New insights on β-glycan synthases using in vitro GT-array (i-GT-ray) platform

Cellulose and hemicellulose are the major structural β-glycan polysaccharides in cell walls of land plants. They are characterized by a backbone of β-(1,3)- and/or β-(1,4)-linked sugars such as glucose, mannose, or xylose. The backbones of these polymers are produced by processive glycosyltransferases (GTs) called synthases having multiple transmembrane domains anchoring them to the membrane. Thus, they are among the most difficult membrane proteins to test in vitro and to purify. Recently, we developed an in vitro GT-array (i-GTray) platform and showed that non-processive type II membrane GTs could be produced via cell-free system in a soluble and active form and tested in this platform. To determine whether i-GT-ray platform is adequate for the production and testing of β-glycan synthases, we tested five synthases involved in cellulose, xyloglucan, (gluco)mannan, and β-(1,3)(1,4)-mixed-linkage glucan synthesis. Our results revealed unsuspected features of these enzymes. For example, all these synthases could be produced in a soluble and active form and are active in the absence of detergent or membrane lipids, and none of them required a primer for initiation of synthesis. All synthases produced ethanol-insoluble products that were susceptible to the appropriate hydrolases (i.e., cellulase, lichenase, mannanase). Using this platform, we showed that AtCslC4 and AtXXT1 interact directly to form an active xyloglucan synthase that produced xylosylated cello-oligosaccharides (up to three xylosyl residues) when supplied with UDP-Glc and UDP-Xyl. i-GTray platform represents a simple and powerful functional genomics tool for discovery of new insights of synthase activities and can be adapted to other enzymes.