Diagnostic potential of baculovirus-expressed rubella virus envelope proteins.

The envelope glycoproteins E1 and E2 of rubella virus were abundantly expressed in Spodoptera frugiperda Sf9 insect cells by using a baculovirus expression vector. The recombinant protein products were purified by immunoaffinity chromatography and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and enzyme immunoassay (EIA). The purified recombinant antigen consisted of the envelope polypeptides, corresponding to the viral E1 and E2 proteins, and a polyprotein precursor (molecular mass, 90 to 95 kDa). The antigen was reactive with human convalescent-phase sera in immunoblot analysis, and the reactivity correlated well (r = 0.861) with that of a whole-virus antigen when tested by EIA by using a total of 106 rubella virus immunoglobulin G-positive and -negative serum specimens. When the sera from patients with recent rubella virus infection were tested with the recombinant glycoproteins by EIA, the correlation was not as close (r = 0.690). However, all of the 26 serum specimens were reactive with the recombinant antigen. The results demonstrate that these bioengineered antigens have a potential for use in routine diagnostic assays of rubella virus immunity and recent infection.

Human colon carcinoma, fibrosarcoma and leukemia cell lines produce tumor-associated trypsinogen.

Previous studies have indicated that cyst fluid of ovarian tumors contains 2 trypsinogen isoenzymes, called tumor-associated trypsinogen-I (TAT-I) and trypsinogen-2 (TAT-2), the levels of which correlate with the degree of malignancy of the tumors. In addition, these cyst fluids contain large amounts of tumor-associated trypsin inhibitor (TATI), which is also expressed in many other human tumors. In the present study we examined the production of TAT-I, TAT-2 and TATI in 9 established tumor-cell lines. TAT-2 was produced by 5 cell lines. Its concentration in the conditioned medium of COLO 205 colon adenocarcinoma cells, K-562 erythroleukemia cells and fibrosarcoma cell lines HT 1080, 8387 and A 9733 was 460 micrograms/l, 9.8 micrograms/l, 21 micrograms/l, 8.8 micrograms/l and 0.24 micrograms/l, respectively. TAT-I was detectable in the conditioned medium of COLO 205 and HT 1080 cells at concentrations of 64 micrograms/l and 0.5 micrograms/l, respectively. TATI was detected only in the media of COLO 205 cells at a concentration of 23 micrograms/l. TAT-2 zymogen was purified from the conditioned medium of COLO 205 and HT 1080 cells by immunoaffinity chromatography. According to its aminoterminal amino acid sequence, a molecular mass of 28 kDa by SDS-PAGE, elution pattern in ion-exchange chromatography and ability to be activated by enteropeptidase, the zymogen is identical to that previously isolated from cyst fluid of ovarian tumors. In addition, we found that TAT-2 secretion could be down-regulated by dexamethasone in HT 1080 cells but not in COLO 205 cells. The abundant production of TAT-2 isoenzyme in different cancer cell lines suggests that it could contribute to the increased proteolytic activity of many human tumors.

Antigenic regions of poliovirus type 3/Sabin capsid proteins recognized by human sera in the peptide scanning technique.

We used the peptide scanning technique to identify regions of poliovirus type 3/Sabin capsid proteins that bind antibodies from human immune sera. Several reactive regions were seen in VP1, VP2, and VP3 while peptides resembling VP4 did not bind antibodies. Peptides derived from sequences of the previously known antigenic sites 1 and 3 were recognized to a moderate degree. Peptides imitating the four loops in the closed ends of the beta barrels or the alpha helical CD insertions of VP1, VP2 or VP3, whether exposed in the crystal structure or not, all represented major reactivity in the scans. In VP1 several additional reactive regions were found in the amino terminal quarter of the protein, which is buried in the crystal structure, and in a partially exposed region close to but separated from the carboxy terminus. In VP2 the nonexposed peak activities clustered in a bridge-like structure spanning from the outer to the inner surface of the capsid shell. Likewise, most of the novel antigenic regions of VP3 clustered in an internal location and partially composed of beta sheets with a conserved amino acid sequence. Whether any of the novel antigenic sites is capable of inducing neutralizing antibodies is not known.

Synthetic peptides in HIV antibody screening and typing.

We have defined continuous native epitopes of HIV proteins by using a systematic epitope-scanning technology. We have demonstrated that there is a highly immunoreactive continuous native epitope region in the transmembrane protein gp41 of HIV-1 that is immunoreactive with all studied HIV-1 antibody-positive sera. The corresponding region in HIV-2 gp34 behaves similarly. There is a clear difference, however, between HIV type 1 and type 2 transmembrane proteins in the number of highly immunoreactive regions, when presented properly as synthetic antigens in solid-phase EIA, can provide tests unusually suitable for early and reliable diagnosis of HIV-1 and HIV-2 infections and for type-specific distinction of the two types of HIV infections.

PIP: This article reviews the basic method used to define native epitopes from transmembrane proteins and the function of synthetic peptides in HIV screening and typing. Identification of continuous native epitopes from structural protein sequences of HIV-1 and HIV-2 involves the use of systematic scanning epitope technology. Scanning profiles of these two types of HIV demonstrated that there is a highly immunoreactive continuous native epitope region in the transmembrane protein gp41 of HIV-1 as well as in the corresponding region in HV-2 gp34. However, the number of highly immunoreactive regions differs in the structural proteins of the two types of HIV infections. These highly immunoreactive regions, when presented accurately as synthetic antigens in solid-phase enzyme immunoassay, can provide tests that are remarkably appropriate for the early and reliable diagnosis and type-specification of HIV-1 and HIV-2 infections.

Purification and characterization of endometrial protein PP14 from mid-trimester amniotic fluid.

Two methods are described for the purification of placental protein 14 from human mid-trimester amniotic fluid. The first includes gel filtration, anion exchange chromatography, and reversed-phase high performance liquid chromatography. The second method employs octyl-Sepharose chromatography instead of high performance liquid chromatography, and it also includes an anti-hCG adsorption step in order to remove the remaining traces of hCG from the purified PP14. In the first method, 362 micrograms of PP14 was recovered from 26 ml amniotic fluid with a final recovery of 25%. In the second method, 745 micrograms of PP14 was recovered from 200 ml amniotic fluid with a final recovery of 9.8%. As a result of either method sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified protein showed one band at 28 kDa. Polyclonal antibodies against placental PP14 reacted with this band in immunoblot analysis and radioimmunoassay. A single N-terminal amino acid sequence of M D I P Q T K Q D L E L P K L A G T W H S M A was obtained for the isolated protein. This sequence is identical to that previously reported for human placental PP14. Due to its high PP14 concentration amniotic fluid serves as an excellent starting material for purification of this protein.

Human ovarian tumor-associated trypsin. Its purification and characterization from mucinous cyst fluid and identification as an activator of pro-urokinase.

In search of the target protease for the tumor-associated trypsin inhibitor TATI we recently identified a trypsin-like protease in cyst fluid of mucinous ovarian tumors (Stenman, U.-H., Koivunen, E., and Vuento, M. (1988) Biol. Chem. Hoppe-Seyler 369, 9-14). We have now purified this protease and demonstrate that it represents isoenzyme forms of trypsinogen, here called tumor-associated trypsin(ogen)s (TAT). The purification procedure comprised batchwise anion exchange chromatography, immunoaffinity chromatography with antibodies to trypsin, and separation of the two isoenzymes by reverse phase chromatography. In sodium dodecyl sulfate (SDS)-gel electrophoresis, the TAT-1 and TAT-2 isoenzymes have relative molecular weights (Mr) of 25,000 and 28,000, respectively, TAT-2 being the major component. The amino-terminal amino acid sequences correspond to those of pancreatic trypsinogen-1 and -2, respectively, and activation of the zymogens results in cleavage of a NH2-terminal activation peptide of 8 residues characteristic of trypsinogen. Isoelectric focusing in the presence of urea gives pI values of about 5 and 4 for TAT-1 and -2, respectively. The substrate specificities of the two TAT isoenzymes are very similar to, but not identical with, those of trypsin-1 and trypsin-2, respectively, suggesting slight differences in substrate binding site. TAT was found to be an efficient activator of pro-urokinase. Hence, TAT could take part in the protease cascade associated with tumor invasion.

The adhesive and neurite-promoting molecule p30: analysis of the amino-terminal sequence and production of antipeptide antibodies that detect p30 at the surface of neuroblastoma cells and of brain neurons.

A membrane-bound adhesive protein that promotes neurite outgrowth in brain neurons has been isolated from rat brain (Rauvala, H., and R. Pihlaskari. 1987. J. Biol. Chem. 262:16625-16635). The protein is an immunochemically distinct molecule with a subunit size of approximately 30 kD (p30). p30 is an abundant protein in perinatal rat brain, but its content decreases rapidly after birth. In the present study the amino-terminal sequence of p30 was determined by automated Edman degradations. A single amino-terminal sequence was found, which is not present in previously studied adhesive molecules. This unique sequence has a cluster of five positive charges within the first 11 amino acid residues: Gly-Lys-Gly-Asp-Pro-Lys-Lys-Pro-Arg-Gly-Lys. Antisynthetic peptide antibodies that recognize this sequence were produced in a rabbit, purified with a peptide affinity column, and shown to bind specifically to p30. The antipeptide antibodies were used, together with anti-p30 antibodies, to study the localization of p30 in brain cells and in neuroblastoma cells as follows. (a) Immunofluorescence and immunoelectron microscopy indicated that p30 is a component of neurons in mixed cultures of brain cells. The neurons and the neuroblastoma cells expressed p30 at their surface in the cell bodies and the neurites. In the neurites p30 was found especially in the adhesive distal tips of the processes. In addition the protein was detected in ribosomal particles and in intracellular membranes in a proportion of cells. (b) The antibodies immobilized on microtiter wells enhanced adhesion and neurite growth indicating that p30 is surface exposed in adhering neural cells. (c) Immunoblotting showed that p30 is extracted from suspended cells by heparin suggesting that a heparin-like structure is required for the binding of p30 to the neuronal cell surface. A model summarizing the suggested interactions of p30 in cell adhesion and neurite growth is presented.

Synthetic env gp41 peptide as a sensitive and specific diagnostic reagent in different stages of human immunodeficiency virus type 1 infection.

An enzyme immunoassay (EIA) for serum antibodies to human immunodeficiency virus type 1 (HIV-1), based on the synthetic pentadecapeptide SGKLICT-TAVPWNAS, a segment of the transmembrane glycoprotein (gp41) of the virus, was developed and tested for sensitivity and specificity. Sera of 152 individuals at various stages of HIV-1 infection, including two prospectively and six retrospectively studied patients exposed to HIV-1 but seronegative on initial testing in whole-virus EIA and immunoblotting, were screened with the gp41 peptide antibody EIA. The reference population consisted of 1,000 healthy HIV-1 antibody-negative blood donors. In addition, five individuals with antibodies to HIV-2 were studied. Antibodies to the synthetic peptide were detected in 100% of those with asymptomatic infection. Only one patient with LAS failed to react in the peptide EIA. Patients with HIV-2 infection did not react in this test. The peptide antibodies appeared rapidly after infection, were detectable at the time when seroconversion was observed by immunoblotting, and preceded reactivity in whole-virus EIA. Sera of seven patients with verified HIV-1 infection did not react with gp41 in immunoblotting, although antibodies were readily detectable in the gp41 peptide EIA.

Molecular cloning and sequence analysis of cDNA encoding human prostatic acid phosphatase.

lambda gt11 clones encoding human prostatic acid phosphatase (PAP) (EC 3.1.3.2) were isolated from human prostatic cDNA libraries by immunoscreening with polyclonal antisera. Sequence data obtained from several overlapping clones indicated that the composite cDNAs contained the complete coding region for PAP, which encodes a 354-residue protein with a calculated molecular mass of 41,126 Da. In the 5'-end, the cDNA codes for a signal peptide of 32 amino acids. Direct protein sequencing of the amino-terminus of the mature protein and its proteolytic fragments confirmed the identity of the predicted protein sequence. PAP has no apparent sequence homology to other known proteins. However, both the cDNA clones coding for human placental alkaline phosphatase and PAP have an alu-type repetitive sequence about 900 nucleotides downstream from the coding region in the 3'-untranslated region. Two of our cDNA clones differed from others at the 5'-ends. RNA blot analysis indicated mRNA of 3.3 kb. We are continuing to study whether acid phosphatases form a gene family as do alkaline phosphatases.

Highly immunoreactive antigenic site in a hydrophobic domain of HIV-1 gp41 which remains undetectable with conventional immunochemical methods.

A synthetic pentadecapeptide (A15; env residues 599-613: SGKLICTTAVPWNAS), derived from a hydrophobic region in the transmembrane protein gp41 of HIV-1 and comprising a highly immunoreactive antigenic site in eliciting antibody responses during HIV-1 infection in humans, was used to purify, by affinity, the corresponding anti-peptide antibodies from HIV-1-infected patient sera. The purified antibodies to peptide A15 reacted specifically with the peptide in EIA, but not in whole virus EIA. These antibodies were immunoreactive with the corresponding peptide-albumin conjugates in immunoblotting but not with gp41 molecules. The results suggest that the peptide A15 sequence is not exposed in intact gp41, but will be exposed and is antigenic in the course of HIV-1 infection in humans.

Structural studies, localization in tissue and clinical aspects of human endometrial proteins.

Two human endometrial proteins, PP12 and PP14, are abundant in human amniotic fluid which is an excellent source for purification. In SDS-PAGE, purified PP12 migrates as several immunoreactive bands from 17,000 to 34,000, all having the same N-terminal amino acid sequence of Ala-Pro-Trp-Gln-Cys-Ala-, and all of them binding IFG-I. PP14 migrates at 28,000, and its N-terminal sequence is Met-Asp-Ile-Pro-Gln-Thr-Lys-Gln-Asp-Leu-Gln-Leu-Pro-Lys-Leu-Ala-Gly-Thr- Trp-His-Ser-Met-. There is a 59% identity between this sequence and that of horse beta-lactoglobulin, and also between PP14 and beta-lactoglobulins of various other species. PP14 and human retinol-binding protein show a 23% sequence identity, and the amino acid residues -Gly-Thr-Trp- at positions 17-19 of PP14 are identical with the corresponding residues of human retinol-binding protein. This site is assumed to play a part in the binding of retinol. An additional sequence identity (32%) is reported here for PP14 and protein BG, a 182 amino acid protein deduced from a 700-base pair cDNA clone isolated from the olfactory neuroepithelium of the frog. Sequence homology is also reported here between PP14 and insecticyanin, a camouflage-associated biliprotein in insects. The sequence of PP14 is therefore homologous to members of a family of proteins that bind and transport biologically active small molecules. Clinical studies have indicated an increase of PP12/IGF-bp and PP14 in the endometrium with advancing secretory changes. PP12/IGF-bp is also found in preovulatory follicular fluid. In hyperstimulated cycles of infertile women undergoing in-vitro fertilization, the serum PP12/IGF-bp concentration rises as multiple follicles mature, and luteinized granulosa cells contain this protein. In non-pregnant women, elevated values have been found in patients with advanced ovarian cancer and primary liver cancer. During pregnancy the serum PP12/IGF-bp concentration rises above the level in non-pregnant women around Week 8 of gestation. Abnormally high levels are seen in patients with pre-eclampsia and, in the third trimester, there is an inverse correlation between the maternal serum PP12/IGF-bp level and fetal weight. From these studies it is likely that a relationship exists between PP12/IGF-bp, the metabolism of IGFs and fetal growth. In non-pregnant women, serum PP14 concentrations appear to reflect endometrial secretory function. This is indicated by cyclic changes in the PP14 concentration in endometrial tissue and by the rising PP14 values in the late luteal phase.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification and characterization of placental protein 5.

This report describes the purification of placental protein 5, PP5, from the human placenta by two affinity chromatography steps, the first with Heparin-Sepharose and the second with Sepharose-linked monoclonal anti-PP5 antibody. The final purification is achieved by reversed-phase high performance liquid chromatography. In SDS-polyacrylamide gel electrophoresis under reducing or nonreducing conditions, PP5 purified in this study migrates as one major band at 36 kD. The previously purified PP5 is more heterogeneous: under nonreducing conditions it migrates at 30 kD and, after reduction, it gives three bands at 16.8 kD, 18.3 kD, and 19.0 kD. In Western blot analysis, both purified proteins react with polyclonal and monoclonal anti-PP5 antibodies. Three N-terminal amino acid sequences are obtained for the previously purified PP5, whereas the N-terminal of PP5 purified in this study is blocked. These results suggest that PP5 previously purified in the absence of protease inhibitors, does not represent the native form of PP5. Computer comparison of the obtained amino acid sequences revealed no significant homology to known protein sequences.

Tumour-associated trypsin inhibitor (TATI) in human ovarian cyst fluid. Comparison with CA 125 and CEA.

The levels of tumour-associated trypsin inhibitor (TATI), CA 125 and CEA were measured in ovarian cyst fluids from 21 patients. TATI in cyst fluid was immunologically and physicochemically similar to the peptide originally isolated from the urine of a patient with ovarian cancer. Mucinous cysts contained significantly higher levels of TATI than did serous cysts. Immunohistochemically TATI was localized in the apical parts of cells of mucinous ovarian cysts. These results suggest that this tumour-associated peptide is actually produced by a tumour. Like TATI, CEA occurred at higher concentrations in mucinous than in serous cyst fluids, whereas CA 125 was found in higher concentrations in serous than in mucinous cyst fluids. The concentrations of these tumours markers in cyst fluids did not correlate with circulating levels of the same markers. In spite of the very high levels of all these tumour markers in benign cyst fluids, serum levels were normal or only slightly elevated. Clearly elevated serum levels occurred only in patients with malignant tumours. Cyst fluid levels of these tumour markers could not be used to distinguish between benign and malignant tumours.

Localization of insulin-like growth factor-binding protein and endometrial beta-lactoglobulin in cultured decidual and chorionic villus cells.

Immunofluorescence staining was used to localize two 'endometrial' proteins, insulin-like growth factor-binding protein and human beta-lactoglobulin homologue, in cultured cells of chorionic villi and decidua. Both cultured cell types were positive for each protein. These results indicate that immunohistochemical detection of these two proteins cannot be used to distinguish between cultured chorionic villus and decidual cells.

Amino acid sequence homology between human placental protein 14 and beta-lactoglobulins from various species.

The primary structure of 22 N-terminal amino acid residues of placental protein 14 was determined by automated Edman degradation with a gas-phase sequencer. This protein, isolated from the human placenta and its membranes, was considered pure as evidenced by a single N-terminal amino acid sequence M D I P Q T K Q D L E L P K L A G T W H S M. It shows significant sequence homology with horse, bovine, buffalo, sheep and goat beta-lactoglobulins. We found 13 identities out of 22 possible matches with horse beta-lactoglobulin. beta-lactoglobulins from several animal species have been found to bind retinol. Among the identical residues there is one tryptophan at position 19 which is conserved in beta-lactoglobulins and is also found in the human retinol-binding protein at the corresponding position. These data suggest a common origin of PP14 and beta-lactoglobulins.

Purification of placental protein PP12 from human amniotic fluid and its comparison with PP12 from placenta by immunological, physicochemical and somatomedin-binding properties.

Human amniotic fluid was found to contain a protein which is immunochemically indistinguishable from placental protein PP12. This protein was purified by gel filtration, hydrophobic interaction high performance liquid chromatography and anion-exchange chromatography. The relative molecular mass as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 34,000, and the isoelectric point was 4.9. Tryptic peptides of amniotic fluid PP12 as determined by reversed phase high performance liquid chromatography were similar to those of placental PP12. Both had the N-terminal amino acid sequence Ala-Pro-Trp-Gln-, which is the same as previously reported for a somatomedin-binding protein. Both placental PP12 and amniotic fluid PP12 were found to bind somatomedin C (IGF-I) with high affinity, Ka = 1 X 10(9) l/mol). Amniotic fluid is an ideal source of this somatomedin-binding protein, and the purification method described allows rapid isolation of PP12 under mild conditions which are essential for studies on its biological function.

Molecular cloning of the beta-subunit of human prolyl 4-hydroxylase. This subunit and protein disulphide isomerase are products of the same gene.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyses the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here the isolation of cDNA clones coding for the beta-subunit of prolyl 4-hydroxylase from a human hepatoma lambda gt11 library and a corresponding human placenta library. Five overlapping clones covering all the coding sequences and almost all the non-coding sequences were characterized. The size of the mRNA hybridizing with these clones in Northern blotting is approximately 2.5 kb. The clones encode a polypeptide of 508 amino acid residues, including a signal peptide of 17 amino acids. These human sequences were found to be very similar to those recently reported for rat protein disulphide isomerase (EC 5.3.4.1). The degree of homology between these two proteins was 84% at the level of nucleotide sequences or 94% at the level of amino acid sequences. Southern blot analyses of human genomic DNA with a cDNA probe for the beta-subunit indicated the presence of only one gene containing these sequences. The product of a single gene thus appears to possess two different enzymatic functions depending on whether it is present in cells in monomer form or in the prolyl 4-hydroxylase tetramer.

Progesterone-associated proteins PP12 and PP14 in the human endometrium.

Two proteins, designated as PP12 and PP14 were originally isolated from soluble extracts of the human placenta and its adjacent membranes. We have shown that they are synthesized by decidualized/secretory endometrium and not by placenta. Both proteins occur at high concentrations in human amniotic fluid, which is therefore an excellent source for purification. PP12 is a 34-kDa glycoprotein, which has an N-terminal amino acid sequence of Ala-Pro-Trp-Gln-Cys-Ala-Pro-Cys-Ser-Ala. This is identical with that of somatomedin-binding protein purified from the amniotic fluid. PP12 too binds somatomedin-C, or IGF-I (insulin-like growth factor-I). Human secretory endometrium synthesizes and secretes PP12, and progesterone stimulates its secretion. PP14 is a 28-kDa glycoprotein. Its N-terminal sequence shows homology to that of beta-lactoglobulins from various species. We have found PP14 in the human endometrium, serum and milk. Immunologically, PP14 is related to progestagen-associated endometrial protein (PEP), alpha-2 pregnancy-associated endometrial protein (alpha-2, PEG), endometrial protein 15 (EP15), alpha-uterine protein (AUP) and chorionic alpha-2 microglobulin (CAG-2). In ovulatory menstrual cycles, the concentration of PP14 increases in endometrial tissue as the secretory changes advance. In serum, the PP14 concentration begins to rise later than the progesterone levels, and high serum PP14 levels are maintained for the first days of the next cycle. By contrast, no elevation of serum PP14 level is seen in anovulatory cycles. Our results show that progesterone-associated proteins are synthesized by the human endometrium and appear in the peripheral circulation, where they can be quantitatively measured using immunochemical techniques.