Assessment of N-glycan heterogeneity of cactus glycoproteins by one-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
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.
Balen
Biljana
B
Department of Molecular Biology, Faculty of Science, University of Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia. bbalen@zg.biol.pmf.hr
Krsnik-Rasol
Marijana
M
Zamfir
Alina D
AD
Zadro
Ivana
I
Vakhrushev
Sergey Y
SY
Peter-Katalinic
Jasna
J
D029421:Cactaceae / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D046509:Tissue Culture Techniques
D006023:Glycoproteins; D011134:Polysaccharides