Synthesis and characterization of extended and deleted recombinant analogues of parathyroid hormone-(1-84): correlation of peptide structure with function.
Recombinant analogues of human parathyroid hormone [hPTH-(1-84)] were expressed in Escherichia coli harboring plasmids containing synthetic genes under the control of the lac promoter. The level of expression of the gene encoding the truncated analogue, hPTH-(3-84), was greater than that of the gene encoding full-length hPTH-(1-84) but less than that of the gene encoding proparathyroid hormone (hProPTH). This may be due in part to the relative efficiency of translation of the mRNA as suggested by secondary structure analysis and in part because of enhanced stability of the extended peptide. Formylmethionyl derivatives of hProPTH and of hPTH-(3-84) and underivatized hPTH-(3-84) were purified by HPLC, and their identity was confirmed by NH2-terminal sequencing and amino acid analysis. The bioactivity of these recombinant peptides was then tested in skeletal and renal adenylate cyclase assays in vitro and in assays examining effects on plasma and urine calcium and phosphate levels and on urine cyclic AMP levels in vivo. The NH2-terminally extended analogue fMet-hProPTH displayed 10% of the in vitro activity of hPTH-(1-84) and was a partial agonist in vivo. The peptides hPTH-(3-84) and fMet-hPTH-(3-84) were inert in vitro and were very weak in vitro antagonists when compared to the NH2-terminal analogue bovine [Nle8,18Tyr34]PTH-(3-34)-NH2. In vivo, hPTH-(3-84) and the bPTH-(3-34) analogue, when assayed at a 10:1 molar ratio relative to bPTH-(1-84), were each inert, and neither demonstrated antagonist activity at these concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
Rabbani
S A
SA
Department of Medicine, McGill University, Montreal, Quebec, Canada.
Kaiser
S M
SM
Henderson
J E
JE
Bernier
S M
SM
Mouland
A J
AJ
Roy
D R
DR
Zahab
D M
DM
Sung
W L
WL
Goltzman
D
D
Hendy
G N
GN
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000652:urine; D005786:Gene Expression Regulation; D005813:Genes, Synthetic; D006801:Humans; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D011498:Protein Precursors / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D019379:Teriparatide / Q000031:analogs & derivatives
D010281:Parathyroid Hormone; D010446:Peptide Fragments; D010710:Phosphates; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D019379:Teriparatide; C057206:parathyroid hormone (3-84); C066833:parathyroid hormone (3-84), formylmethionyl-; C066832:proparathyroid hormone (-6-+84), formylmethionyl-; C061438:parathyroid hormone (1-34)amide, Nle(8), Nle(18), Tyr(34)-; D000242:Cyclic AMP; D002118:Calcium