Isolation and characterization of VGF peptides in rat brain. Role of PC1/3 and PC2 in the maturation of VGF precursor.
The neurotrophin responsive gene vgf is widely expressed in central and peripheral neurones, and in certain neuroendocrine cell populations. Its encoded VGF precursor protein (proVGF1: 617 amino acids in rat, 615 in man, > 85% homology) gives rise to several low molecular weight species. We studied a range of neuroendocrine and neuronal cells, in which VGF-processing products were prominent with an apparent molecular weight of 20 and 10 kDa (VGF20 and VGF10, respectively). Such peptides were recognized by antibodies specific for the C-terminal rat VGF nonapeptide, thus indicating that they included the C-terminus of proVGF. Ectopic expression of the neuroendocrine-specific prohormone convertases PC1/3 or PC2 in GH3 cells showed that both could generate VGF20, while VGF10 was preferentially produced by PC1/3. Site-directed mutagenesis was used to identify the KRKRKK(488) motif as the target within VGF sequence which leads to the production of VGF20. Molecular characterization of rat VGF10, on the other hand, revealed that this peptide is produced by cleavage at the RPR(555) site. By the combined use of high-resolution separation techniques, matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectrometry and manual Edman degradation we identified in rat brain a VGF fragment analogous to bovine peptide V and two novel peptides also derived from the C-terminal region of proVGF.
Trani
E
E
Istituto di Neurobiologia e Medicina Molecolare, CNR, Roma, Italy.
Giorgi
A
A
Canu
N
N
Amadoro
G
G
Rinaldi
A M
AM
Halban
P A
PA
Ferri
G L
GL
Possenti
R
R
Schininà
M E
ME
Levi
A
A
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D001921:Brain / Q000166:cytology / Q000378:metabolism; D001923:Brain Chemistry; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009414:Nerve Growth Factors; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009479:Neuropeptides; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D043484:Proprotein Convertases; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D009414:Nerve Growth Factors; D009479:Neuropeptides; D010446:Peptide Fragments; D011498:Protein Precursors; D011506:Proteins; C512697:Vgf protein, mouse; C062040:Vgf protein, rat; D043484:Proprotein Convertases; D013381:Subtilisins; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D016282:Aspartic Acid Endopeptidases