Tissues of the clawed frog Xenopus laevis contain two closely related forms of UDP-GlcNAc :alpha 3-D-mannoside beta-1,2-N-acetylglucosaminyttransferase I

UDP-GlcNAc:alpha3-D-mannoside beta -1,2-N-acetylglucosaminyl-transferase I (GnTI; EC 2.4.1.101) is a medial-Golgi enzyme that is essential for the pro cessing of oligomannose to hybrid and complex N-glycans. On the basis of hi ghly conserved sequences obtained from previously cloned mammalian GnTI gen es, cDNAs for two closely related GnTI isoenzymes were isolated from a Xeno pus laevis ovary cDNA library. As typical for glycosyltransferases, both pr oteins exhibit a type II transmembrane protein topology with a short N-term inal cytoplasmic tail (4 amino acids); a transmembrane domain of 22 residue s; a stem region with a length of 81 (isoenzyme A) and 77 (isoenzyme B) ami no acids, respectively; and a catalytic domain consisting of 341 residues. The two proteins differ not only in length but also at 13 (stem) and IS (ca talytic domain) positions, respectively. The overall identity of the cataly tic domains of the X. laevis GnTI isoenzymes with their mammalian and plant orthologues ranges from 30% (Nicotiana tabacum) to 67% (humans). Isoenzyme s A and B are encoded by two separate genes that were both found to be expr essed in all tissues examined, albeit in varying amounts and ratios. On exp ression of the cDNAs in the baculovirus/insect cell system, both isoenzymes were found to exhibit enzymatic activity. Isoenzyme B is less efficiently folded in vivo and thus appears of lower physiological relevance than isoen zyme A. However, substitution of threonine at position 223 with alanine was sufficient to confer isoenzyme B with properties similar to those observed for isoenzyme A.