Chemical synthesis of La1 isolated from the venom of the scorpion Liocheles australasiae and determination of its disulfide bonding pattern.
La1 is a 73-residue cysteine-rich peptide isolated from the scorpion Liocheles australasiae venom. Although La1 is the most abundant peptide in the venom, its biological function remains unknown. Here, we describe a method for efficient chemical synthesis of La1 using the native chemical ligation (NCL) strategy, in which three peptide components of less than 40 residues were sequentially ligated. The peptide thioester necessary for NCL was synthesized using an aromatic N-acylurea approach with Fmoc-SPPS. After completion of sequential NCL, disulfide bond formation was carried out using a dialysis method, in which the linear peptide dissolved in an acidic solution was dialyzed against a slightly alkaline buffer to obtain correctly folded La1. Next, we determined the disulfide bonding pattern of La1. Enzymatic and chemical digests of La1 without reduction of disulfide bonds were analyzed by liquid chromatography/mass spectrometry (LC/MS), which revealed two of four disulfide bond linkages. The remaining two linkages were assigned based on MS/MS analysis of a peptide fragment containing two disulfide bonds. Consequently, the disulfide bonding pattern of La1 was found to be similar to that of a von Willebrand factor type C (VWC) domain. To our knowledge, this is the first report of the experimental determination of the disulfide bonding pattern of peptides having a single VWC domain as well as their chemical synthesis. La1 synthesized in this study will be useful for investigation of its biological role in the venom.
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan.
Junya
J
Nagao
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan.
Masahiro
M
Miyashita
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan.
Yoshiaki
Y
Nakagawa
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan.
Hisashi
H
Miyagawa
D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification; D015394:Molecular Structure; D012604:Scorpion Venoms / Q000138:chemical synthesis / Q000737:chemistry*; D012605:Scorpions / Q000737:chemistry* / Q000502:physiology; D013440:Sulfides / Q000737:chemistry*; D053719:Tandem Mass Spectrometry; D014841:von Willebrand Factor / Q000737:chemistry
D060829:Arthropod Proteins; D012604:Scorpion Venoms; D013440:Sulfides; D014841:von Willebrand Factor
MS/MS; disulfide bond; native chemical ligation; peptide synthesis; scorpion venom