N-glycosylation and dimerization regulate the PtrMAN6 enzyme activity that may modulate generation of oligosaccharide signals

PtrMAN6 is a plant mannan endo-hydrolase involved in modulating cell expansion and cell wall thickening in Populus developing xylem. N-glycosylation and dimerization affect the PtrMAN6 enzymatic activity, which is crucial for production of the endogenous galactoglucomannan oligosaccharide signal molecule in plants. There are 5 potential N-glycosylation sites and 6 cysteines in PtrMAN6 sequence. Each of the N-glycosylation or cysteine sites was site-direct mutagenized individually as well as in combination to analyze their effects on the PtrMAN6 N-glycosylation or dimerization status and the enzyme activity. Our results demonstrated that all 5 potential N-glycosylation sites are involved in the N-glycosylation, which is essential for PtrMAN6 enzyme activity. Meanwhile, we found only 3 carboxyl-terminal cysteines are involved in formation of disulfide-linked dimer to regulate PtrMAN6 activity. The 3 carboxyl-terminal cysteines were conserved in the wall-bounded mannan endo-hydrolases, and this structure may play a role in regulating the PtrMAN6 activity through interaction with redox signals such as reactive oxygen species (ROS) and hydrogen sulfide (H(2)S) for GGMOs signal generation.