GlcNAc-TV is a key regulator of polylactosamino-N-glycan chain formation and is causally involved in the metastasis of tumours in mice and humans. We have characterised the C. elegans orthologue (
gly-2) originally identified via
yk126h8. Northern analysis indicates a single rare ~2.2kb species of mRNA. 5' RACE indicates that the transcript is exclusively trans -spliced to SL1 at the acceptor site immediately upstream of the probable initiator methionine in the genomic sequence (C55B7.2). The deduced polypeptide is 669 residues with 37% identity and 60% overall similarity to mammalian homologues. Golgi glycosyltransferases are type II transmembrane protein, and as expected, the relatedness is notably stronger in the C-terminal portion of the alignment (catalytic domain) and hydropathy analysis indicated a single potential transmembrane domain at the N-terminal. Constructs driving the secretion of soluble forms of GLY-2 were transfected into Lec4 cells, a GlcNAc-TV deficient mutant CHO line. We found that the CHO-expressed gene-product is enzymatically active and the catalytic domain starts at I 138 , after which there is high similarity with mammalian homologues. Interestingly, the genomic gene is split by an in-frame intron boundary between K 137 and I 138 . We observed that
gly-2 can rescue the cell surface phenotype of Lec4 cells which lack endogenous GlcNAc-TV. Lec4A (CHO) cells have active enzyme which is mislocalised due to a missense mutation. We introduced the equivalent mutation, GLY2-L 116 R, and abolished the phenotype rescue of Lec4 cells. We infer that the nematode enzyme can be correctly localised to the medial Golgi and acceptor specificity is well conserved with mammalian enzyme. We have isolated a C. elegans line carrying an allele where a Tc1 transposon is inserted into an intron in the 3' region of
gly-2 and a sub-library is being screened for a deletion allele. GlcNAc-TV null mice have suppressed tumour induction and metastasis. We aim to exploit C. elegans as a genetic tool to analyse the pathways by which these phenomena are mediated.