Structure/activity relationship of eel calcitonin. A study using a newly devised method for designing analogs.
A series of analogs of eel calcitonin (eCT) was synthesized according to a newly devised scheme, 'the insertion-inactivation method', to clarify the structure/activity relationship of a given peptide. This method consists of two steps: the deletion of a residue of the peptide is first chosen and then a series of analogs with the residue reinserted into serial positions is synthesized and biological activities are assessed in each step. An analog lacking Lys18 (dK), selected as a deleted analog for the first step, showed marked loss of activities determined by inhibition of 125I-eCT binding, growth inhibition, and cAMP production in a porcine kidney cell line LLC-PK1. Activities of a set of 20 analogs with the reinserted lysine residue at serial positions from 12 to 32 (K12-K32) were then evaluated. The results showed the following three patterns of the expression of activities according to the position of the reinsertion: (a) analogs K12-K16 (positions 12-16) and K25 (position 25) showed lower activities than eCT in all assays; (b) K17-K24 (positions 17-24) showed slightly lower activities than eCT in the receptor binding and the growth inhibition and similar level in cAMP production; (c) K26-K32 (positions 26-32) showed considerably lower activities in the former two assays and slightly lower activity in cAMP production. Further, analogs considerably less active than eCT showed unchanged alpha-helix contents and destroyed amphiphilicity by the insertion of a lysine residue, indicating that amphiphilicity is one of important factors for expressing the activity. The results obtained here lead to a conclusion on the significance of each region of eCT molecule as follows: (a) the presence of Lys18 is necessary for the complete expression of biological activity; (b) the length of amphiphilic alpha-helix to be required for the activity is at most 10 residues ranging from position 8 to position 17; (c) the receptor binding region is located within 9 residues ranging from position 24 to position 32.
Inoue
A
A
Protein Engineering Section, Sagami Chemical Research Center, Kanagawa, Japan.
Shikano
M
M
Komatsu
Y
Y
Obata
J
J
Ochiai
J
J
Nishide
H
H
Ito
N
N
Nagao
H
H
Kondo
K
K
Tunemoto
D
D
D000595:Amino Acid Sequence; D000818:Animals; D002116:Calcitonin / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000494:pharmacology / Q000502:physiology*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D000242:Cyclic AMP / Q000096:biosynthesis; D004524:Eels; D008239:Lysine; D008969:Molecular Sequence Data; D018003:Receptors, Calcitonin; D011956:Receptors, Cell Surface / Q000378:metabolism; D013329:Structure-Activity Relationship
D018003:Receptors, Calcitonin; D011956:Receptors, Cell Surface; D002116:Calcitonin; D000242:Cyclic AMP; D008239:Lysine