Characterization of the disulfide bonds and the N-glycosylation sites in the glycoprotein from Rathke's gland secretions of Kemp's ridley sea turtle (Lepidochelys kempi).

ABSTRACT

The disulfide bonds and N-glycosylation sites in a glycoprotein from the Rathke's gland secretion of the Kemp's ridley turtle (Lepidochelys kempi) have been characterized with respect to peptide sequences and glycan structures. The glycoprotein constitutes about 70% of the total protein in the secretion, and based on partial sequence information, it shows more than 20% identity with both the catalytic (esterases) and the noncatalytic (thyroglobulin) members of the esterase/lipase family of proteins. For the determination of the disulfide locations, the glycoprotein was digested with chymotrypsin, and the three HPLC peptide peaks yielding fluorescent products after treatment with tributylphosphine (Bu3P) and 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) were collected. The three fractions were treated with the same reagents in separate experiments, the resulting pairs of ABD-Cys-containing peptides were separated by HPLC, and the sequence of each individual peptide was determined. The peptide identity established that three disulfide bonds existed in the glycoprotein Cys 65-Cys 91, Cys 254-Cys 265, and Cys 130-Cys 404; the first two of these are conserved in all the members of the esterase family. For the study of the glycosylation sites, the glycoprotein was reduced with Bu3P and the SH groups covalently blocked with ABD-F, and the resulting product was digested with chymotrypsin. The glycopeptides were isolated by affinity chromatography, separated by reverse-phase HPLC, and subjected to sequence analysis and fast atom bombardment mass spectrometry before and after separation of the glycans and the peptides through the action of glycoamidase. Three separate glycosylation sites were identified, each containing multiple glycans. The sugar analyses of the hydrolysates of the glycoprotein indicated that only GlcNAc and Man were present as building blocks, and the mass spectrometric data showed that Man3GlcNAc2-, GlcNAc2-4Man3GlcNAc2-, and possibly GlcNAc2Man2GlcNAc2- were the major glycan structures, distributed differently at the three sites. The three glycosylation sites match three of the nine sites glycosylated in human serum choline esterase, and one of them, Asn 106, is also found as one of two glycosylation sites in the homologous segment of thyroglobulin.