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Large variations in the proteolytic formation of a chromogranin A-derived peptide (GE-25) in neuroendocrine tissues.

ABSTRACT: We have established a radioimmunoassay for GE-25, a peptide present in the C-terminal end of the primary amino acid sequence of chromogranin A where it is flanked by typical proteolytic cleavage sites. Gel-filtration HPLC was used to characterize the molecular sizes of the immunoreactive molecules. The antiserum recognized not only the free peptide but also larger precursors including the proprotein chromogranin A. The tissues with the highest levels of GE-25 immunoreactivity were in decreasing order: the adrenal medulla, the three lobes of the pituitary gland, intestinal mucosa, pancreas and various brain regions. In adrenal medulla and parathyroid gland most of the immunoreactivity was found to be present as intact chromogranin A and some intermediate-sized peptides, without significant amounts of the free peptide. In anterior pituitary, and even more so in intestine, a shift to smaller peptides was seen. In the posterior and intermediate pituitary and in pancreas the predominant immunoreactive material was apparently represented by the free peptide GE-25. In reverse-phase chromatography this peptide eluted exactly like the synthetic standard, which allows a tentative identification as GE-25. In brain tissue the processing of chromogranin A was intermediate, with significant amounts of immunoreactivity corresponding to GE-25 as well as precursor proteins being present. We suggest that in those organs (endocrine pancreas, intermediate and posterior pituitary) where the major hormones are proteolytically processed there is also a concomitant proteolysis of further susceptible peptides. Since GE-25 is apparently formed in vivo and is well conserved between species it seems a good candidate for having specific physiological functions.

SUBMITTER: Kirchmair R

PROVIDER: S-EPMC1135892 | biostudies-other | 1995 Aug

REPOSITORIES: biostudies-other

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Project description:Objective:Currently, serum chromogranin A is a well-established biomarker for pancreatic neuroendocrine tumors; however, other pancreatic diseases, oral use of a proton pump inhibitor and renal impairment can affect chromogranin A. Meanwhile, chromogranin B, belonging to the same granin family as chromogranin A, is not fully examined in these conditions. The present study aimed to evaluate the utility of chromogranin B as a pancreatic neuroendocrine tumor biomarker. Methods:Serum chromogranin B levels were determined by radioimmunoassay and serum chromogranin A levels by enzyme-linked immunosorbent assay in pancreatic neuroendocrine tumor (n = 91) and other pancreatic conditions, and in healthy people (n = 104), to assess the relationships with clinical features. Results:The diagnostic ability of chromogranin B was as good as chromogranin A. The area under the curve was 0.79 for chromogranin B (sensitivity/specificity: 72%/77%), and 0.78 for chromogranin A (sensitivity/specificity: 79%/64%). Chromogranin B was not affected by proton pump inhibitor use and age, which affected chromogranin A. The number of cases without liver metastases was larger in pancreatic neuroendocrine tumor patients with positive chromogranin B and negative chromogranin A. Though chromogranin A significantly elevated cases with proton pump inhibitor treatment and had positive correlation with age, chromogranin B did not have the tendencies. However, both chromogranin B and chromogranin A elevated in the case with renal impairment. In addition, the logistic regression analysis showed that chromogranin B was superior to chromogranin A in differentiation of pancreatic neuroendocrine tumor from other pancreatic diseases. Conclusions:Compared with chromogranin A, chromogranin B may be more useful during proton pump inhibitor treatment and can detect tumors without liver metastases. In addition, chromogranin B may be an excellent biomarker when differentiation of pancreatic neuroendocrine tumor from other pancreatic diseases is required.

Project description:Secretion of proteins and peptides from eukaryotic cells takes place by both constitutive and regulated pathways. Regulated secretion may involve interplay of proteins that are currently unknown. Recent studies suggest an important role of chromogranin A (CHGA) in the regulated secretory pathway in neuroendocrine cells, but the mechanism by which CHGA enters the regulated pathway, or even triggers the formation of the pathway, remains unclear. In this study, we used a transcriptome/proteome-wide approach, to discover binding partners for CHGA, by employing a phage display cDNA library method. Several proteins within or adjacent to the secretory pathway were initially detected as binding partners of recombinant human CHGA. We then focused on the trans-Golgi protein SCLIP (STMN3) and its stathmin paralog SCG10 (STMN2) for functional study. Co-immunoprecipitation experiments confirmed the interaction of each of these two proteins with CHGA in vitro. SCLIP and SCG10 were colocalized to the Golgi apparatus of chromaffin cells in vivo and shared localization with CHGA as it transited the Golgi. Downregulation of either SCLIP or SCG10 by synthetic siRNAs virtually abolished chromaffin cell secretion of a transfected CHGA-EAP chimera (expressing CHGA fused to an enzymatic reporter, and trafficked to the regulated pathway). SCLIP siRNA also decreased the level of secretion of endogenous CHGA and SCG2, as well as transfected human growth hormone, while SCG10 siRNA decreased the level of regulated secretion of endogenous CHGB. Moreover, a dominant negative mutant of SCG10 (Cys 22,Cys 24-->Ala 22,Ala 24) significantly blocked secretion of the transfected CHGA-EAP chimera. A decrease in the buoyant density of chromaffin granules was observed after downregulation of SCG10 by siRNA, suggesting participation of these stathmins in granule formation or maturation. We conclude that SCLIP and SCG10 interact with CHGA, share partial colocalization in the Golgi apparatus, and may be necessary for typical transmitter storage and release from chromaffin cells.

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Large variations in the proteolytic formation of a chromogranin A-derived peptide (GE-25) in neuroendocrine tissues.

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