Expression and characterization of a recombinant human parathyroid hormone partial agonist with antagonistic properties: Gly-hPTH(-1-->+84).
We have produced and characterized a hPTH analogue with an amino-terminal extension of glycine, Gly-hPTH(-1-->+84) (denoted Gly-hPTH). The hormone analogue was synthesized in E. coli strain BJ5183 transformed with the expression plasmid pKKPTH, extracted from the bacterial pellet and purified by reverse-phase high performance liquid chromatography. Its chemical nature, as determined by amino acid composition analysis, N-terminal amino acid analysis, and mass spectrometry, showed the 9480-Da Gly-hPTH as the predominant species. Because f-Met-Gly-hPTH was the expected form encoded by the plasmid construct, the results indicate that the f-Met residue was efficiently removed from the precurser form. The following functional characteristics of Gly-hPTH were demonstrated. 1) In cells transfected with the human PTH/PTHrP receptor, the receptor binding affinity was reduced threefold compared to the authentic hPTH(1-84) produced by Saccharomyces cerevisiae (apparent Kds: 8.4 and 2.7 nM, respectively). 2) Using the same cells, Gly-hPTH showed 27-fold reduced potency compared to hPTH(1-84) in stimulating intracellular cAMP production (EC50: 32 and 1.2 nM, respectively). 3) Gly-hPTH demonstrated antagonist activity by reducing hPTH-induced cAMP production by 33 +/- 5% (mean +/- SD) when tested at a 1:1 molar ratio. In these studies the recombinant authentic hPTH(1-84) was used as standard for comparisons, and it showed an equal receptor binding affinity and cAMP production as the chemically synthesized peptide [Nle8,18,Tyr34]bovinePTH(1-34)-NH2.
Olstad
O K
OK
Institute of Medical Biochemistry, University of Oslo, Norway.
Jemtland
R
R
Loseth
O P
OP
Bringhurst
F R
FR
Gautvik
K M
KM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D018374:LLC-PK1 Cells; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000235:genetics; D018016:Receptors, Parathyroid Hormone / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D013552:Swine; D014162:Transfection
D000596:Amino Acids; D010281:Parathyroid Hormone; D018016:Receptors, Parathyroid Hormone; D011994:Recombinant Proteins; D000242:Cyclic AMP