Cell Wall- and Glycolipid-related Functions of Arabidopsis Glycosyltransferase Family Eight Proteins

Glycosyltransferases catalyze the transfer of sugars from activated phosphate sugar substrates to various acceptor molecules ranging from primary metabolites to glycolipids to plant cell walls. The model plant Arabidopsis encodes about 500 glycosyltransferase proteins. Forty-one of these are members of Glycosyltransferase Family 8. These enzymes are thought to function in synthesis of diverse compounds including the disaccharide galactinol, and the cell wall polymer pectin, and the energy storage polysaccharide starch. This thesis will describe evidence that allowed us to assign additional functions of members of this family: five of these enzymes synthesize the cell wall polymer glucuronoxylan, while one synthesizes glycosyl inositol phosphoryl-ceramide sphingolipids. The eight Plant Glycogenin-like Starch Initiation Proteins (PGSIPs) were named based on their homology to glycogenin, a protein in fungi and animals that initiates glycogen synthesis by glucosylating itself. The PGSIP proteins were proposed to initiate starch synthesis in a similar manner. However, two of these proteins, PGSIP1 and PGSIP3, are highly coexpressed with glucuronoxylan biosynthetic proteins. Mutations in PGSIP1 cause a decrease in cell wall glucuronoxylan content and microsomal xylan:glucuronosyltransferase activity, leading to the re-annotation of five of the PGSIP proteins as Glucuronic Acid Substitution of Xylan (GUX). Heterologously expressed and purified GUX1, GUX2, and GUX4 show xylan glucuronosyltransferase activity in vitro. These proteins transferred glucuronic acid to a xylooligomer acceptor with different patterns, showing that these related proteins have subtle differences in specificity.Another protein, PGSIP6, is related to both the GUX proteins and the galactinol synthase (GolS) proteins, which transfer galactose onto the acceptor inositol. The phylogenetic placement of PGSIP6 between the GUX and GolS proteins led us to suspect that this enzyme catalyzes the transfer of glucuronic acid to the inositol moiety of glycosyl inositol phosphorylceramides, a class of glycosphingolipids that are abundant in plant plasma membranes. Expression of PGSIP6 in yeast led to production of glucuronic acid linked to inositol phosphorylceramide. Overexpressing PGSIP6 in Nicotiana benthamiana plants led to an increase in inositol phosphorylceramide glucuronosyltransferase activity, while silencing expression of PGSIP6 led to a decrease in activity. PGSIP6 has now been renamed Inositol Phosphorylceramide Glucuronosyltransferase 1 (IPGT1).