An efficient cellular system for mutational analysis of prohormone processing.
A novel system for heterologous expression of prohormones based on transient transfection of the HIT beta-cell line was established using human progastrin as a model. Progastrin was expressed at high levels compared to other gene transfer systems in endocrine cells, and the processing pattern was similar to that of normal antral gastrin cells. Thus, gastrin was partially tyrosine O-sulfated and carboxyamidated. Cell extracts contained mainly gastrin-17 and gastrin-34 and the corresponding glycine-extended forms. In contrast, the media contained more incompletely processed gastrin forms. This suggests that gastrin was directed to the regulated secretory pathway but that some progastrin products were constitutively secreted. Glucose increased both the level of gastrin gene expression and maturation to carboxyamidated peptides, indicating that glucose influences the activity of the amidation enzyme complex, peptidylglycine alpha-amidating mono-oxygenase (PAM), in insulin cells. Mutational analysis of tyrosine sulfation of gastrin demonstrated that substitution of the uncharged residue carboxy-terminal to the tyrosine with an acidic residue does not increase sulfation in contrast to previous results, where the amino-terminal residue was replaced with an acidic residue. The mutant peptides displayed sulfation-dependent processing, supporting our recent suggestion that tyrosine sulfation increases the proteolytic processing of prohormones.
Bundgaard
J R
JR
Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Denmark.
Cowland
J B
JB
Vuust
J
J
Rehfeld
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002766:Cholecystokinin / Q000378:metabolism; D006224:Cricetinae; D004252:DNA Mutational Analysis; D005755:Gastrins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007515:Islets of Langerhans / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013455:Sulfur / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D005755:Gastrins; D009097:Multienzyme Complexes; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C013706:big gastrin; D014443:Tyrosine; D013455:Sulfur; D002766:Cholecystokinin; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D005947:Glucose