α1,2-linked fucose can be found on xyloglucans which are the main hemicellulose compounds of dicotyledons. The fucosylated nonasaccharide XXFG derived from xyloglucans plays a role in cell signaling and is active at nanomolar concentrations.
The plant enzyme acting on this α1,2-linked fucose residues has been previously called fucosidase II; here we report on the molecular identification of a gene from Arabidopsis thaliana (At4g34260 hereby designed AtFuc95A) encoding this enzyme. Analysis of the predicted protein composed of 843 amino acids shows that the enzyme belongs to the glycoside hydrolase family 95 and has homologous sequences in different monocotyledons and dicotyledons. The enzyme was expressed recombinantly in Nicotiana bentamiana, a band was visible by Coomassie blue staining and its identity with the α1,2-fucosidase was assessed by an antibody raised against a peptide from this enzyme as well as by peptide-mass mapping. The recombinant AtFuc95A is active towards 2-fucosyllactose with a Km of 0.65 mM, a specific activity of 110 mU/mg and a pH optimum of 5 but does not cleave α1,3, α1,4 or α1,6-fucose containing oligosaccharides and p-nitrophenyl-fucose. The recombinant enzyme is able to convert the xyloglucan fragment XXFG to XXLG, and is also active against xyloglucan polymers with a Km value for fucose residues of 1.5 mM and a specific activity of 36 mU/mg. It is proposed that the AtFuc95A gene has a role in xyloglucan metabolism.

Analysis of the numerous possible, often isobaric structures of protein-bound oligosaccharides calls for a high-performance two-dimensional method that combines liquid chromatography's ability to separate isomers and mass spectrometry's ability to determine glycan composition. Here we investigate the usefulness of porous graphitic carbon columns coupled to ESI-MS for the separation of N-glycans with two or more sialic acids. Internal standards helped to rectify retention time fluctuations and thus allowed elution times to play an essential role in the structural assignment of peaks. For generation of a retention time library, standards representing the possible isomers of diantennary non-, mono-, and disialylated N-glycans, differing in the linkage of galactose and sialic acids as well as isobaric hybrid-type N-glycans, were produced using recombinant glycosyltransferases. Once the retention times library was established, isomers could be identified by LC-ESI-MS in the positive mode without additional MS/MS experiments. The method was applied for the detailed structural analysis of fibrin(ogen) N-glycans from various species (human, cow, pig, mouse, rat, cat, dog, Chinese hamster, horse, sheep, and chicken). All fibrins contained diantennary N-glycans. They differed in the occurrence of β1,3-linked galactose, α2,3-linked sialic acids, and N-glycolylneuraminic acid, in the mono/diantennary glycan ratio, and in the O-acetylation of neuraminic acids. The separation system's potential for analyzing tri- and tetrasialylated N-glycans was demonstrated.