The somatostatin-28(1-12)-NPAMAP sequence: an essential helical-promoting motif governing prosomatostatin processing at mono- and dibasic sites

Biochemistry. 2002 Feb 5;41(5):1630-9. doi: 10.1021/bi011928m.

Abstract

Proline residues, known to have special structural properties, induce particular conformations which participate in some biological functions. Two prolines (Pro(-9), Pro(-5)) located near the processing sites (Arg(-15) and Arg(-2)Lys(-)(1)) of human prosomatostatin were previously shown to be important for cleavage of the precursor into somatostatin-28 (S-28) and somatostatin-14 (S-14) [Gomez et al. (1989) EMBO J. 8, 2911-2916]. In this study, the importance of the pentapeptide P-A-M-A-P sequence (P-(X)(3)-P pattern), located in the S-28(1-12) segment connecting the mono- and dibasic cleavage sites, was investigated by using site-directed mutagenesis. Analysis of prosomatostatin-derived peptides produced by expression of mutated cDNA species in Neuro2A cells indicated that (i) deletion of PAMAP decreased S-14 production, (ii) deletion of the two Pro residues almost abolished the cleavage at the dibasic site, and (iii) Pro displacement generating the AMAPP motif resulted in a decrease of S-28 production. Moreover, both theoretical and spectroscopic studies of synthetic peptides reproducing the S-28(1-12) sequence bearing critical mutations showed that this sequence can organize as an alpha helical structure. These observations demonstrate that NPAMAP constitutes an accurate alpha-helix nucleation motif allowing for the generation of equal amounts of S-28 and S-14 from their common precursor in Neuro2A cells. Moreover, they emphasize the importance of the S-28(1-12) segment joining Arg(-15) and Arg(-2)Lys(-1) cleavage sites whose conformational organization is essential for controlling their accessibility to the appropriate processing proteases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs / genetics
  • Amino Acid Sequence
  • Amino Acid Substitution / genetics
  • Animals
  • Arginine / metabolism
  • Circular Dichroism
  • Fishes
  • Humans
  • Hydrolysis
  • Lysine / metabolism
  • Mice
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oligopeptides / chemistry*
  • Oligopeptides / genetics
  • Oligopeptides / metabolism*
  • Protein Conformation
  • Protein Precursors / chemistry*
  • Protein Precursors / genetics
  • Protein Precursors / metabolism*
  • Protein Processing, Post-Translational* / genetics
  • Protein Structure, Secondary / genetics
  • Protein Structure, Tertiary / genetics
  • Sequence Deletion
  • Somatostatin / chemistry*
  • Somatostatin / genetics
  • Somatostatin / metabolism*
  • Somatostatin-28
  • Spectroscopy, Fourier Transform Infrared
  • Tumor Cells, Cultured

Substances

  • Oligopeptides
  • Protein Precursors
  • Somatostatin
  • prosomatostatin
  • Somatostatin-28
  • Arginine
  • Lysine