Mass spectrometric analysis of posttranslational modifications of a carrot extracellular glycoprotein.
Expression of extracellular dermal glycoprotein (EDGP) is induced by biotic or abiotic stress. The amino acid sequence alignment showed that EDGP shared significant homology with proteins from legumes, tomato, Arabidopsis, wheat, and cotton. These proteins are involved in signal transduction or stress response systems. Most of the Cys residues in these proteins are conserved, suggesting that they share similar tertiary structures. Surface plasmon resonance (SPR) analysis shows that EDGP binds a soybean 4-kDa hormone-like peptide (4-kDa peptide) in vitro and reduction of EDGP decreased significantly the binding activity, implying that posttranslational modifications are important for its function. Therefore, we investigated the posttranslational modifications in EDGP using mass spectrometry. As the result, six disulfide bonds in EDGP were identified: Cys(70)-Cys(158), Cys(84)-Cys(89), Cys(97)-Cys(113), Cys(100)-Cys(108), Cys(201)-Cys(426), and Cys(332)-Cys(378). In addition, the N-terminal glutamine was cyclized into pyroglutamic acid. All four putative glycosylation sites were occupied by N-linked glycans, which have similar masses of m/z 1171. Finally, measuring the mass of the native protein showed that the posttranslational modifications of EDGP (pI 9.5) involved only disulfide bonds, N-terminal modification, and glycosylation.
Shang
Chengwei
C
Kihara Institute for Biological Research, Graduate School of Integrated Science, Yokohama City University, Maioka-cho 641-12, Totsuka, Yokohama 244-0813, Japan.
Shibahara
Tadashi
T
Hanada
Kazuki
K
Iwafune
Yuko
Y
Hirano
Hisashi
H
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D018552:Daucus carota / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D005110:Extracellular Space / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D004220:Disulfides; D006023:Glycoproteins; D010940:Plant Proteins