Reliable sequence determination of ribosome- inactivating proteins by combining electrospray mass spectrometry and Edman degradation.
The primary structure of saporin-S9 and MAP-S, two type-1 ribosome-inactivating proteins isolated from the seeds of Saponaria officinalis L. and Mirabilis jalapa, respectively, was determined using a combined approach based on Edman degradation and electrospray ionization mass spectrometry (ESMS). Saporin-S9 has 253 amino acids with a calculated molecular mass of 28,492.99, which is in good agreement with that determined by ESMS (28 495 +/- 2 Da). Unlike other saporins with known primary structure, saporin-S9 contains four histidinyl residues (positions 111, 121, 216 and 248). By comparing the amino acid sequence of saporin-S9 with that of saporin-S6, we found 22 amino acid substitutions (8.7%), 13 of which are conservative and nine non-conservative. The residues known to be involved in the definition of the active site and with RNA base recognition are conserved. The four histidinyl residues and especially Lys for Gln203 contribute to the higher calculated pI value (10.17) of saporin-S9 compared with saporin-S6 (9.98). MAP-S contains 250 amino acid residues with a calculated molecular mass of 27,789.49, in good agreement with that determined by ESMS (27,789 +/- 2). Cys36 and Cys220 form a disulphide bridge and only four amino acid residues are different from the amino acid sequence of MAP, isolated from the roots of the same plant, i.e. Leu34 (Glu), Ile161 (Leu), Asp185 (Glu) and Asp191 (Glu) (in parentheses, the residues present in MAP). The reported approach can provide rapid and reliable sequence screening in the analysis of homologous proteins, including the presence of disulphide bridges.
Di Maro
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A
Dipartimento di Chimica Organica e Biologica, Università di Napoli Federico II, Via Mezzocannone 16, I-80134 Naples, Italy.
Ferranti
P
P
Mastronicola
M
M
Polito
L
L
Bolognesi
A
A
Stirpe
F
F
Malorni
A
A
Parente
A
A
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D003488:Cyanogen Bromide; D010450:Endopeptidases; D000922:Immunotoxins; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D010446:Peptide Fragments / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins; D020539:Sequence Analysis, Protein; D017385:Sequence Homology; D021241:Spectrometry, Mass, Electrospray Ionization; D014357:Trypsin
D000922:Immunotoxins; D010446:Peptide Fragments; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D009699:N-Glycosyl Hydrolases; C059351:MAP protein, Mirabilis jalapa; D000076985:Saporins; D010450:Endopeptidases; D014357:Trypsin; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N; D003488:Cyanogen Bromide