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

Abstract

UDP-GlcNAc:alpha3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnTI; EC 2.4.1.101) is a medial-Golgi enzyme that is essential for the processing of oligomannose to hybrid and complex N-glycans. On the basis of highly conserved sequences obtained from previously cloned mammalian GnTI genes, cDNAs for two closely related GnTI isoenzymes were isolated from a Xenopus laevis ovary cDNA library. As typical for glycosyltransferases, both proteins exhibit a type II transmembrane protein topology with a short N-terminal cytoplasmic tail (4 amino acids); a transmembrane domain of 22 residues; a stem region with a length of 81 (isoenzyme A) and 77 (isoenzyme B) amino acids, respectively; and a catalytic domain consisting of 341 residues. The two proteins differ not only in length but also at 13 (stem) and 18 (catalytic domain) positions, respectively. The overall identity of the catalytic domains of the X. laevis GnTI isoenzymes with their mammalian and plant orthologues ranges from 30% (Nicotiana tabacum) to 67% (humans). Isoenzymes A and B are encoded by two separate genes that were both found to be expressed in all tissues examined, albeit in varying amounts and ratios. On expression 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 isoenzyme 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.

Molecular basis of N-acetylglucosaminyltransferase I deficiency in Arabidopsis thaliana plants lacking complex N-glycans

“…All GnTI constructs were expressed in Spodoptera frugiperda Sf21 cells as described previously [8]. Cells and conditioned media were harvested and subjected to enzymic analysis and immunoblotting.…”

“…GnTI activity assays were performed as described in [8] using as acceptor substrates either 0.5 mM Man 3 -octyl [Manα1-6(Manα1-3)Manβ-1-O-octyl; Toronto Research Chemicals] or 0.25 mM Man 5 GlcNAc 2 -glycopeptide (prepared from Aspergillus oryzae α-amylase; [9]), and as donor substrate 0.1 mM UDP-[ 14 C]GlcNAc (3000-4000 c.p.m./nmol; Amersham Biosciences). After incubation at 37 • C for 1 h, reactions were stopped by the addition of 0.5 ml of 20 mM sodium tetraborate containing 2 mM EDTA.…”

Arabidopsis thaliana β1,2-xylosyltransferase: an unusual glycosyltransferase with the potential to act at multiple stages of the plant N-glycosylation pathway

“…After maintaining for 5 days at 27 • C, the supernatant containing recombinant baculovirus was used for the infection of Sf21 cells (we prefer this cell line to Sf9 cells for the high-yield production of recombinant proteins in the baculovirus system). Cells and conditioned media were harvested and subjected to enzyme analysis and immunoblotting [10].…”

“…GnGn-GP [where GnGn stands for GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)Manβ1-4Glc-NAcβ1-4GlcNAc and GP for glycopeptide] was prepared as described previously [9]. Purified recombinant rabbit GnT I (β1,2-N-acetylglucosaminyltransferase I) and human GnT II were obtained by the method described in [8,10]. Recombinant A. thaliana XylT and FucT (core α1,3-fucosyltransferase) produced in Pichia pastoris were purified as described previously [11].…”

Biological and biochemical properties of two Xenopus laevis N-acetylgalactosaminyltransferases with contrasting roles in embryogenesis

“…In amphibians, the O-glycan chains of a range of amphibian egg mucins have been shown in a number of studies to be rather complex and to display a range of unusual and extended oligosaccharide chains ( Guerardel et al, 2000 ). In terms of glycosyltransferases, amongst the few from an amphibian to be previously characterised are two N- acetylglucosaminyltransferases (GlcNAc-TI and -TII) and one sialyltransferase (xSTX) involved in N-glycan (and not O-glycan) biosynthesis ( Kudo et al, 1998; Mucha et al, 2001, 2002 ); only recently was a paper including enzymatic data on an amphibian N- acetylgalactosaminyltransferase published ( Boskovski et al, 2013 ). On the other hand, enzymes which catalyse the first step in mucin biosynthesis have been studied from a number of mammalian and invertebrate sources; obviously, the first studies were on activities in crude tissue extracts, but interestingly the basic properties (cation dependency and pH optimum) are similar to those defined in the present study ( McGuire and Roseman, 1967 ).…”