2001 Volume 13 Issue 73 Pages 447-462
UDP-N-acetylglucosamine: α-3-D-mannoside β-1, 2-N-acetylglucosaminyltransferase I (GnT I) is a key enzyme in the synthesis of Asn-linked complex and hybrid glycans. We have cloned cDNAs for three predicted C. elegans genes homologous to mammalian GnT I (designated gly-12, gly-13 and gly-14). All three cDNAs encode proteins with the domain structure typical of previously cloned Golgi-type glycosyltransferases. Expression in transgenic worms showed that they all encode active GnT I. GLY-13 is unique in that it utilizes only the physiological substrate Man5GlcNAc2-R as sugar acceptor. We have isolated mutants lacking these three genes by the method of UV irradiation in the presence of trimethylpsoralen (TMP). The gly-12 and gly-14 mutants as well as the gly-14; gly-12 double mutant displayed wild type phenotypes indicating that neither gly-12 nor gly-14 is necessary for worm development under standard laboratory conditions. This finding and other data indicate that GLY-13 is the major functional GnT I in C. elegans. The mutation lacking the gly-13 gene is partially lethal and the few survivors display severe morphological and behaviorial defects. We have shown that the observed phenotype co-segregates with the gly-13 deletion in genetic mapping experiments although a second mutation near the gly-13 gene cannot as yet be ruled out. Studies on mice with a null mutation in the GnT I gene have indicated that complex and hybrid N-glycans play critical roles in mammalian morphogenesis. Our data indicate that this may also be true for the nematode Caenorhabditis elegans. There are limitations to the use of null mutations of GnT I and other glycosyltransferases in determining the function of glycans in metazoan development. An alternative approach to the problem (functional post-translational proteomics) is required.
