Location of cysteine and cystine residues in S-ribonucleases associated with gametophytic self-incompatibility.
S-Ribonucleases (S-RNases) that cosegregate with S-alleles in the styles of solanaceous and rosaceous plants are associated with gametophytic self-incompatibility (GSI). The amino acid sequences of many S-RNases have been derived from cDNA sequences, but the state of half-cystines has not been clarified. We report the locations of the two free cysteine residues and four disulfide bridges of tobacco S6-RNase and of the four disulfide bridge of Japanese pear S4-RNase. The protein was first S-pyridylethylated at a low pH to selectively modify the free cysteines without thiol-disulfide exchange. The S-pyridylethylated protein (PE-protein) was digested with Achromobacter protease I (API) at pH 6.5 then analyzed by liquid chromatography/electrospray-ionization mass spectrometry (LC/ESI-MS). This analysis showed that tobacco S6-RNase has two free cysteine residues, Cys77 and Cys95, and four disulfide bonds at Cys16-Cys21, Cys45-Cys94, Cys153-Cys182 and Cys165-Cys176. Similarly, four disulfide bonds were identified for pear S4-RNase, which bears no free cysteine, at Cys15-Cys22, Cys48-Cys92, Cys156-Cys195 and Cys172-Cys183. The eight cysteines forming these four disulfide bonds are conserved in all the known S-RNases, indicative that these cross-links are important in stabilizing the tertiary structures of the self-incompatibility-associated glycoproteins in the solanaceous and rosaceous families. The LC/ ESI-MS analysis also provided some structural informations regarding sugar chains attached to the S-RNases.
Ishimizu
T
T
Division of Protein Chemistry, Osaka University, Japan.
Norioka
S
S
Kanai
M
M
Clarke
A E
AE
Sakiyama
F
F
D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D005638:Fruit / Q000201:enzymology*; D006020:Glycopeptides / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D010940:Plant Proteins / Q000737:chemistry; D010947:Plants, Toxic; D012098:Reproduction; D012260:Ribonucleases / Q000737:chemistry*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000201:enzymology*
D004220:Disulfides; D006020:Glycopeptides; D006023:Glycoproteins; D010940:Plant Proteins; D003553:Cystine; D012260:Ribonucleases; D003545:Cysteine