeared smaller than controls at 35, 50 and 62 days (P<0.05). At 62 days of pregnancy, CRL in cloned fetuses that died before 90 days was smaller compared to the other cloned fetuses (P<0.05) whereas the width of the fetal sack and the biparietal diameter (BPD) was larger in fetuses that developed LOS in late gestation (P<0.05). Maternal PAGs concentrations were statistically different between controls and all clone recipients as early as Day 34, suggesting early abnormal placental glycoprotein synthesis for clone pregnancies regardless of pregnancy outcome. This work provides a practical, non-invasive tool to follow up clone pregnancies and suggests that primary growth retardation and abnormal placental function precedes excessive fetal and placental growth at later stages of pregnancy.     

Autoimmunization of ewes against pregnancy-associated glycoproteins does not interfere with the establishment and maintenance of pregnancy

Pregnancy-associated glycoproteins (PAGs) are a large grouping of placental proteins that belong to the aspartic peptidase gene family. Although useful to detect pregnancy in ruminant species, the function of these molecules is unclear. Several PAGs expressed by trophoblast binucleate cells can enter the maternal circulation, suggesting that they could have a systemic role in altering maternal physiology. The objective of this work was to examine whether these circulating placental antigens were important in pregnancy by actively immunizing ewes against them. PAGs were purified by pepstatin-affinity chromatography and conjugated to the immunogenic protein, keyhole limpet hemocyanin (KLH). Ewes were immunized with PAG-KLH conjugate (n = 22) or with KLH alone (n = 9), and bred to intact rams. Blood samples, collected on Day 0 (day of estrus), Day 10, Days 15 to 25 and weekly throughout pregnancy, were analyzed for PAG by an ELISA. On Day 30, pregnancy was confirmed by ultrasound. Ewes immunized against PAG-KLH produced a range of reactive anti-PAG titers, whereas all immunized ewes had high anti-KLH immunoreactivity. PAGs became detectable in the anti-KLH (control) ewes at Day 21.6 ± 2.2 of pregnancy. Those ewes immunized against PAGs (n = 7), that had very low immunoreactivity toward PAGs, had measurable PAG by Day 22.9 ± 1.3, and their PAG serum profiles throughout pregnancy did not differ from the controls. Those exhibiting moderate to high anti-PAG immunoreactivity (n = 15), had significantly lower PAG concentrations than controls, with antigen not becoming detectable until Day 48.1 ± 15.6. The decrease in circulating PAG in the immunized animals did not correlate with changes in pregnancy rates, lamb number or lamb birth weight. These results suggest that while PAGs may play a role in maintaining pregnancy, their major contribution is likely to be at the fetal-maternal interface. Their actions at extra-placental sites are presumably of more secondary importance.     

Synthesis of surfactant-associated glycoprotein A by rat type II epithelial cells. Primary translation products and post-translational modification

Surfactant-associated glycoproteins A, 38 (A3), 32 (A2) and 26 (A1) kDa, pI (4.2-4.8), were identified as related proteins present in surfactant isolated from rat lung lavage fluid. Differences in size and charge among surfactant-associated glycoproteins A were related to differences in glycosylation as determined by reduction of the larger forms (38 and 32 kDa) to 26 kDa by endoglycosidase F and by increased isoelectric points of the glycosylated forms after treatment with neuraminidase. Synthesis and secretion of surfactant-associated glycoproteins A and precursors were demonstrated in purified rat Type II epithelial cells by immunoprecipitation of [35S]methionine-labelled proteins with anti-surfactant-associated glycoprotein A antisera. In pulse-chase experiments, labelled proteins 26-34 kDa, appeared within 10 min and smaller forms co-migrated with surfactant-associated glycoprotein A from alveolar lavage. The relative abundance of the larger molecular mass forms (30-34 kDa, pI 4.8) increased at later times up to 3 h. More acidic mature forms, which co-migrated with surfactant-associated glycoproteins A2 and A3 in surfactant (38 and 32 kDa), were readily detectable in the media, but were not abundant forms in lysates of labelled Type II cells after 1-3 h of incubation. Primary translation products of surfactant-associated glycoprotein A were immunoprecipitated with monospecific anti-surfactant-associated glycoprotein A antiserum after in vitro translation of poly(A)+ mRNA isolated from adult rat lung. The immunoprecipitated translation product migrated at 26 kDa, pI 4.8, and migrated slightly faster than surfactant-associated glycoprotein A1 from surfactant. Treatment of surfactant-associated glycoprotein A with bacterial collagenase resulted in proteolytic fragments 23-20 kDa, pI 4.2-4.8, which no longer underwent sulfhydryl-dependent cross-linking, suggesting that the collagen-like domain was required for the sulfhydryl-dependent oligomerization. Surfactant-associated glycoproteins A are synthesized by rat Type II epithelial cells as pre-proteins, 26-34 kDa. Larger forms result primarily from N-linked glycosylation of the 26 kDa primary translation product. Mature, more acidic forms result from further addition of sialic acid.     

Identification and NH2-terminal amino acid sequence of three insulin-like growth factor-binding proteins in porcine serum.

Three distinct species of IGFBP in porcine serum were identified by NH2-terminal amino acid sequence analysis. The IGFBPs identified include pIGFBP-2 (34 kDa), three isoforms of pIGFBP-3 (43, 40 and 30 kDa) and two isoforms of pIGFBP-4 (30 and 26 kDa). The three isoforms of pIGFBP-3 were found to have a common NH2-terminal amino acid sequence, as were the two isoforms of pIGFBP-4. These results indicate that porcine serum contains a truncated form of IGFBP-3 and two forms of pIGFBP-4, similar to those previously isolated from human and rat serum. Furthermore, the presence of a truncated form(s) of the GH-dependent IGFBP-3 in porcine serum suggests that elucidating its origin and function may be important in understanding how IGFBPs affect the somatogenic actions of GH.     

Identification and characterization of a novel outer membrane protein (OMP J) of Moraxella catarrhalis that exists in two major forms

Moraxella catarrhalis is a common commensal of the human respiratory tract that has been associated with a number of disease states, including acute otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. During studies to investigate the outer membrane proteins of this bacterium, two novel major proteins, of approximately 19 kDa and 16 kDa (named OMP J1 and OMP J2, respectively), were identified. Further analysis indicated that these two proteins possessed almost identical gene sequences, apart from two insertion/deletion events in predicted external loops present within the putative barrel-like structure of the proteins. The development of a PCR screening strategy found a 100% (96/96) incidence for the genes encoding the OMP J1 and OMP J2 proteins within a set of geographically diverse M. catarrhalis isolates, as well as a significant association of OMP J1/OMP J2 with both the genetic lineage and the complement resistance phenotype (Fisher's exact test; P < 0.01). Experiments using two DeltaompJ2 mutants (one complement resistant and the other complement sensitive) indicated that both were less easily cleared from the lungs of mice than were their isogenic wild-type counterparts, with a significant difference in bacterial clearance being observed for the complement-resistant isolate but not for its isogenic DeltaompJ2 mutant (unpaired Student's t test; P < 0.001 and P = 0.32). In this publication, we characterize a novel outer membrane protein of Moraxella catarrhalis which exists in two variant forms associated with particular genetic lineages, and both forms are suggested to contribute to bacterial clearance from the lungs.     

Apolipoprotein A-IGiessen (Pro143----Arg). A mutant that is defective in activating lecithin:cholesterol acyltransferase

Apolipoprotein A-IGiessen is a variant form of apo A-I that is displaced from the corresponding normal A-I isoforms on isoelectric focusing gels by a single charge unit towards the cathode [Utermann et al. (1982) J. Biol. Chem. 257, 501-507]. Three subjects heterozygous for the variant were detected in one family. The percentage of the total A-I in plasma represented by the A-IGiessen in these subjects ranged over 25-30%. The variant and normal major A-I isoforms from the proband (Y.J.) were purified by preparative isoelectric focusing and cleaved with CNBr. Analytical focusing of CNBr fragments demonstrated a charge difference between CB3Giessen and normal CB3. Sequence analysis of CB3Giessen revealed that a proline existing in normal A-I was replaced by an arginine in the variant A-I at residue 143. The ability of the mutant A-I to activate purified lecithin:cholesterol acyltransferase was determined in vitro. The cofactor activity of [Arg143]apolipoprotein A-I was about 60-70% of that demonstrated by control A-I. Residue 143 is in a putative beta-turn between two of the repeating amphiphilic helices in apolipoprotein A-I and may be a critical determinant of the protein's structure and function.     

Identification of a new aspartic proteinase expressed by the outer chorionic cell layer of the equine placenta

The pregnancy-associated glycoproteins (PAGs) are placental antigens that were initially characterized as pregnancy markers in the maternal circulation of domestic ruminant species. They are members of the aspartic proteinase gene family, having greatest sequence identity with pepsinogens. However, some are not capable of functioning as enzymes. The PAGs are associated with a large gene family within the Artiodactyla order (cattle, camels, pigs). So far, no members of this family have been characterized in species outside this order. This report describes the cloning and initial characterization of a PAG-like protein (equine PAG or ePAG) expressed in the placenta of the horse and zebra (order Perrisodactyla). Equine PAG is a proteinase capable of degrading 14C-hemoglobin and catalyzing the removal of its own pro-peptide. The ePAG mRNA is restricted to the chorion both prior to implantation and in the term placenta. Equine PAG is secreted from cultured placental tissue as both a processed (mature) and unprocessed (zymogen) form. Equine PAG shares similar identity with the PAGs and pepsinogens and probably arose from a pepsinogen-like precursor that gained the ability to be expressed in the placenta. The promoter of the ePAG gene shares sequence identity with the promoter from a bovine PAG gene but not with promoters of other aspartic proteinases. Therefore, we hypothesize that ePAG is a remnant of the pepsinogen-like progenitor gene that was expanded within the Artiodactyla to create the large and highly diverse PAG family.