Conformationally constrained human calcitonin (hCt) analogues reveal a critical role of sequence 17–21 for the oligomerization state and bioactivity of hCt

Abstract

Calcitonin (Ct) is a 32-residue peptide hormone that is mainly known for its hypocalcemic effect and the inhibition of bone resorption. Our previous studies have led to ]hCt (4), corresponding partial sequence peptides containing the lactam-bridged region 16-22, and nonbridged control peptides. Only 1 showed a higher Ct receptor binding affinity than hCt, whereas analogues 2-4 had similar receptor affinities to hCt.In the in vivo hypocalcemic assay, 3 and 4 were as potent as 1, whereas 2 completely lost the high potency of 1, suggesting that type I (and II¢) b turn-promoting substituents are fully compatible with in vivo bioactivity. CD spectroscopy showed that analogues 1-4 were markedly b sheet-stabilized compared to hCt and indicated the presence of distinct b turn conformeric populations in each of the analogues. Unexpectedly, the D-amino acid-or Aib-containing cyclic analogues 2-4 but not 1 or hCt self-associated into SDS denaturation-stable dimers. Our results demonstrate a crucial role of the conformational and topological features of the residues in sequence 17-21 and in particular of residues 18 and 19 for human Ct receptor binding and in vivo bioactivity and also for the self association state of hCt. These results may assist to delineate the structure-function relationships of hCt and to design novel hCt agonists for the treatment of osteoporosis and other bone-disorder-related diseases.

Rational design of aggregation-resistant bioactive peptides: Reengineering human calcitonin

“…To test the ability to predict the effect of multiple amino acid substitutions on the aggregation rate of a polypeptide sequence, a series of short peptides were designed by using amino acids 10-21 of hCT as an initial template (these peptides are collectively named CT 10 -21 ). Studies on peptide fragments of hCT have suggested that residues in this region could play an important role in the activity and aggregation behavior of the full-length peptide (35,36). It has been shown in particular that five residues corresponding to positions 15-19 of hCT (DFNKF) play an active role in oligomerisation and fibril formation by hCT in vitro (35), and Lys-18 and Phe-19 have been identified as key residues in both the bioactivity and self-assembly of hCT (36).…”

Converting the Highly Amyloidogenic Human Calcitonin into a Powerful Fibril Inhibitor by Three-dimensional Structure Homology with a Non-amyloidogenic Analogue

“…For this, we used an in silico approach applying the Waltz software (23). We first tested the software on hCT and sCT wild-type sequences, which were recognized as fibril-forming and aggregation-resistant according to reported experimental findings (11 (38,39) and the C-terminal region previously suggested as the fibril core (22,39,40).…”

Structural Determinants of Salmon Calcitonin Bioactivity

“…Cell Culture-Human breast cancer T 47D cells were cultured as described (20) in RPMI 1640 containing 10% heat inactivated fetal bovine serum, 1% streptomycin/penicillin, 0.1 M insulin, and 0.1 M hydrocortisone in 5% CO 2 and 310 K. The latter hormones were omitted from the medium when subculturing cells to be used 1-3 days later for the receptor binding assay. Subculturing was performed with trypsin/EDTA as described (41,42), and for the binding experiments cells were subcultured in 12-well dishes. Receptor binding experiments were performed when cells reached 90% confluence (1-3 days after subculture).…”

“…Receptor Binding Assay-The assay was performed according to established protocols (41)(42). Briefly, cells in the 12-well dishes were washed with NaCl/P i (1 ml) at room temperature, and then prewarmed (310 K) assay buffer (RPMI 1640 and 0.1% (w/v) bovine serum albumin) was added to the cells (950 l).…”