Activity and Action of Cell-Wall Transglycanases

Transglycanases (endotransglycosylases) are enzymes that "cut and paste" polysaccharide chains. Several transglycanase activities have been discovered which can cut (i.e., use as donor substrate) each of the major hemicelluloses [xyloglucan, mannans, xylans, and mixed-linkage β-glucan (MLG)], and, as a recent addition, cellulose. These enzymes may play interesting roles in adjusting the wall's physical properties, influencing cell expansion, stem strengthening, and fruit softening.Activities discussed include the homotransglycanases XET (xyloglucan endotransglucosylase, i.e., xyloglucan-xyloglucan endotransglycosylase), trans-β-mannanase (mannan -mannan endotransglycosylase), and trans-β-xylanase (xylan -xylan endotransglucosylase), plus the heterotransglycanases MXE (MLG -xyloglucan endotransglucosylase) and CXE (cellulose -xyloglucan endotransglucosylase).Transglycanases acting on polysaccharide donor substrates can utilize small, labeled oligosaccharides as acceptor substrates, generating easily recognizable polymeric labeled products. We present methods for extracting transglycanases from plant tissues and assaying them in vitro, either quantitatively in solution assays or by high-throughput dot-blot screens. Both radioactively and fluorescently labeled substrates are mentioned. A general procedure (glass-fiber blotting) is illustrated by which proposed novel transglycanase activities can be tested for.In addition, we describe strategies for detecting transglycanase action in vivo. These methods enable the quantification of, separately, XET and MXE action in Equisetum stems. Related methods enable the tissue distribution of transglycanase action to be visualized cytologically.