Chromogranin A peptide-specific antisera and high-performance size exclusion chromatography demonstrate amino-terminal and carboxy-terminal fragments of the native molecule in human cell lines.
We have studied the pattern of amino-terminal and carboxy-terminal protein processing of chromogranin A (CgA) in five human cell lines, four derived from lung cancers: NCI-H478, NCI-H1011, NCI-H727, and BEN; and one derived from human medullary thyroid carcinoma: TT. This was accomplished by fractionation of cell extracts by high-performance size exclusion chromatography (HPSEC) and measurement of CgA in the fractions by radioimmunoassay (RIA). Three RIA's were used, one specific for the amino-terminus of CgA, one specific for the carboxy-terminal region, and a monoclonal antibody-based assay that recognizes only the native CgA molecule. We demonstrated the presence of different amino- and carboxy-terminal immunoreactive species of CgA in the different cell lines. The amino-terminal assay demonstrated distinct low-molecular-size species in the NCI-H478 and NCI-H1011 cell lines, and a similar peak in the TT cells. The amino-terminal assay did not recognize any distinct species in BEN and NCI-H727 cell lines. The carboxy-regional assay demonstrated distinct low-molecular-size species in the NCI-H478 and NCI-H101 cell lines and high-molecular-size species in the NCI-H727 and BEN cells. Our studies demonstrate with region-specific RIA's the presence of both amino- and carboxy- forms of CgA in human cells that secrete this protein. These results provide direct evidence that CgA-producing cells produce, probably through endoproteolytic processing of the native molecule, amino- and carboxy-terminal forms of the protein.
Brandt
D W
DW
Department of Medicine, University of California, San Diego.
Burton
D W
DW
Hogue-Angeletti
R
R
Deftos
L J
LJ
D000595:Amino Acid Sequence; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D053379:Chromogranin A; D002864:Chromogranins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D008175:Lung Neoplasms; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011863:Radioimmunoassay; D013964:Thyroid Neoplasms; D014407:Tumor Cells, Cultured
C506150:CHGA protein, human; D053379:Chromogranin A; D002864:Chromogranins; D007106:Immune Sera; D010446:Peptide Fragments