Preparation, purification, and determination of the biological activities of 12 N terminus-truncated recombinant analogues of bovine placental lactogen

• Published in: The Journal of biological chemistry Vol. 267; no. 18; pp. 12655 - 12659
• Main Authors: GERTLER, A, HAUSER, S. D, SAKAL, E, VASHDI, D, STATEN, N, FREEMAN, J. J, KRIVI, G. G
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25.06.1992
• Subjects: amino acid removal; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; binding; Biological and medical sciences; Cattle; Cells, Cultured; Circular Dichroism; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; growth hormone receptors; lactogen; Molecular Sequence Data; placenta; Placental Lactogen - analogs & derivatives; Placental Lactogen - chemistry; Placental Lactogen - isolation & purification; Placental Lactogen - metabolism; Protein hormones. Growth factors. Cytokines; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; tertiary structure; Tumor Cells, Cultured; Protein hormone; Placental lactogen; Vertebrata; Mammalia; Bovine; Structure activity relation; Artiodactyla; Recombinant protein; N terminal peptide; Secondary structure; Ungulata; Hormonal receptor
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)42327-7

Removal of 13 to 15 amino acids from the N terminus of bovine placental lactogen (bPL), which according to the three-dimensional structure of pGH corresponds to a nonhelical part of bPL, did not effect its secondary structure or change the monomer content of the protein preparation. However, it remarkably decreased the binding of the prolactin (PRL) type of receptors on Nb2 cells with subsequent reduction in bioactivity. The binding to the growth hormone (somatogen) receptors either did not change or was increased, resulting in an increase of somatogen receptor-mediated bioactivity. Further truncation (17-18 amino acids) resulted in a decrease of alpha-helical content and loss of binding properties and biological activity mediated through interaction of the analogues with both somatogen (3T3-F442A cells) and lactogen (PRL) receptors (NB2-11C cells). Truncation of 19-27 amino acids caused additional loss in activity, without further change in the secondary structure. Replacement of Leu28 by a more hydrophobic Phe has only minor, if any, effect on the bioactivity of bPL. Occasional point mutations due to polymerase chain reaction errors in several analogues did not seem to have any major effect on the hormone properties. It can thus be suggested that the N-terminal part of the nonhelical portion of bPL, which corresponds to the portion of the molecule that does not exist in growth hormones, is required for efficient binding to the lactogen (PRL) but not to the somatogen or unique bPL receptors. Removal of the N-terminal part of pBL changed the specificity of bPL by decreasing its PRL receptor-mediated activities and increasing its somatogen receptor-mediated activities.