Rat placental lactogen-I variant (rPL-Iv), product of an unique gene, is biologically different from rPL-I.
The rat placenta expresses two rat placental lactogen-I (rPL-I) proteins: the normal rPL-I in the first half of pregnancy and a variant (rPL-Iv) in the second half of pregnancy. They are 70% identical at the amino acid level but arise from different cell types: rPL-I from giant cells and rPL-Iv from cytotrophoblasts. To assess whether rPL-Iv originates from alternative splicing of the rPL-I gene or is the product of a separate gene, genomic clones of rPL-I and rPL-Iv were isolated from a lambda EMBL-3 rat genomic library. Restriction enzyme analysis of the 14-kilobase full-length genomic clones of rPL-I and rPL-Iv indicated that the two genes are distinct. To assess the biological activity of the variant protein relative to other members of the rat PL/PRL/GH family, two expression systems were chosen to obtain the purified recombinant protein: 1) a secreted form of rPL-Iv was obtained from Chinese hamster ovary (CHO) cells transfected with rPL-Iv-complementary DNA; and 2) a rPL-Iv fusion protein (Bac-rPL-Iv) was obtained from Spodoptera frugiperda (Sf9) insect cells that had been infected with a recombinant baculovirus generated from rPL-Iv complementary DNA. An antibody was generated to the purified Bac-rPL-Iv fusion protein and used for affinity chromatography to purify recombinant rPL-Iv from the CHO cell media. The mitogenic activity of rPL-Iv was monitored in the Nb2 lymphoma cell bioassay. The relative potency of rPL-Iv compared with other members of the PL/PRL/GH family follows: ovine PRL 100, rPL-I 200, rPL-II 160, rPRL 40, CHO-rPL-Iv 0.7, and Bac rPL-Iv 0.4. Iodinated CHO-rPL-Iv showed minimal binding to Nb2 lymphoma cells, but at a 500-fold protein concentration rPL-I was able to displace [125I]rPL-I from the lymphoma cell PRL receptor. Using recombinant CHO-derived rPL-Iv as standard and antisera against the Bac-rPL-Iv fusion protein, a RIA was developed for rPL-Iv. In pregnant rats rPL-Iv appeared in the serum at day 14, rising to a peak of 2080 +/- 440 ng/ml at day 18, followed by a slight decline. These values reflect the levels of messenger RNA for rPL-Iv in rat placenta noted previously. In summary, rPL-Iv arises from a gene different from rPL-I and the rPL-I protein displays minimal binding and mitogenic activity in the Nb2 lymphoma cells.
Robertson
M C
MC
Department of Physiology, University of Manitoba, Winnipeg, Canada.
Cosby
H
H
Shiu
R P
RP
D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D005260:Female; D005796:Genes; D014644:Genetic Variation; D016678:Genome; D008969:Molecular Sequence Data; D010928:Placental Lactogen / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011247:Pregnancy; D011863:Radioimmunoassay; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; C085699:placental lactogen I, rat; D010928:Placental Lactogen