engleski

Sialylated Cellular Glycoproteins in Mammillaria gracillis Pfeiff. Tissues Grown in vitro

The structure elucidation of protein-linked N-glycans in plants has raised interest in the past years due to remarkable physiological roles attributed to these modifications: control of protein folding, biological activity and stability of proteins. However, little information about the glycoprotein patterns related to plant cell differentiation, dedifferentiation and transformation is available. A spontaneous switch from an organized to unorganized way of growth in the Mammillaria gracillis Pfeiff. (Cactaceae) tissue culture makes this system suitable to study plant morphogenesis. We probed glycoproteins from four different Mammillaria tissues (shoot, callus, hyperhydric regenerants and tumour) with a biotin- and digoxigenin-labeled lectins from Sambucus nigra (SNA I) and Maackia amurensis (MAA), revealing terminal sialic acid-α 2, 6-galactose and sialic acid-α 2, 3-galactose structures, respectively. After getting positive cross-reaction with biotin-labeled SNA and MAA, the digoxigenin-labeled lectins were used in order to confirm previous results due to existence of endogenous biotin in various tissues that can lead to false-positive staining. Obtained data clearly indicated the presence of sialylated glycoproteins in cactus tissues, although there are only a few data about the presence of sialic acids and sialoconjugates in plant cells. The 42 kDa glycoprotein, which was present in all cactus tissues and reacted with both applied lectins, was excised from the gel and deglycosylated by PNGase F. Oligosaccharide mixtures were analyzed by HPAEC-PAD as well as by ESI-Q-TOF MS. The presence of one type of sialic acid, NeuAc, was confirmed by HPAEC-PAD analysis of oligosaccharides from hyperhydric regenerant and tumour while the other type, NeuGc, could not be detected. N-glycans detached from the selected glycoprotein, analyzed by ESI-Q-TOF MS, revealed similarities of assigned structures in callus and tumour while the highest number of different oligosaccharide structures was found in the hyperhydric regenerant. Molecular mapping by electrospray mass spectrometry revealed three oligosaccharide structures which are probably sialylated (Hex4HexNAc4NeuAc2, Hex6HexNAc4NeuAc2, and Hex6HexNAc5NeuAc2). From the data obtained so far, it could be deduced, that the complexity of the N-glycan expression is increasing, if the characteristic tissue organization pattern is lost. Hyperhydric shoot regenerated from callus, which morphologically resemble cactus plants, but are more swollen with disarranged spines, is shown to contain a high number and diversity of detected oligosaccharide structures in comparison to the normal shoots. From callus and TW tumour, however, as two types of unorganised tissues, similar structures of N-linked glycans could be postulated, although the glycan mixture from tumour tissue, which has no regeneration potential, revealed structures not present in the callus sample. Novel insights in correlation of different morphological levels in plant tissues with N-glycan composition of the analyzed cellular glycoprotein were delivered in this study, although here presented glycan pattern data require further detailed analysis by sequencing.

plant development; glycosylation; glycoconjugates; sialylation; tissue culture

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