A chemically synthesized radiolabeled signal peptide: design, preparation, and biological evaluation of an iodinated analog of preproparathyroid hormone.
The chemically synthesized signal peptide (native-sequence signal peptide) of preproparathyroid hormone exhibits signal sequence-like activity by inhibiting the translocation/processing of precursor proteins to their mature forms in an in vitro translation system. In order to prepare a biologically functional radiolabeled form of this peptide, we undertook structure-function studies of the native-sequence signal peptide. Since conventional iodination of peptides is performed under oxidizing conditions, chemical design efforts were focused on the oxidation-labile residues, methionine and cysteine, present in the native sequence. Substitution of the three methionines with norleucine and the single cysteine with alanine yielded a surfur-free analog, [Nle-(-25), Nle-(-21),Nle-(-18),Ala-(-14),D-Tyr-(+1)]pre-proPTH-(-29-+1)amide, which is resistant to oxidation and active in the inhibition of processing assay. An interaction between the signal region and one of the components of the intracellular secretory apparatus, signal recognition particle (SRP), was demonstrated: iodinated sulfur-free analog was cross-linked (using the homo-bifunctional reagent disuccinimidyl suberate) to the 54 kilodalton (kDa) subunit of SRP. The 68 kDa and 72 kDa subunits of SRP were also labeled, but to a lesser extent, by the iodinated peptide.
Department of Biological Research and Molecular Biology, Merck, Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
M P
MP
Caulfield
L T
LT
Duong
R
R
O'Brien
J A
JA
Majzoub
M
M
Rosenblatt
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007455:Iodine / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000138:chemical synthesis* / Q000494:pharmacology; D011498:Protein Precursors / Q000138:chemical synthesis* / Q000494:pharmacology; D021382:Protein Sorting Signals / Q000138:chemical synthesis* / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013455:Sulfur / Q000494:pharmacology
D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D010281:Parathyroid Hormone; D011498:Protein Precursors; D021382:Protein Sorting Signals; C057075:pre-proparathyroid hormone (29+1) amide, Tyr(1)-Ala(14)-Nle(18,21,25)-; D013455:Sulfur; D007455:Iodine