engleski

Assessment of N-glycan Heterogeneity of Cactus Glycoproteins by One Dimensional Gel Electrophoresis and Matrix Assisted Laser/Desorption Time-of-Flight Mass Spectrometry

Artificial environmental conditions in the tissue culture, such as elevated relative humidity and rich nutrient medium, can influence and modify tissue growth and induce spontaneous changes from characteristic organisation pattern to unorganised callus. As succulent plants with Crassulacean Acid Metabolism (CAM), cacti are particularly susceptible to this altered growth environment. Glycosylated proteins of Mammillaria gracillis tissues cultivated in vitro, separated by the 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 RCA120). The obtained results revealed significant differences in glycoprotein pattern among the investigated cactus tissues (shoot, callus, hyperhydric regenerant and tumour). 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-TOFMS 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 tumour shared glycans of complex-type. The hybrid oligosaccharide structures were found only in tumour 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.

cacti; glycoproteins; MALDI-TOFMS; N-glycans; plant tissue culture

nije evidentirano