Assessment of N-glycan heterogeneity of cactus glycoproteins by one-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Abstract

Artificial environmental conditions in tissue culture, such as elevated relative humidity and rich nutrient medium, can influence and modify tissue growth and induce spontaneous changes from characteristic organization pattern to unorganized callus. As succulent plants with crassulacean acid metabolism, cacti are particularly susceptible to this altered growth environment. Glycosylated proteins of Mammillaria gracillis tissues cultivated in vitro, separated by SDS-PAGE, were detected with Con A after the transfer of proteins onto the nitrocellulose membrane. The glycan components were further characterized by affinity blotting with different lectins (GNA, DSA, PNA, and RCA(120)). The results revealed significant differences in glycoprotein pattern among the investigated cactus tissues (shoot, callus, hyperhydric regenerant, and tumor). To test whether the N-glycosylation of the same protein can vary in different developmental stages of cactus tissue, the N-glycans were analyzed by MALDI-TOF MS after in-gel deglycosylation of the excised 38-kDa protein band. Paucimannosidic-type N-glycans were detected in oligosaccharide mixtures from shoot and callus, while the hyperhydric regenerant and tumor shared glycans of complex type. The hybrid oligosaccharide structures were found only in tumor tissue. These results indicate that the adaptation of plant cells to artificial environment in tissue culture is reflected in N-glycosylation, and structures of N-linked glycans vary with different developmental stages of Mammillaria gracillis tissues.

FIGURE 1

Soluble cellular proteins of Mammillaria tissues separated by SDS-PAGE in 10% gel and stained with Coomassie blue R-250. Arrows indicate the position of the excised 38-kDa glycoprotein band. Lane 1, protein molecular-weight marker; lane 2, shoot; lane 3, callus; lane 4, hyperhydric regenerant; and lane 5, tumor.

FIGURE 2

Con A-glycoprotein pattern after SDS-PAGE and transfer onto a nitrocellulose membrane. Lane 1, carboxypeptidase Y; lane 2, transferrin; lane 3, fetuin; lane 4, asialofetuin; lane 5, protein molecular-weight marker; lane 6, shoot; lane 7, callus; lane 8, hyperhydric regenerant; and lane 9, tumor.

FIGURE 3

Glycoprotein pattern after SDS-PAGE and transfer onto a nitrocellulose membrane according to lectin binding (a) GNA, (b) PNA, (c) DSA, and (d) RCA₁₂₀ I. Lane 1, carboxypeptidase Y; lane 2, transferrin; lane 3, fetuin; lane 4, asialofetuin; lane 5, protein molecular-weight marker; lane 6, shoot; lane 7, callus; lane 8, hyperhydric regenerant; and lane 9, tumor.

FIGURE 4

Positive-ion MALDI-TOF mass spectrum of the paucimannosidic type N-glycans released with PNGase A from the 38-kDa shoot glycoprotein band: assigned structures in (a) the mass range of m/z 850–970 and (b) m/z 1040–1090. The labels in the spectra correspond to the N-glycoforms in Table 1. All ions labeled 1p–5p were also detected in the mass spectra of callus tissue.

FIGURE 5

Positive-ion MALDI-TOF mass spectrum of the N-glycans obtained by in-gel deglycosylation with PNGase A from the 38-kDa glycoprotein band of tumor: assigned structures in the mass range of (a) m/z 1100–1500 and (b) m/z 1580–1630. The labels in the spectra correspond to the N-glycoforms in Table 2 and Table 3. All ions labeled 1c–4c were also detected in mass spectra of hyperhydric regenerant.