Structural studies of a neuropeptide precursor protein with an RGD proteolytic site.
The snail Lymnaea stagnalis produces a neuropeptide precursor protein that contains seven Arg-Gly-Asp (RGD) sites. These sites are recognized and cleaved by one or more prohormone convertases in the first processing step to yield mature neuropeptides in the secretory pathway. Conformations of two synthetic RGD-containing peptides derived from the L. stagnalis precursor protein were determined by NMR spectroscopy. The peptides were tested in a platelet aggregation assay for RGD activity and were processed in vitro by PC2 and furin. The native peptide with a proline following the RGD site has minimal structure around the RGD region, does not inhibit platelet aggregation, and is properly processed by the enzymes PC2 and furin. A variant of the native fragment with a serine following the RGD sequence has a significant amount of a reverse turn around the RGD region, is a potent inhibitor of platelet aggregation, and is processed with the same specificity as the native fragment. The large conformational differences between the two peptides provide a molecular mechanism for effects of proline residues following the RGD site and suggest that precursor processing is influenced more by flexibility than by the conformation of the processing site.
Zachariah
C
C
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, USA.
Cameron
A
A
Lindberg
I
I
Kao
K J
KJ
Beinfeld
M C
MC
Edison
A S
AS
D000595:Amino Acid Sequence; D000818:Animals; D010450:Endopeptidases / Q000378:metabolism*; D045683:Furin; D006801:Humans; D006868:Hydrolysis; D008195:Lymnaea; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism*; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000378:metabolism
D009479:Neuropeptides; D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D011498:Protein Precursors; C047981:arginyl-glycyl-aspartic acid; D010450:Endopeptidases; D013381:Subtilisins; D045683:Furin