METHODS AND APPLICATIONS Identifying N-terminal peptides by a combination of the edman procedures with a bromine isotope tag: Application to the silicateins

摘要

Silicateins are proteins found within spicules of siliceous sponges. They are analogs of proteinases cathepsins; they catalyze the transformation of silicic acid esters into biogenic silica (SiO2.nH(2)O), and are believed to take part in the processes of silicification in marine and freshwater sponges. Earlier studies by Kalyuzhnaya et al. revealed that the Baikal Sponge Lubomirskia baicalensis Pallas, 1773 (L. baicalensis) contains a gene 1988 bp long, which hosts four sequences that encode four mRNAs giving rise to silicateins alpha 1, alpha 2, alpha 3 and alpha 4 (SIL alpha 1, SIL alpha 2, SIL alpha 3, SIL alpha 4) whose predicted amino acid sequences are similar to those of the predicted sequences of marine sponge silicateins. However, the sequences of mature silicateins of L. baicalensis remained unknown, since their N-terminal peptides were not identified. We found the sequences of these N-terminal peptides using a combination of the Edman procedure, which involved reaction with phenylisothiocyanate, treatment with trifluoroacetic acid and trypsinolysis followed by treatment with 4bromine-phenylisothiocyanate performed directly within polyacrylamide gel bands, and subsequent mass spectrometry. The N-terminal peptides are YAESIDWR (SIL alpha 1), YVDSIDWR (SIL alpha 2 and alpha 4), and YADSLDWR (SIL alpha 3). All mature silicateins of L. baicalensis had a length 217 amino acid residues.