Active variants of human parathyroid hormone (1-34) with multiple amino acid substitutions

John F. Reidhaar-Olson, Robyn M. Davis, Janet A. De Souza-Hart, Harold E. Selick

Research output: Contribution to journalArticle

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Abstract

We used site-directed mutagenesis to construct 55 single-site variants of rhPTH, a recombinantly-expressed form of human parathyroid hormone (1-34) containing three amino acid changes compared to the natural sequence (ML8, ML18 and FY34). We identified several mutations, at residues Lys13, Glu19, Val21, Glu22, Lys27 and Asp30, that increase biological activity by up to 2.5-fold, as measured by stimulation of adenylate cyclase activity in rat UMR-106 cells. We constructed a series of 15 variants in which two to eight substitutions at these positions were combined, and found that the mutations behaved additively, leading to peptides with significantly enhanced potency. The most active combination variant, with six substitutions (KS13, ES19, VQ21, ES22, KQ27 and DN30), is 15 times more active than the parent molecule. However, the extent to which such combinations increase the activity of the peptide depends critically on the identity of the residues at positions 8 and 18. We constructed two of the combination variants in a variety of sequence backgrounds containing different combinations of leucine, methionine and norleucine at positions 8 and 18. Enhancements in potency were significantly reduced when Met or Nle was present at either of these positions, both in UMR-106 cells and human SaOS-2 cells. A corresponding non-additivity was observed in direct measurements of receptor binding affinity on UMR-106 cells. These results suggest that interactions, either direct or indirect, between certain PTH side chains prevent these mutations from behaving in an additive manner. Copyright (C) 2000 Elsevier Science Ireland Ltd.

Original languageEnglish (US)
Pages (from-to)135-147
Number of pages13
JournalMolecular and Cellular Endocrinology
Volume160
Issue number1-2
DOIs
StatePublished - Feb 25 2000
Externally publishedYes

Fingerprint

Teriparatide
Amino Acid Substitution
Substitution reactions
Norleucine
Amino Acids
Mutagenesis
Peptides
Bioactivity
Adenylyl Cyclases
Leucine
Methionine
Mutation
Rats
Molecules
Site-Directed Mutagenesis

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Active variants of human parathyroid hormone (1-34) with multiple amino acid substitutions. / Reidhaar-Olson, John F.; Davis, Robyn M.; De Souza-Hart, Janet A.; Selick, Harold E.

In: Molecular and Cellular Endocrinology, Vol. 160, No. 1-2, 25.02.2000, p. 135-147.

Research output: Contribution to journalArticle

Reidhaar-Olson, John F. ; Davis, Robyn M. ; De Souza-Hart, Janet A. ; Selick, Harold E. / Active variants of human parathyroid hormone (1-34) with multiple amino acid substitutions. In: Molecular and Cellular Endocrinology. 2000 ; Vol. 160, No. 1-2. pp. 135-147.
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