CHROMOGRANIN-B, A PUTATIVE PRECURSOR OF 8 NOVEL RAT GLUCAGONOMA PEPTIDES THROUGH PROCESSING AT MONOBASIC, DIBASIC, OR TRIBASIC RESIDUES

Chromogranin-B (CgB), a secretory granule protein, is normally synthesized in a variety of neuroendocrine tissues, including the pancreatic islet alpha-cells. We have demonstrated that rat CgB is expressed and extensively processed by limited proteolysis in a transplantable glucagonoma tumor line. Eight peptides (fragments 1-8) purified by HPLC from acidic tumor extracts were partially sequenced and showed homology to CgB amino acid sequences deduced from rat, mouse, and human cDNA. Similar peptides were not found in insulinomas of common origin. The determined amino acid sequence represents approximately 35% of the rat precursor CgB. Ten of a total of 231 sequenced residues deviated from the published rat cDNA sequence. The differences were clustered in 3 fragments, suggesting allelic polymorphism. Five of the 8 peptides could be derived from the precursor by processing at paired basic amino acids, but processing N-terminally at a single basic residue was also seen. One peptide equivalent to the previously reported C-terminal CgB (CCB) is released by processing at a tribasic segment. Fragment 1 containing the N-terminal sequence Ala-Pro-Val-Asp represents the actual N-terminus of CgB after removal of the putative signal peptide sequence. All processing sites used in glucagonoma tissue to derive the 8 isolated fragments were conserved between murine and human CgB. At least 3 dibasic sites present in rat, but not human, CgB sequence were actually not used. A previously reported CgB-derived pituitary peptide, GAWK, was further processed at a conserved internal dibasic site to yield fragment 6, indicating alternative processing in different tissues. The small undecapeptide, fragment 7, is 100% conserved among murine and human CgB and, thus, may have an important biological function. We conclude that CgB is extensively processed in glucagonoma tissue by limited proteolysis as prohormones at conserved basic residues. The proglucagon-converting enzymes present in transformed alpha-cells are likely candidates to be involved in tissue-specific CgB processing. Destinct bioligical activities of any of the CgB-derived fragments remain to be identified.