Class Of Glycosyltransferases Research Articles

Use of Click Chemistry to Define the Substrate Specificity ofLeishmaniaβ‐1,2‐Mannosyltransferases

Leishmania spp. are human pathogens that utilize a novel beta-1,2-mannan as their major carbohydrate reserve material. We describe a new approach that combines traditional substrate-modification methods and "click chemistry" to assemble a library of modified substrates that were used to qualitatively define the substrate tolerance of the Leishmania beta-1,2-mannosyltransferases responsible for beta-1,2-mannan biosynthesis. The library was assembled by using the highly selective copper(I)-catalysed cycloaddition reaction of azides and alkynes to couple an assortment of azide- and alkyne-functionalized small molecules with complementary alkyne- and azide-functionalized mannose derivatives. All mannose derivatives with alpha-orientated substituents on the anomeric carbon were found to act as substrates when incubated with a Leishmania mexicana particulate fraction containing GDP-mannose. In contrast, 6-substituted mannose derivatives were not substrates. Representative products formed from the library compounds were analysed by mass spectrometry, methylation linkage analysis and beta-mannosidase digestions and showed extension with up to four beta-1,2-linked mannosyl residues. This work provides insights into the substrate specificity of this new class of glycosyltransferases that can be applied to the development of highly specific tools and inhibitors for their study.

Endo-xyloglucan transferase, a novel class of glycosyltransferase that catalyzes transfer of a segment of xyloglucan molecule to another xyloglucan molecule.

Xyloglucans are the major component of plant cell walls and bind tightly to the surface of individual cellulose microfibrils, thereby cross-linking them into a complex polysaccharide network of the cell wall. The cleavage and reconnection of xyloglucan cross-links are considered to play the leading role during chemical processes essential for wall expansion and, therefore, cell growth and differentiation. Although it is hypothesized that some transglycosylation is involved in these chemical processes, the enzyme responsible for the reaction was not identified. We have now purified a novel class of endo-type glycosyltransferase to apparent homogeneity from the extracellular space or the cell wall of the epicotyls of Vigna angularis, a bean plant. The enzyme is a glycoprotein with a molecular mass of about 33 kDa. The enzyme catalyzes both 1) endo-type splitting of a xyloglucan molecule and 2) linking of a newly generated reducing end of the xyloglucan to the nonreducing end of another xyloglucan molecule, thereby mediating the transfer of a large segment of the xyloglucan to another xyloglucan molecule. The transferase exhibits no glycosidase or glycanase activity. Substrate specificity of the enzyme was investigated using several polysaccharides with different glycosidic linkages as donor substrates and pyridylamino oligosaccharides as acceptor substrates, in which the reducing end of the carbohydrate was tagged with a fluorescent group. The enzyme required a basic xyloglucan structure, i.e. a beta-(1-->4)-glucosyl backbone with xylosyl side chains, for both acceptor and donor activity. Galactosyl or fucosyl side chains on the main chain were not required for the acceptor activity. The enzyme exhibited higher reaction rates when xyloglucans with higher M(r) were used as donor substrates. Xyloglucans smaller than 10 kDa were no longer the donor substrate. On the other hand, pyridylamino heptasaccharide acted as a good acceptor as did xyloglucan polymers. Based on these results we propose to designate this novel enzyme a xyloglucan: xyloglucano-transferase, to be abbreviated endo-xyloglucan transferase (EXT) or xyloglucan recombinase. This enzyme is the first enzyme identified that mediates the transfer of a high M(r) segment between polysaccharide molecules to generate chimeric polymers. We conclude that endo-xyloglucan transferase functions as a reconnecting enzyme for xyloglucans and is involved in the interweaving or reconstruction of cell wall matrix, which is responsible for chemical creepage that leads to morphological changes in the cell wall.

Sperm-associated sialyltransferase activity.

FERTILISATION begins with a specific adhesion between sperm and egg and culminates with the fusion of the male and female pronuclei. In mammals, sperm seem to recognise the zona pellucida surrounding the egg1. This adhesive interaction is the first of many that will take place during subsequent embryogenesis. In molecular terms, these later cell–cell interactions are not generally understood, and the mechanism of sperm–egg recognition is unknown. Conceptually, advances in this area have been rare since Lillie presented the fertilisin hypothesis, and even this has been questioned2. There is evidence to support a model for intercellular recognition that utilises the molecular complementarity inherent in cell surface glycosyltransferases and their respective acceptor molecules3,4. The transferases catalyse the addition of sugars, from sugar nucleotides, to the non-reducing termini of saccharide acceptors. The enzymes are highly specific for both the donor sugar nucleotide and the acceptor, usually an oligosaccharide. For example, sialyltransferases are the class of glycosyltransferases that transfer sialic acid (N-acetylneuraminic acid) from cytidine monophosphate-N-acetylneuraminic acid (CMPNAN) to either glycoprotein, glycolipid, or free oligosaccharide acceptors4. Glycosyltransferases have been reported on the external surfaces of many different cell types and, in some cases, enzyme–substrate complexes between the transferases and their acceptors may be instrumental in the cellular recognition that occurs (reviewed in ref. 5). We report here a study of mouse sperm–egg interactions in terms of the transferase–acceptor hypothesis.