Site-specific mutagenesis identifies amino acid residues critical in prohormone processing.
Peptide hormones are generally synthesized as inactive higher mol. wt precursors. Processing of the prohormone into biologically active peptides by specific proteolytic cleavages occurs most often at pairs of basic amino acids but also at single arginine residues. To study the role of protein secondary structure in this process, we used site-directed mutagenesis to modify the predicted secondary structure around the cleavage sites of human prosomatostatin and monitored the processing of the precursor after introduction of the mutated cDNAs in Neuro2A cells. Amino acid substitutions were introduced that affected the possibility of forming beta-turn structures in the immediate vicinity of the somatostatin-28 (S-28) and somatostatin-14 (S-14) cleavage sites. Infection of Neuro2A cells with a retrovirus carrying a human somatostatin cDNA resulted in the expression of prosomatostatin and its processing into S-28 and S-14, indicating that these cells have the necessary enzymes to process prohormone at both single and paired amino acid residues. Disruption of the different beta-turns had various effects on prosomatostatin processing: substitution of Ala for Pro-5 drastically decreased prosomatostatin processing and replacement of Pro-9 by Ala led to the accumulation of the intermediate maturation product [Arg-2Lys-1]-S-14. In contrast, substitution of Ala for Asn-12, Gly+2 and Cys+3 respectively had only very little effect on the proteolytic processing of prosomatostatin. Our results show that amino acids other than the basic amino acid residues are required to define the cleavage sites for prohormone proteolytic processing and suggest that higher orders of protein structure are involved in substrate recognition by the endoproteases.
Gomez
S
S
Unité Associée au Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, Paris, France.
Boileau
G
G
Zollinger
L
L
Nault
C
C
Rholam
M
M
Cohen
P
P
D000217:Acyltransferases; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D005786:Gene Expression Regulation; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013004:Somatostatin / Q000235:genetics / Q000378:metabolism*; D053561:Somatostatin-28; D014162:Transfection
D001426:Bacterial Proteins; D008565:Membrane Proteins; D011498:Protein Precursors; C061842:NodE protein, Rhizobium; D013004:Somatostatin; D053561:Somatostatin-28; D000217:Acyltransferases