Expression and characterization of novel ovine orthologs of bovine placental prolactin-related proteins.
BACKGROUND
The prolactin-related proteins (PRPs) are non-classical placental-specific members of the prolactin/growth hormone family. Among ruminants, they are expressed in the cotyledonary villi of cattle and goat. We investigated placental PRP in sheep in order to gain a comprehensive understanding of the function and evolution of these molecules. We also examined the sequence properties, expression and lactogenic activation of the cloned genes.


RESULTS
We cloned two novel ovine PRPs, named oPRP1 and oPRP2. oPRP2 had a typical PRP sequence similar to bovine PRP1 (bPRP1). oPRP1 had a short sequence identical with bovine or caprine type PRP but the reading frame was shifted. Both oPRPs were expressed in trophoblast giant binucleate cells (BNC) as in cattle and goat. oPRP1 expression declined from the early to the middle stage of gestation. In contrast, oPRP2 expression remained constant throughout the gestation period. oPRP2 was translated to form a mature protein in a mammalian cell expression system. Western blotting showed a molecular mass of 35 kDa for the FLAG-tag fusion oPRP2 protein. This recombinant protein and bPRP1 were bioassayed using Nb2 lymphoma cells; it was confirmed that neither ruminant PRP had lactogenic activity because the Nb2 lymphoma cells did not proliferate.


CONCLUSIONS
We have identified two novel PRPs, oPRP1 and oPRP2, in ovine placenta. Both these ovine PRPs were localized and quantitatively expressed in BNC. Absence of lactogenic activity was confirmed for the oPRP2 molecule. It is anticipated that novel and known ruminant PRPs have common functions, except for lactogenic activity.
Ushizawa
Koichi
K
Reproductive Biology Research Unit, Division of Animal Sciences, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki 305-8602, Japan. ushizawa@affrc.go.jp
Takahashi
Toru
T
Hosoe
Misa
M
Ohkoshi
Katsuhiro
K
Hashizume
Kazuyoshi
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D018388:Codon, Terminator; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D010920:Placenta / Q000378:metabolism; D010928:Placental Lactogen / Q000378:metabolism; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011388:Prolactin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D034561:Sheep, Domestic / Q000378:metabolism*
D018388:Codon, Terminator; D011257:Pregnancy Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D011388:Prolactin; D010928:Placental Lactogen