Measurement of nonsulfated cholecystokinins

Abstract

Most proteins undergo posttranslational modifications that govern the function of the protein. In synchrony, correspondingly unmodified proteins that are functionally silent or act differently may also be synthesized. The gut hormone precursor, procholecystokinin (proCCK) is an example of a protein that is heavily modified. An essential modification is O-sulfation of Y₇₇, which is necessary for the gallbladder emptying effect of CCK peptides via the CCK_A-receptor. In order to examine possible in vivo synthesis also of nonsulfated CCK, we have established a two-step analysis that requires tryptic cleavage at a defined processing site in proCCK (R₇₅-D₇₆) followed by monospecific RIA-measurement of the then exposed nonsulfated N-terminal sequence of CCK-8 (DYMGW…). The analysis shows that endocrine cells in the gut synthesize nonsulfated CCK peptides (−58, −33, −22, and −8) in the order of 20–35% of the corresponding sulfated CCKs. Since nonsulfated CCK peptides are full agonists of the CCK_B-receptor, the assay has revealed a hitherto unrecognized gut hormonal peptide system. The assay may prove useful in the diagnosis and control of diseases with hyperCCKemia. This includes CCK-producing neuroendocrine tumors such as the recently described CCKomas and medullary thyroid C-cell carcinomas.

Key Words::

• Cholecystokinin (CCK)
• CCKomas
• hormone biosynthesis
• peptide hormones
• processing-independent analysis
• sequence-specific radioimmunoassay (SS-RIA)
• tyrosyl O-sulfation

Acknowledgements

The skillful secretarial assistance of Connie Bundgaard and the technical assistance of Alice Lieth and Rikke Kröncke are gratefully acknowledged.

Declaration of interest: The authors have no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This study was financially supported by the Research Foundation of Rigshospitalet (Copenhagen) by a scholarship to one of us (MA). Moreover, support was obtained from the Danish Biotechnology Center of Cellular Communication and the Lundbeck Foundation.