Glycosylation is an important post translational modification in plants. First analysis of N-linked glycosylated proteins of Dioscorea alata using Concanavalin A lectin affinity chromatography enrichment coupled with label free quantification is presented. In total, 114 enriched glycoproteins were detected. Signal P and sub-cellular localization showed 42.2% of proteins to be secretory. These included peroxidases, endochitinases, calreticulin, calnexin, thaumatins and lipid transfer proteins. Gene Ontology and MapMan analysis predicted the enriched glycoproteins to be involved in processes essential for tuber maturation namely: signal transduction, lignification, protein trafficking, endoplasmic reticulum quality control and cell wall remodeling. This was supported by biochemical validation of the essential glycoproteins. Interestingly, out of the two dioscorin isoforms, Dio B was the only N-glycosylated form. In silico analysis showed O-glycosylation sites in the other form, Dio A suggesting its similarity with sporamin, the storage protein of sweet potato. Absence of signal peptide in Dio B and the presence of non-canonical motif hints towards its atypical glycosylation. The analysis revealed that N-glycosylation of Dio B isoform maintains the activities associated with Dioscorin at maturity and provides an overview of protein N-glycosylation, enriching the glycoproteome database of plants especially tubers.

The study of interactions between proteins and surfactants is of relevance in a diverse range of applications including food, enzymatic detergent formulation, and drug delivery. In spite of sodium dodecyl sulfate (SDS)-induced unfolding has been studied in detail at the protein level, deciphering the conformation-activity relationship of a recombinant γ-glutamyltranspeptidase (BlrGGT) from Bacillus licheniformis remains important to understand how the transpeptidase activity is related to its conformation. In this study, we examined the enzyme catalysis and conformational transition of BlrGGT in the presence of SDS. Enzymatic assays showed that the transpeptidase activity of BlrGGT was greatly affected by SDS in a concentration-dependent manner with approximately 90% inactivation at 6 mM. Native polyacrylamide gel electrophoresis of SDS-treated samples clearly revealed that the heterodimeric enzyme was apparently dissociated into two different subunits at concentrations above 2 mM. The study of enzyme kinetics showed that SDS can act as a mixed-type inhibitor to reduce the catalytic efficiency of BlrGGT. Moreover, the t1/2 value of the enzyme at 55 °C was greatly reduced from 495.1 min to 7.4 min in the presence of 1 mM SDS. The I3/I1 ratio of pyrene excimer fluorescence emission changed around 3.7 mM SDS in the absence of BlrGGT and the inflection point of enzyme samples was reduced to less than 2.7 mM. The Far-UV CD spectrum of the native enzyme had two negative peaks at 208 and 222 nm, respectively; however, both negative peaks increased in magnitude with increasing SDS concentration and reached maximal values at above 4.0 mM. The intrinsic fluorescence spectra of tryptophan further demonstrated that the SDS-induced enzyme conformational transition occurred at approximately 5.1 mM. Tween 20 significantly suppressed the interaction of BlrGGT with SDS by forming mixed micelles at a molar ratio of 1.0. Taken together, this study definitely promotes our better understanding of the relationship between the conformation and catalysis of BlrGGT.