Determining sequences and post-translational modifications of novel conotoxins in Conus victoriae using cDNA sequencing and mass spectrometry.
A combination of cDNA cloning and detailed mass spectrometric analyses was employed to identify novel conotoxins from Conus victoriae. Eleven conotoxin sequences were determined using molecular methods: one belonging to the A superfamily (Vc1.1), six belonging to the O superfamily (Vc6.1-Vc6.6) and four members of the T superfamily (Vc5.1-Vc5.4). In order to verify the sequences and identify the post-translational modifications (excluding the disulfide connectivity) of three Conus victoriae conotoxins, vc1a, vc5a and vc6a, deduced from sequences Vc1.1, Vc5.1, and Vc6.1, respectively, liquid chromatography/electrospray ionization ion trap mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanospray ionization ion trap mass spectrometry with collisionally induced dissociation were performed on reduced and alkylated venom fractions. We report that vc1a, the native form of alpha-conotoxin Vc1.1 (an unmodified 16 amino acid residue peptide that has notable pain-relieving capabilities), includes a hydroxyproline and a gamma-carboxyglutamate residue. Conotoxin vc5a is a 10-residue peptide with two disulfide bonds and a hydroxyproline and vc6a is a 25 amino acid peptide with three disulfide bonds.
Jakubowski
Jennifer A
JA
Department of Chemistry and the Beckman Institute, University of Illinois, Urbana-Champaign, Illinois 61801, USA.
Keays
David A
DA
Kelley
Wayne P
WP
Sandall
David W
DW
Bingham
Jon-Paul
JP
Livett
Bruce G
BG
Gayler
Ken R
KR
Sweedler
Jonathan V
JV
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D020916:Conotoxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008978:Mollusk Venoms / Q000737:chemistry / Q000235:genetics; D011499:Protein Processing, Post-Translational; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D020916:Conotoxins; D018076:DNA, Complementary; D008978:Mollusk Venoms