Marja-Liisa Huhtala

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.

Rapid chromatographic quantitation of glycosylated haemoglobins.

We have developed a rapid chromatographic method for determination of glycosylated haemoglobins by high-performance liquid chromatography with a new cation-exchange column. The haemoglobins are eluted with a three-step gradient in 7 min, and total assay time including re-equilibration of the column is 15 min. The method permits separation and quantitation of HbAlc, even in the presence of elevated levels of HbF. HbA1a, A1b and A2 can also be determined. The results correlate well (r = 0.94) with those obtained by the macro-column method of Trivelli et al. [New Engl. J. Med., 284 (1971) 353] for determination of HbA1c. The method has been fully automated by the use of an automatic injector. The within-assay and between-assay coefficient of variation of the method is 2-3%.

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.

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.

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.

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.

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.

Rapid extraction and separation of plasma β-endorphin by cation-exchange high-performance liquid chromatography

Cation-exchange high-performance liquid chromatography (HPLC) was used to increase the sensitivity and specificity of the radioimmunoassay of plasma beta-endorphin. Proteins were precipitated from a 0.5 to 2.5 ml sample of plasma with 60% acetonitrile at pH 4.7. The supernatant was subjected to cation-exchange HPLC. Gradient elution with volatile buffers was used to separate beta-endorphin from beta-lipotropin. The beta-endorphin fraction (1.8 ml) was concentrated by lyophilization and subjected to radioimmunoassay. In healthy pregnant women at labour plasma concentration of beta-endorphin varied from 105 to 403 pg/ml. In healthy non-pregnant women plasma concentration of beta-endorphin was low, exceeding the detection limit (4 pg/ml) of the assay in only one of the 7 subjects studied.

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.

Rapid concentration of urinary peptides and proteins

Abstract A method for rapid concentration and dialysis of urinary peptides and proteins is described. The method is based on the use of a cheap disposable dialyzer and a two-channel peristaltic pump. One to twenty liters of urine can be concentrated to 0.3–0.5 liter in 2–24 h. The recovery of peptides larger than 6000 daltons is 80–95%. The recovery of smaller peptides varies depending on the conditions used.