Human Placental Protein Research Articles

Tac promoter vectors incorporating the bacteriophage T7 gene 10 translational enhancer sequence for improved expression of cloned genes in Escherichia coli

Two new plasmid expression vectors, pTacT7 and pTacT7L, have been constructed, which incorporate between the tac promoter and a downstream NcoI- HindIII polylinker sequence a synthetic sequence derived from the region up-stream from gene 10 of bacteriophage T7 ( g10-L). This sequence was recently shown to act as a translational enhancer (Olins et al., 1988) and was termed “Epsilon” ( Enhancer of Protein Synthesis Initiation) element (Olins and Rangwala, 1989). In this communication we describe in detail the construction of ptacT7 and ptacT7L. Furthermore, we present evidence that the “Epsilon” element is able to enhance 3 to 20-fold the expression levels of two poorly expressed test genes encoding the human placental proteins PP9 and PP15. On the other hand, the expression levels of two highly expressed test genes encoding the human placental proteins PP4 and FXIIIa could not be further enhanced by the presence of the “Epsilon” element. These experiments show that the T7 gene 10 leader sequence can be utilized to improve the expression yields of otherwise poorly expressed heterologous genes in Escherichia coli.

Structure and function of CD45: a leukocyte-specific protein tyrosine phosphatase.

CD45 is a large abundant leukocyte-specific cell surface glycoprotein 1. It is structurally heterogeneous consisting of a family of isoforms (Mr ~ 180-220K) that are distributed in characteristic cell-type specific patterns on different leukocyte sUbpopulations. Because of its leukocyte-specific tissue distribution, CD45 is a useful marker for the differential diagnosis of undifferentiated lymphoma 2. The primary structures of rat, mouse and human CD45 deduced from cDNA sequencing in the mid-1980’s revealed that the cytoplasmic domain of CD45 was unusually large 1, confirming earlier studies of a mutant CD45 molecule 3. Charbonneau et al. showed in 1988 that two tandem imperfect repeats within the large cytoplasmic domain of CD45 had highly significant sequence similarity with PTP1B, a soluble ~37K human placental protein tyrosine phosphatase (PTP) 4. Subsequently, CD45 was shown to have intrinsic PTP activity 5, suggesting that transmembrane PTPs might represent a novel class of receptors that oppose the action of protein tyrosine kinases (PTKs) and playa fundamental role in the regulation of cell growth and differentiation. Other putative transmembrane and intracellular PTPs in organisms as diverse as higher eukaryotes, Drosophila, yeast, bacteria, and viruses have now been identified and the complete primary structures of at least twenty PTPs have been determined (reviewed in Ref 6). CD45 is the best characterized of the transmembrane PTPs and serves as a prototype for this class of enzyme. An understanding of the function of CD45 in leukocyte physiology requires identification of its substrates, elucidation of the structuralfeatures of the molecule essential for PTP activity, and information about how its activity is regulated. This paper will review recent progress in defining the role of CD45 in lymphocyte signal transduction and summarize the current status of the rapidly growing PTP family.

Suppression by Human Placental Protein 14 of Natural Killer Cell Activity

Human decidua of early pregnancy contains considerable numbers of CD3-CD56+ natural killer (NK) cells. In this study, two major protein products of the decidua, placental protein 14 (PP14) and placental protein 12 (PP12), were tested for the ability to regulate human NK cell activity. In vitro overnight exposure to PP14 of blood lymphocytes or purified large granular lymphocytes (LGL) resulted in suppression of cytotoxicity against K562 target cells in a 4-h 51Cr release assay. The NK inhibition was dependent on concentrations of PP14, being detectable at 5 micrograms/ml and reaching maximum at 50 micrograms/ml. Manifestation of PP14-induced NK suppression required 18-h contact with NK cells. The suppression of NK activity by PP14 was not abolished by indomethacin. In a target binding assay the number of PP14-treated LGL binding to K562 was comparable to that of untreated ones. By contrast with PP14, PP12 produced no effects on NK cells. These results indicate that PP14 suppresses the function of NK cells, which might be involved in prevention of maternal immune rejection of fetus at the fetomaternal interface.

The human placental protein 14 (PP14) gene is localized on chromosome 9q34

PP14 protein (placental protein 14) is abundantly secreted by the human endometrium under the influence of progesterone. Human PP14 is homologous to beta-lactoglobulin, the main component of equine, bovine, and ovine milk whey. A genomic PP14 probe (PP14G1) was used for the chromosome assignment of the PP14 gene. Somatic hybrid cells enabled PP14G1 to be assigned to chromosome 9. In situ hybridization further refined this assignment to 9q34. The localization of the PP14 gene in the region of the ABO locus is consistent with the linkage described in bovines between beta-lactoglobulin and the J blood group (homologous to the human ABO group).

Zn +2 induces reversible cross-linking of human placental thyroid hormone nuclear receptor with no effect on hormone binding

Zn+2 is required for specific binding of c-erbA proteins to the hormone response elements of target genes. It is unclear whether Zn+2 is important for the binding of ligand to c-erbA proteins. The present study evaluated the effect of Zn+2 and other divalent cations on the binding of 3,3',5-triiodo-L-thyronine(T3) to the purified human placental c-erbA protein (h-TR beta 1). Zn+2 induced cross-linking of h-TR beta 1 to form aggregates in a dose-dependent manner with an apparent half-maximal concentration of approximately 200 microM at 22 degrees C. Cross-linking was reversible by the addition of 5 microM EDTA or 10 mM dithiothreitol. The cross-linked h-TR beta 1 bound T3. These results indicated Zn+2 had no effect on T3 binding and suggested that the cysteines and histidines involved in cross-linking are not essential for T3 binding.

Lung calcium-dependent phospholipid-binding proteins: structure and function

Distinct peptide maps of two rabbit lung Ca 2+-dependent phospholipid-binding proteins (PLBPs), 36 000 and 33 000, were generated by cyanogen bromide (CNBr) cleavage, trypsin or Staphylococcus aureus V8 proteinase digestion. The amino acid sequence of a CNBr-cleaved peptide of the 36 000 PLBP was aligned to the amino terminus of human lipocortin I with more than 77% identity, but had no identity with the known amino terminal sequence of other known annexins. Partial amino acid sequence of a 33 000 PLBP peptide demonstrated a close (56%) relationship to endonexin II, human placental anticoagulant protein, and porcine intestine protein II, but shared only 32% identity with lipocortin I, 30% with lipocortin II. Antiserum generated against purified 36 000 PLBP reacted strongly with the 33 000 PLBP, but did not react with any other rabbit lung cytosolic proteins. Both PLBPs inhibited the phospholipase A 2 reaction when dioleoyl phosphatidylcholine and phosphatidylglycerol vesicles or monolayers were used as substrates. In the vesicle assay, the phospholipase A 2 reaction was inhibited at lower substrate phospholipid concentrations but not at nearly saturating substrate concentrations. In the monolayer assay, the phospholipid-binding proteins did not inhibit phospholipase A 2 at a low phospholipid surface concentration of 3.8·10 −3 molecules/Å 2, but they did at higher surface concentrations between 1.1·10 −2 and 3.8·10 −2 molecules/Å 2. The inhibition of phospholipase A 2 by rabbit lung phospholipid-binding proteins is most likely due to the prevention of penetration by phospholipase A 2 into the interface, a requirement for the enzyme to act on the substrate.

Human placental protein methylase—I. Purification and characterization

1. 1. Protein methylase I (S-adenosylmethionine[:]protein-arginine N-methyltransferase; EC 2.1.1.23) which methylates protein-bound arginine residues has been purified from human term placenta 400-fold with an approximate yield of 6%. 2. 2. When histone was used as in vitro substrate, the methylation products were found to be N G-mono-, N G, N G-di- and N G, N 'G-dimethylarginine. The enzyme was found to be sensitive toward Cu 2+ with K i, value of 8 x lO -5M. The K m value for S-adenosyl-L-methionine was 5 x lO -6M. 3. 3. When this partially purified protein methylase I was incubated with isolated human placental nuclei and S-adenosyl-L-Imethyl-'Hlmethionine, the major endogenous [methyl- 3H]-labeled proteins were protein species of 23,38,45 and 68 kDa, the 23 kDa species being the most predominant. 4. 4. The endogenous enzyme activity during the pregnancy increased significantly, reaching more than 4 times the initial activity at the end of term.

Evolutionary conservation and structural determinants of the calelectrins (annexins)

Evolutionary conservation and structural determinants of the calelectrins (annexins)

Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42.

We have isolated cDNA clones from a human placental library that code for a low molecular weight GTP-binding protein originally designated Gp (also called G25K). This identification is based on comparisons with the available peptide sequences for the purified human Gp protein and the use of two highly specific anti-peptide antibodies. The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins (approximately 50% identical), and the rac proteins (approximately 70% identical). The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell-division-cycle protein CDC42. The human placental gene, which we designate CDC42Hs, complements the cdc42-1 mutation in S. cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein.

Annexin proteins PP4 and PP4-X. Comparative characterization of biological activities of placental and recombinant proteins

The human placental proteins PP4 and PP4-X, belonging to the annexin protein family, were expressed in Escherichia coli at high yield. The proteins were purified to homogeneity. The physicochemical parameters of the recombinant proteins were determined and compared with those of their natural placental counterparts. Except for a minor change in the pI, the proteins appeared to be indistinguishable by several criteria. Both recombinant PP4 and recombinant PP4-X were biologically active in a thromboplastin inhibition test and in a phospholipase A2 inhibition test.

Unique guanine nucleotide binding properties of the human placental GTP-binding protein Gp

Gp is a major GTP-binding protein of human placenta and platelets [Evans, T., Brown, M. L., Fraser, E. D., & Northup, J. K. (1986) J. Biol. Chem. 261, 7052-7059]. High-affinity guanine nucleotide binding is associated with a polypeptide migrating identically with H-ras on SDS-PAGE. We have characterized the interactions of preparations of purified human placental Gp with guanine nucleotides in detergent solution. Equilibrium binding studies with [35S]GTP gamma S, [3H]Gpp(NH)p, and [3H]GTP identified a single class of sites with a dissociation constant of 10 +/- 1, 153 +/- 61, and 125 +/- 77 nM for the ligands, respectively. These three ligands were mutually competitive with Ki values consistent with the Kd values from direct binding experiments. Competition for the binding of [3H]Gpp(NH)p was used to determine the specificity of the site. Ki values determined from this assay were 14 nM for GTP gamma S, 143 nM for Gpp(NH)p, 3.3 microM for GDP beta S, 69 nM for GTP, and 64 nM for GDP. ATP, ADP, cAMP, cGMP, and NAD+ had no detectable affinity for this site. While the equilibrium binding data fit well to a single class of sites, association kinetics of these ligands were better fit to two rate constants. Dissociation kinetics, however, were not clearly resolved into two rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Human Placental Protein 14 Gene: Sequence and Characterization of a Short Duplication

Differential hybridization of cDNAs corresponding to mRNAs expressed in the human endometrium during the secretory phase or during the first trimester of pregnancy, but not during the proliferative phase, allowed us to isolate and characterize cDNAs encoding human placental protein 14 (PP14). The cDNA was used to isolate the PP14 gene from a human genomic library. The entire gene encompasses 5.05 kb divided into seven exons by six introns. The human PP14 gene shows identical organization with the ovine beta-lactoglobulin gene, as expected from protein homology. Sequencing of 3 kb of the 5'-flanking region of the gene allowed us to characterize a 400-bp duplication of the PP14 gene lying at position -2,660. This duplication was homologous to 100 bp of exon 4 and 300 bp of intron 4, including 180 bp corresponding exactly to the right arm of an Alu element lying on the complementary strand. This homology suggests that this duplication may have arisen through a retroposition event.

Cloning and Expression of a cDNA Encoding Human Placental Protein 11, a Putative Serine Protease with Diagnostic Significance as a Tumor Marker

The placental protein 11 (PP11) can act as a tumor marker because of its specific association with various forms of cancer. A lambda gt11 cDNA library prepared from human placenta was screened with a polyclonal anti-PP11 antiserum. Out of 10(6) independent clones, only one clone reacted with the anti-PP11 antiserum. The isolated cDNA coded only for the carboxy-terminal part of PP11 and was subsequently used to rescreen a lambda gt10 placental cDNA library. Two cDNA clones out of 10(6) screened were identified encoding the entire protein of 369 amino acids, including a typical hydrophobic signal sequence of 18 amino acids. Expression of the PP11 cDNA coding sequence in Escherichia coli resulted in the synthesis of a protein with the expected size which can be specifically immunoprecipitated with anti-PP11 antiserum. Fractionation experiments revealed that two forms of the protein are present in the bacterial cell: a higher-molecular-weight form of approximately 42 kD in the cytoplasm and a smaller-molecular-weight form of approximately 42 kD in the periplasm. This result indicates that PP11 can be synthesized in E. coli and is process by removal of the hydrophobic signal sequence. Both the placental and the processed recombinant PP11 protein exhibit a protease activity.

Hormone and DNA binding activity of a purified human thyroid hormone nuclear receptor expressed in Escherichia coli.

Using a T7 expression system, large amounts of the human placental c-erbA protein (h-TR beta 1) were expressed. From 1 liter of Escherichia coli culture, approximately 50-100 micrograms of purified h-TR beta 1 were obtained. Analysis of the binding data indicated that the purified h-TR beta 1 binds to 3,3',5-triiodo-L-thyronine (T3) with a Ka = 2.8 x 10(9) M-1. It binds to 3,3',5-triiodo-L-thyropropionic acid, 3,3',5-triiodo-L-thyroacetic acid, D-T3, L-thyroxine (T4), and 3',5',3-triiodo-L-thyronine with 475, 120, 39, 7, and 0.1%, respectively, of the activity of L-T3. This order of binding activity to T3 analogs is similar to that reported for the T3 nuclear receptor identified in tissues or cultured cells. Furthermore, the purified h-TR beta 1 binds to the T3 response element of the rat growth hormone gene. Thus, the purified h-TR beta 1 is active. To identify the hormone binding domain, the purified h-TR beta 1 was affinity labeled with underivatized [3',5'-125I]T4. A partial digestion by trypsin yielded a 125I-labeled 25-kDa fragment which was identified to be the domain Phe240-Asp456 by amino acid sequencing. Thus, the purified h-TR beta 1 appears suitable for other structural and functional studies.

Expression of CD45 alters phosphorylation of the lck-encoded tyrosine protein kinase in murine lymphoma T-cell lines.

CD45 is a family of high molecular weight leukocyte cell surface glycoproteins. Recently, two related subregions of the cytoplasmic domain of CD45 have been shown to have 30-40% amino acid identity with a human placental protein phosphotyrosine phosphatase, and CD45 isolated from human spleen was found to exhibit intrinsic protein phosphotyrosine phosphatase (EC 3.1.3.48) activity. In the present studies, we demonstrate that each of the known isoforms of murine CD45 has an equivalent basal level of protein phosphotyrosine phosphatase activity and establish that this enzymatic activity is associated with the cytoplasmic domain of the glycoprotein. Studies with three independent sets of well-characterized parental CD45+, mutant CD45-, and revertant CD45+ lymphoma cell lines indicate that loss of CD45 increases the phosphorylation of the src-related leukocyte-specific tyrosine protein kinase p56lck on tyrosine-505, a putative negative regulatory site. This suggests that CD45 may play a role in leukocyte growth regulation by altering the kinase activity of p56lck.

Structure and Expression of cDNA for Calphobindin II, a Human Placental Coagulation Inhibitor

Calphobindin II, with Mr 73,000, is one of the human placental anticoagulant proteins. The cDNA encoding calphobindin II was obtained by screening a human placental lambda gt11 cDNA library using a specific antibody as a probe. The longest cDNA insert consisted of 2,361 nucleotides and a 64-nucleotide-long poly(A) tract. An open reading frame encoding 673 amino acids was predicted. The deduced sequence includes an 8-fold repeat of a conserved 70-amino-acid-long segment that has a high degree of sequence identity with the repeated segments in members of the Ca2+-dependent phospholipid binding protein family. The cDNA fragment including the open reading frame was introduced into the expression vector pKK223-3 and subsequently expressed in Escherichia coli JM105 cells. The resulting recombinant protein reacted with the specific monoclonal antibodies to calphobindin II and prolonged the blood coagulation time as did placental calphobindin II.

Characterisation and covalent labeling of the human placental methyltrienolone binding protein

Human placental cytosol contains an androgen binding protein which binds the synthetic androgen methyltrienolone (R1881) with high affinity ( K d 8.7 nM) and with an average binding capacity of 518 fmol/mg cytosol protein. This study provides further evidence that this protein is distinguishable from classical androgen receptors on the basis of steroid specificity and sulphydryl group sensitivity. Covalent labeling studies have shown this protein, which we have called “the methyltrienolone binding protein”, to have a mol. wt of 67,000 daltons.

Antipeptide antibodies recognize c-erbA and a related protein in human A431 carcinoma cells.

Peptides corresponding to the deduced amino acid residues 15-29 of the amino-terminal region and 445-456 of the carboxyl-terminal region of the human placental c-erbA protein (hc-erbA-beta) were synthesized and used to produce site-specific rabbit polyclonal antipeptide sera. Antibodies to the carboxyl-terminal peptide (C-91) and the amino-terminal peptide (N-98) specifically immunoprecipitated the hc-erbA-beta proteins synthesized in vitro. Furthermore, 68% and 48% of the T3-binding activity of the hc-erbA-beta protein were immunoabsorbed by antibodies C-91 and N-98, respectively. These results indicate that C-91 and N-98 recognized hc-erbA-beta proteins. These antibodies were used to study the subcellular distribution of hc-erbA-beta protein in human cultured cells. Nuclear extracts were prepared from human A431 carcinoma cells; C-91 immunoabsorbed 50% of the specific T3-binding activity in these extracts. These results provide structural evidence to confirming that hc-erbA-beta is the T3 nuclear receptor. Cells were metabolically labeled with [35S]methionine, and the cytosolic extracts were immunoprecipitated by C-91 or N-98. A protein with a mol wt of 58,000 (Cp58) was specifically immunoprecipitated by N-98 or C-91. Peptide mapping by V8 digestion and cyanogen bromide cleavage showed that the Cp58 molecules immunoprecipitated by N-98 or C-91 was identical. Indirect immunofluorescence using N-98 or C-91 indicated that Cp58 is present exclusively in the cytoplasm. Other human cultured cells, HepG2, MCF-7, IM-9, and KB, were also evaluated, and similar results were found. These results raised the possibility that a precursor of hc-erbA-beta may be present in the cytosol. The functional significance of the hc-erbA-beta-related cytosolic Cp58 remains to be established.

CD45 regulates signal transduction and lymphocyte activation by specific association with receptor molecules on T or B cells.

Evidence is presented that the leukocyte common antigen CD45 can regulate both signal transduction by lymphocyte receptor molecules and T- and B-cell proliferation in a manner dependent on specific interactions between these receptors on the cell surface. Formation of homoaggregates of CD3, CD2, or CD28 on the surface of T cells induced by crosslinking with monoclonal antibodies (mAbs) results in an increase in cytoplasmic free calcium concentration ([Ca2+]i). This increase in [Ca2+]i was abolished when these receptors were crosslinked to CD45 on the cell surface. In contrast, the increase in [Ca2+]i induced by formation of homoaggregates of CD4 was strongly amplified when CD4 was coupled to CD45. T-cell proliferation initiated by immobilized anti-CD3 was inhibited by anti-CD45 or anti-CD45R when immobilized on the same surface, but not when in solution. Similarly, proliferation after stimulation of the CD2 and CD28 receptors was inhibited when a CD45 mAb was crosslinked to either CD2 or CD28 mAbs, but not when a CD45-specific mAb was bound to the cell surface separately. In B cells, the increase in [Ca2+]i and resulting proliferation induced by crosslinking either the CD19 or Bgp95 receptors was inhibited by coupling these molecules to CD45. Thus, CD45 appears to modify other cellular receptors functionally when brought into close physical association with them. The homology of the CD45 conserved cytoplasmic domains with a major human placental protein tyrosine phosphatase suggests that the effects of CD45 described here result from alterations in the phosphorylation state of tyrosyl residues in membrane-associated proteins.

Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli

A series of new plasmid expression vectors (the pTrc series) has been constructed for the regulated expression of genes in Escherichia coli. Based on pKK233-2 [Amann and Brosius, Gene 40 (1985) 183–190], the vectors carry a strong hybrid trp/lac promoter, the lacZribosome-binding site (RBS), the multiple cloning site of pUC18 and the rrnB transcription terminators. With the aid of synthetic oligodeoxynucleotides, the multiple cloning site has been inserted behind an NcoI site in three reading frames. Thus, the vectors are equally useful for the expression of proteins in their authentic, non-fused form (by using the NcoI site) and for the expression of fusion proteins (by choosing any of the cloning sites in the correct translational frame). To ensure complete repression of the hybrid trp/lac promoter during construction and growth in any host strain, the lacI q allele of the lac repressor gene was added to some of the vectors. The complete vector nucleotide sequence and examples of heterologous gene expression (human coagulation factor XIIIa and human placental anticoagulant protein PP4) with the new vectors are presented.