Modificações pós-traducionais revelam que Mo-CBP3, uma albumina 2s ligante de quitina de Moringa oleifera, é uma mistura complexa de isoformas

Abstract

The study of 2S albumin expressed in Moringa oleifera seeds has been a very complex process, especially when using conventional proteomic techniques due to the presence of many isoforms. The great diversity of protein isoforms may be dueto the multigenic origin, in addition to the intense post-translational processing. In this context, the aim of this work was to investigate the occurrence of new Mo-CBP3 isoforms and characterize these proteins for the presence of post-translational modifications (PTMs). The work employed an experimental approach which included (1) cloning and sequencing of genomic DNA fragments encoding Mo-CBP3; (2) molecular mass analysis through LC-ESI-MS; and (3) computational study, with the objective to correlate theoretical and experimental masses. A total of 32 different Mo-CBP3 clones were obtained and completely sequenced. After multiple alignments of the deduced amino acid sequences it was possible to categorize them into eight distinct isoform groups. Four of these isoforms have been previously described (Mo-CBP3-1, Mo-CBP3-2, Mo-CBP3-3 and Mo-CBP3-4), whereas Mo-CBP3-2A (163 amino acid residues), Mo-CBP3-2B (162 residues), Mo-CBP3-3A (160 residues) and Mo-CBP3-3B (160 aa) isoforms were first reported in this work. Analysis of the amino acid sequences, deduced from the genomic DNA, suggested that Mo-CBP3 isoforms are synthesized as preproproteins containing an N-terminal signal peptide, an N-terminal propeptide, a small chain of about 4 kDa, a peptide linkage between the two chains, a large chain with approximately 8 kDa, and a C-terminal extension. ESI-MS experiments revealed 147 distinct values of molecular mass, of which 89 corresponded to variants of the small chain and 58 to variants of the large chain. Molecular mass values were calculated from the amino acid sequences, assuming different degrees of proteolytic processing at the N- and C-terminal ends of each chain. In addition, possible changes produced by different PTMs, such as proline hydroxylation, serine or threonine phosphorylation, methionine oxidation, and the cyclization of the N-terminal glutamine into pyroglutamate (pGlu) were also taken into account. Using this strategy, the majority of the experimental masses of the small chain (72 of a total of 89) and almost all experimental masses corresponding to the large chain (57 out of 58) could be attributed to specific amino acid sequences of the different Mo-CBP3 isoforms. These results suggest that: i) Mo-CBP3 isoforms are primarily derived from closely related members of a multigenic family; ii) once processed, the precursors encoded from the different mRNAs give rise to two chains, one smaller (4 kDa) and one larger (8 kDa), joined by disulfide bonds; the N- and C-terminal ends of both chains can undergo proteolytic processing, resulting in the removal of one or a few residues from each sequence, yielding chains with slightly different masses; iii) post-translational modifications in certain residues of one of the polypeptide chains or both are a third source of variation in the molecular mass, substantially increasing the number of isoforms. In conclusion, an extremely complex mixture of Mo-CBP3 isoforms, derived from a few genes, is produced by different combinations of distinct PTMs. These results constitute important advances in understanding the inherent post-translational mechanisms that operate during the biosynthesis of 2S albumins in seeds of M. oleifera.