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.

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.

Placental protein 12 is a decidual protein that binds somatomedin and has an identical N-terminal amino acid sequence with somatomedin-binding protein from human amniotic fluid.

Placental protein 12 (PP12) was originally isolated from term human placenta and adjacent membranes. Recently we found that the site of PP12 synthesis is decidua but not placenta. In this work, the purity of PP12 was first tested by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis and by reverse phase HPLC, and the N-terminal amino acid sequence of 15 residues was determined by a liquid-phase sequencer. A single amino acid sequence of Ala-Pro-Trp-Gln-Cys-Ala-Pro-Cys-Ser-Ala-Asp-Glu-Leu-Ala-Leu was obtained showing identity to the known N-terminal amino acid sequence of somatomedin-binding protein from human amniotic fluid. Like the latter, PP12 bound somatomedin (insulin-like growth factor I) as demonstrated in gel chromatography by a shift in the elution pattern of [125I]iodo-insulin-like growth factor I after incubation with PP12. These data show that PP12 is a somatomedin-binding protein and extend through previous literature on PP12 the existing knowledge on the physiology and pathophysiology of somatomedin-binding protein(s) in human reproduction and cancer.

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.

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.

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 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.

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.

Pancreatic secretory trypsin inhibitor from human amniotic fluid and fetal and neonatal urine: concentrations and physicochemical characterization.

The levels and physicochemical properties of the pancreatic secretory trypsin inhibitor, also known as Kazal type trypsin inhibitor, were studied in human amniotic fluid. In the second trimester, the median concentration was 160 ng/ml, which exceeds the maternal serum levels 20-fold. Towards term, the amniotic fluid levels declined about 5-fold, whereas the maternal serum values remained constant. In fetal urine, the concentration of the trypsin inhibitor was similar to that in amniotic fluid in early gestation, whereas in newborn urine, the median level was 4-to 5-fold higher than in term amniotic fluid. The physiochemical characteristics of the trypsin inhibitor in amniotic fluid, neonatal urine and cancer urine from an ovarian cancer patient were similar, as studied by gel filtration, high performance reverse phase liquid chromatography, and complete immunological identity in immunodiffusion. The physicochemical similarity and levels in various compartments suggest fetal contribution to amniotic fluid levels of the trypsin inhibitor.

Pancreatic secretory trypsin inhibitor-like immunoreactivity in pancreatectomized patients.

Pancreatic secretory trypsin inhibitor (PSTI) is a 6000-dalton peptide, that occurs in high concentrations in the pancreas and in pancreatic juice. It is thought to be synthesized by pancreatic acinar cells. We have recently reported the findings of an identical trypsin inhibitor at high concentrations in the urine of patients with gynecological malignancy. Therefore, we have named the inhibitor tumor-associated trypsin inhibitor (TATI). We have now studied patients who have undergone total pancreatoduodenectomy for pancreatic cancer or chronic pancreatitis. By radioimmunoassay (RIA), we found normal levels of this inhibitor in the serum and urine of pancreatectomized patients. The absence of pancreas was confirmed by measuring serum trypsin. By gel filtration and HPLC it was found that PSTI/TATI occurring in pancreatectomized patients was indistinguishable from that found in connection with pancreatitis and ovarian cancer.

Demonstration of a new acrosin inhibitor in human seminal plasma.

We have recently described the purification and characterization of a tumor-associated trypsin inhibitor (TATI). Studies on its N-terminal sequence suggested identity with the pancreatic secretory trypsin inhibitor (PSTI) (Huhtala, M.-L., Pesonen, K., Kalkkinen, N. & Stenman, U.-H. (1982) J. Biol. Chem. 257, 13713-13716). I report here the occurrence of a TATI-like activity in human seminal plasma. Concentrations of this inhibitor in seminal plasma varied considerably (4-500 ng/ml, n = 50). In radioimmunoassay the dose-response curves of the new seminal plasma inhibitor and purified TATI were parallel. The similarity between these two inhibitors was demonstrated by gel filtration, reverse phase liquid chromatography and ion-exchange chromatography. By ion exchange chromatography the new inhibitor could be separated from the main seminal plasma trypsin inhibitors. Purified TATI was shown to inhibit human acrosin effectively.

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.

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.

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.

Excretion of a tumor-associated trypsin inhibitor (TATI) in urine of patients with gynecological malignancy.

In earlier studies we reported the finding of a tumor-associated peptide that also occurred at high concentrations in early amniotic fluid. Determination of the N-terminal sequence of this peptide revealed that it is closely related or identical to the pancreatic secretory trypsin inhibitor. Therefore, the peptide is called tumor-associated trypsin inhibitor (TATI). The concentration of TATI was determined by radioimmunoassay in the urine of 148 patients with various forms of gynecologic malignancy and in a reference population consisting of 98 patients with non-malignant gynecologic disease, and also in 40 patients with severe infections or inflammatory disease. In the reference population, the median urinary concentration of TATI was 22 micrograms/g creatinine and the central 95% reference interval was 7-50 micrograms/g creatinine. Elevated urinary levels were observed in 53% of all patients with gynecologic cancer, in 63% of those with active disease and 26% of those in clinical remission. The highest urinary TATI level (11,000 micrograms/g creatinine) was over 200 times the upper limit of the reference range. Patients with cervical cancer had the highest frequency of elevated values. Increased excretion of TATI was also observed in patients with severe bronchopulmonary infections and pancreatitis. Although increased excretion of TATI is not cancer-specific, the distinction by elevated levels of TATI between malignant and nonmalignant gynecologic disease is better than by most other putative tumor markers, and the increased excretion of TATI in patients with active disease can be important for the understanding of tumor biology.

Biologically active domain in somatomedin-binding protein.

We have found that human decidua synthesizes a 34K somatomedin-binding protein PP12. Purification of PP12 by immunochemical techniques from human placenta and adjacent membranes has also yielded lower-molecular weight immunoreactive polypeptides designated as PP12B. An individual 21K fragment of somatomedin-binding protein, and a mixture of fragments with molecular weight from 17K to 20K were isolated from this material using high performance liquid chromatography (HPLC). These fragments reacted with antibodies to native PP12 as shown by Western blotting. They all shared the same N-terminal amino acid sequence: Ala-Pro-Trp-Gln-, which is identical with that obtained for PP12. The 21K fragment was shown to bind somatomedin-C, or IGF-I (insulin-like growth factor-I). Since the N-terminal end of the 21K fragment is identical with that of the 34K somatomedin-binding protein, our results suggest that the 21K fragment is the N-terminal part of somatomedin-binding protein, and the somatomedin-binding domain resides in this N-terminal portion.

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.

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.

Immunochemical demonstration of an ovarian cancer-associated urinary peptide.

Immunization with a peptide fraction from ovarian cancer urine resulted in an antiserum that reacts with a 6,000 dalton (6K) peptide. In urine from some patients with advanced gynaecological cancer, the concentration of 6K peptide was high enough to be detected by immunodiffusion. By radioimmunoassay the levels in normal serum could be determined. The concentrations in serum was 5-20 microgram/l and that in urine 5-50 microgram/l. Elevated levels were observed in urine from 5 out of 8 patients with ovarian cancer, 10 out of 14 patients with cervical cancer and 9 out of 14 patients with endometrial cancer. The highest level observed was 12,000 microgram/l. High concentrations of the 6K peptide (100-300 microgram/l) were observed in amniotic fluid from 14-16 weeks of pregnancy. Some tumor extracts also had a high concentration of the 6K peptide. No immunological relationship was observed between the peptide and known tumor-associated antigens or serum proteins. These results suggest that the peptide is a new oncodevelopmental peptide which may have significance as a tumor marker.