Acceptor requirements for GDP-fucose:xyloglucan 1,2-α- l-fucosyltransferase activity solubilized from pea epicotyl membranes

Abstract

GDP-fucose:xyloglucan (XG) fucosyltransferase from growing Pisum epicotyl tissue was solubilized in detergent and used to examine the capacity of intact XG from Tamarindus seeds, and its partial hydrolysis products, to act as fucose acceptors with GDP-[ 14C]fucose as donor. Native seed XG (M r > 10 6 Da) was partially depolymerized by incubation with Trichoderma cellulase for various periods of time. Cellulase was inactivated and reaction mixtures were incubated with GDP-[ 14C]fucose plus solubilized pea fucosyltransferase and then fractionated on columns of Sepharose CL-6B or Bio-Gel P 4. Specific activities (Bq/μg carbohydrate) of fragments with M r ranging from 10 6 to 10 4 Da were constant throughout the size ranges, indicating that all stretches of the XG chains were available for fucosylation. More complete cellulase hydrolysis yielded subunit oligosaccharides that chromatographed in a cluster of hepta-, octa-, and nonasaccharides, none of which acted as fucosyl acceptors when incubated with pea fucosyltrans-ferase. However, a substantial amount (up to half of hydrolysate) of larger transient oligosaccharides was also formed with a size equivalent to three of the oligosaccharide subunits. Octasaccharide subunits in this trimer were readily fucosylated. This fucosyltransfer was inhibited by uncombined (free) subunit oligosaccharides, which implies that the latter could bind to the transferase and displace at least part of the trimer, even though they could not themselves be fucosylated. Reduction of the trimer oligosaccharide with NaB 3H 4, followed by further hydrolysis with cellulase, resulted in tritiated nonasaccharide and unlabeled octasaccharide in a concentration ratio of 1:2. The tamarind XG trimer which accepts fucose is therefore composed mainly of the subunit sequence: octa-octa-nonasaccharide (reducing). One of the terminal oligosaccharide subunits in this trimer, probably the nonasaccharide, appears to be required as a recognition (binding) site in fucosyltransferase in order for adjacent octasaccharide(s) to be fucosylated by the active (catalytic) enzyme site.