Jan Mucha

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

XylT (beta1,2-xylosyltransferase) is a unique Golgi-bound glycosyltransferase that is involved in the biosynthesis of glycoprotein-bound N-glycans in plants. To delineate the catalytic domain of XylT, a series of N-terminal deletion mutants was heterologously expressed in insect cells. Whereas the first 54 residues could be deleted without affecting the catalytic activity of the enzyme, removal of an additional five amino acids led to the formation of an inactive protein. Characterization of the N-glycosylation status of recombinant XylT revealed that all three potential N-glycosylation sites of the protein are occupied by N-linked oligosaccharides. However, an unglycosylated version of the enzyme displayed substantial catalytic activity, demonstrating that N-glycosylation is not essential for proper folding of XylT. In contrast with most other glycosyltransferases, XylT is enzymatically active in the absence of added metal ions. This feature is not due to any metal ion directly associated with the enzyme. The precise acceptor substrate specificity of XylT was assessed with several physiologically relevant compounds and the xylosylated reaction products were subsequently tested as substrates of other Golgi-resident glycosyltransferases. These experiments revealed that the substrate specificity of XylT permits the enzyme to act at multiple stages of the plant N-glycosylation pathway.

Removal of 106 amino acids from the N-terminus of UDP-GlcNAc :alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I does not inactivate the enzyme

UDP-GlcNAc : α-3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT I, EC 2.4.1.101) plays an essential role in the conversion of oligomannose to complex and hybrid N-glycans. Rabbit GnTI is 447 residues long and has a short four-residue N-terminal cytoplasmic tail, a 25-residue putative signal–anchor hydrophobic domain, a stem region of undetermined length and a large C-terminal catalytic domain, a structure typical of all glycosyltransferases cloned to date. Comparison of the amino acid sequences for human, rabbit, mouse, rat, chicken, frog and Caenorhabditis elegans GnT I was used to obtain a secondary structure prediction for the enzyme which suggested that the location of the junction between the stem and the catalytic domain was at about residue 106. To test this hypothesis, several hybrid constructs containing GnT I with N- and C-terminal truncations fused to a mellitin signal sequence were inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcMNPV), Sf 9 insect cells were infected with the recombinant baculovirus and supernatants were assayed for GnT I activity. Removal of 29, 84 and 106 N-terminal amino acids had no effect on GnT I activity; however, removal of a further 14 amino acids resulted in complete loss of activity. Western blot analysis showed strong protein bands for all truncated enzymes except for the construct lacking 120 N-terminal residues indicating proteolysis or defective expression or secretion of this protein. The data indicate that the stem is at least 77 residues long.

Functional expression of a cDNA encoding pea (Pisum sativum L.) raffinose synthase, partial purification of the enzyme from maturing seeds, and steady-state kinetic analysis of raffinose synthesis

Abstract. Raffinose (O-α-D-galactopyranosyl-(1→6)-O-α-D-glucopyranosyl-(1↔2)-O-β-D-fructofuranoside) is a widespread oligosaccharide in plant seeds and other tissues. Raffinose synthase (EC 2.4.1.82) is the key enzyme that channels sucrose into the raffinose oligosaccharide pathway. We here report on the isolation of a cDNA encoding for raffinose synthase from maturing pea (Pisum sativum L.) seeds. The coding region of the cDNA was expressed in Spodoptera frugiperda Sf21 insect cells. The recombinant enzyme, a protein of glycoside hydrolase family 36, displayed similar kinetic properties to raffinose synthase partially purified from maturing seeds by anion-exchange and size-exclusion chromatography. Apart from the natural galactosyl donor galactinol (O-α-D-galactopyranosyl-(1→1)-L-myo-inositol), p-nitrophenyl α-D-galactopyranoside, an artificial substrate, was utilized as a galactosyl donor. An equilibrium constant of 4.1 was determined for the galactosyl transfer reaction from galactinol to sucrose. Steady-state kinetic analysis suggested that raffinose synthase is a transglycosidase operating by a ping-pong reaction mechanism and may also act as a glycoside hydrolase. The enzyme was strongly inhibited by 1-deoxygalactonojirimycin, a potent inhibitor for α-galactosidases (EC 3.2.1.22). The physiological implications of these observations are discussed.

Two closely related forms of UDP-GlcNAc: α6-D-mannoside β1,2-N-acetylglucosaminyltransferase II occur in the clawed frog Xenopus laevis

UDP-GlcNAc:α6-D-mannoside β1,2-N-acetylglucosaminyltransferase II (GnT II; EC 2.4.1.143) is a medial-Golgi resident enzyme that catalyses an essential step in the biosynthetic pathway leading from high mannose to complex N-linked oligosaccharides. Screening a cDNA library from Xenopus laevis ovary with a human GnT II DNA probe resulted in the isolation of two cDNA clones encoding two closely related GnT II isoenzymes, GnT II-A and GnT II-B. Analysis of the corresponding genomic DNAs revealed that the open reading frame of both X. laevis GnT II genes resides within a single exon. The GnT II-A gene was found to be transcriptionally active in all X. laevis tissues tested. In contrast, expression of the GnT II-B gene was detected only in a limited number of tissues. Both GnT II-A and GnT II-B exhibit a type II transmembrane protein topology with a putative N-terminal cytoplasmic tail of 9 amino acids followed by a transmembrane domain of 18 residues, and a C-terminal luminal domain of 405 residues. The two proteins differ at 28 amino acid positions within their luminal regions. Heterologous expression of soluble forms of the enzymes in insect cells showed that GnT II-A and GnT II-B are both catalytically active and exhibit similar specific activities. Both recombinant proteins are modified with N-linked oligosaccharides. N-terminal deletion studies demonstrated that the first 49 amino acid residues are not essential for proper folding and enzymatic activity of X. laevis GnT II. Published in 2003.

CCDC 1566599: Experimental Crystal Structure Determination

Related Article: Maros Bella, Shi Yan, Sergej Sestak, Stanislav Kozmon, Chun-Hung Lin, Jan Mucha and Miroslav Koos|2017|CSD Communication|||