Expression, purification, and characterization of recombinant rat placental lactogen-I: a comparison with the native hormone.

Abstract

Rat placental lactogen-I (rPL-I), a member of the PRL/GH gene family, is produced by giant cells in the early trophoblast. The small amount of early placental tissue has limited the purification of rPL-I from this source. To obtain sufficient material for in vitro studies we have used a rPL-I cDNA to express this protein in Chinese hamster ovary (CHO) cells and in these studies have compared the recombinant protein with the native rPL-I. Using an affinity column composed of monoclonal antibody to rPL-I coupled to Sepharose 4B, we have purified rPL-I from four sources: 1) recombinant rPL-I produced and secreted in rPL-I-transfected CHO cells, 2) nonglycosylated recombinant PL-I produced by adding tunicamycin (10 microM/ml medium) to rPL-I-transfected CHO cells, 3) native rPL-I secreted by rat choriocarcinoma (RCHO) cells, and 4) serum rPL-I isolated from day 12 pregnant rats. Analysis by two-dimensional polyacrylamide gel electrophoresis and Western blotting revealed nine subforms with increasing mol wt [approximately 34 kilodaltons (kDa)] and acidic pI for recombinant rPL-I and RCHO-derived rPL-I. Four major species of lower mol wt (approximately 23 kDa) were evident in the nonglycosylated rPL-I, suggesting additional peptide cleavage sites. Serum rPL-I contained four additional forms of higher mol wt (approximately 37 kDa) and more acidic pI. When analyzed by the Nb2 lymphoma cell bioassay, RCHO rPL-I, serum rPL-I, and nonglycosylated rPL-I were equipotent with ovine and human PRL. Recombinant rPL-I was 1.5-2.0 times as active as ovine PRL in the Nb2 assay. A RIA was established for rPL-I. The variant rPL-Iv, displayed nonparallel displacement of [125I]rPL-I from the antibody. There was no cross-reactivity with other pituitary or placental members of the GH/PRL family. Measurement of serum levels of rPL-I by RIA after the injection of recombinant-rPL-I into adult female Sprague-Dawley rats revealed a half-life of 9 min for the recombinant protein compared to 7.8 min for the choriocarcinoma-derived hormone. In conclusion, we have shown that although CHO cells will glycosylate the recombinant protein differently than normal placental cells, the biological properties of our recombinant rPL-I are similar to those of the native, placenta cell-derived hormone.

Adaptation of pancreatic islet B-cells during the last third of pregnancy: regulation of B-cell function and proliferation by lactogenic hormones in rats

“…This gestational profile of B-cell proliferation agrees with that found in the previous study (2). During mid-pregnancy, PL-I is reported to be secreted into the maternal circulation to a maximal concentration of approximately 1000 ng/ml in rats (6). As the first peak of lactogenic activity in maternal circulation was observed at the same period in the present study, this peak is considered to be the secretory profile of PL-I at mid-pregnancy.…”

“…Both PL-I and PL-II are lactogenic hormones rather than somatogenic hormones, interact with prolactin (PRL) receptors, and appear to possess at least some of the same biological actions as pituitary PRL (5)(6)(7). Maximum concentrations of both rat (r) PL-I and rPL-II reach approximately 1000 ng/ml during pregnancy (6,8). rPL-I and rPL-II have strong potencies to induce proliferation in Nb 2 lymphoma cells (9).…”

Placental Lactogen-I (PL-I) Target Tissues Identified with an Alkaline Phosphatase-PL-I Fusion Protein

“…PL-I is a PRL receptor agonist expressed from implantation until midgestation by trophoblast giant cells situated at the maternal-placental interface (Colosi et al 1988;MacLeod et al 1989;Faria et al 1990;Freemark et al 1993;Robertson et al 1994;Dai et al 1996;Soares et al 1998). Recombinant PL-I has previously been shown to bind specifically to liver and ovarian membrane preparations (MacLeod et al 1989), most likely via specific interactions with the extracellular domain of the PRL receptor (Sakal et al 1996).…”

“…The specificity of binding was further assessed by the addition of ovine PRL (5 g/ml) to some of the tissue section incubations ( C,F,I ). other ligands capable of interacting with the PRL receptor, such as PL-I (Tanaka et al 1980;Colosi et al 1988;Robertson et al 1994;Cohick et al 1996;Dai et al 1996) (Figure 4). The AP control did not significantly stimulate rat Nb2 lymphoma cell proliferation (Figure 4).…”

“…PL-I is a glycoprotein, as are most members of the PRL family, and is secreted by trophoblast giant cells of the developing placenta from immediately postimplantation until midgestation (Faria et al 1990). The actions of PL-I have been primarily studied via the generation of recombinant PL-I in heterologous cell types (Colosi et al 1988;Robertson et al 1994;Dai et al 1996). PL-I has been shown to bind PRL receptors (MacLeod et al 1989;Freemark et al 1993) and possesses a number of actions previously attributed to pituitary PRL (Soares et al 1998).…”