Selective modification of recombinant bovine placental lactogen by site-directed mutagenesis at its C terminus.
Five recombinant analogues of bovine placental lactogen (bPL) ((bPL(S184H), bPL(S187A), bPL(S187F), bPL(T188F), bPL(T188F,I190F)) were prepared, expressed in Escherichia coli, and purified to homogeneity. Circular dichroism analysis revealed no or minor structural changes, except in bPL(T188F,I190F). Binding and biological activities of bPL(T188F,I190F) were almost completely abolished, whereas bPL analogues mutated at position 187 retained their full activity. Point mutation T188F resulted in selective modification; binding to somatogenic receptors, their extracellular domains (ECDs), and to bPLR in the endometrium as well as somatogenic receptor-mediated biological activities were reduced or abolished, whereas binding to lactogenic receptors, their ECDs, and subsequent biological activity was fully or almost fully retained. This selective modification most likely results from a steric hindrance induced by a bulky Phe-188 chain of bPL which interacts with the Arg-43 of the human or Leu-43 of the non-human GHRs. Point mutation S184H abolished the interaction with hGHR, most likely due to the unfavorable charge-charge interaction, possibly accompanied by steric hindrance between Arg-43 of the receptor and the newly introduced His-184 and possible interference with the putative interaction between the alkyl portion of Thr-188 and Lys-185 of bPL with Trp-104 of hGHR. In contrast, bPL(S184H) retained its capacity to interact with nonhuman GHRs. Decrease in the biological activity of bPL(S184H) was also observed in two lactogenic receptor-mediated bioassays most likely due to the elimination of the intermolecular hydrogen bond of Ser-184 with a side chain of Tyr-127, which appears in all lactogenic receptors.
Vashdi-Elberg
D
D
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel.
Staten
N R
NR
Sakal
E
E
McKinnie
R E
RE
Djiane
J
J
Krivi
G G
GG
Gertler
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008223:Lymphoma; D008862:Microsomes, Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010928:Placental Lactogen / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D017354:Point Mutation; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D011981:Receptors, Prolactin / Q000378:metabolism*; D011986:Receptors, Somatotropin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
D011981:Receptors, Prolactin; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D013006:Growth Hormone; D010928:Placental Lactogen