Effect of O-linked glycosylation on the antigenicity, cellular uptake and trafficking in dendritic cells of recombinant Ber e 1
Fig 1
Detection of glycosylation on 2S albumin proteins by periodic acid Schiff’s (PAS) staining.
(A.) A positive glycoprotein control, CPDY (1.), rSFA8 (2.), rBer e 1 (3.), and nBer e 1 (4.) were separated on a 12% BisTris NuPAGE gel under denaturing conditions (300 mM DTT) and PAS stained to detect glycoproteins. Only the ~15 kDa band of rBer e 1 and the positive control, CPDY, reacted with the PAS stain, indicating the presence of glycan residues. nBer e 1 and rSFA8 did not react with PAS stain, thus indicate the absence of glycosylation according to this assay. (B.) An identical NuPAGE gel stained with Coomassie brilliant blue shows all of the proteins present. Raw images of Commassie- and PAS-stained gels are presented in (S3 Fig). (C.) rBer 1, nBer e 1 and rSFA8 protein bands were excised for peptide identification. Samples of rBer e 1(I and II), and nBer e 1 (III and IV) and rSFA8 (V) were trypsinised and analysed by LC-MSMS analysis. (D.) Amino acid sequence of precursor native Ber e 1 in planta. Italic fonts represent the N-terminal signal sequence, the underlined fonts represent the pre-prosignal sequences, and the italic and underlined fonts represent the short linker and C-terminal linker. These sequences were cleaved and removed to produce mature Ber e 1 in planta that consists of small subunit (yellow highlight) and large subunit. (E.) Amino acid sequence of mature recombinant Ber e 1 retained the spacer sequence (bold fonts) downstream of the Kex2 cleavage site, spacer, and C-terminal vacuolar targeting signal sequence (italic and underlined fonts) and it is made up of small subunit (yellow highlight) and large subunit [16, 24].