Addition Of Fucose Research Articles

Abnormal glycosylation of human cellular fibronectin in the presence of swainsonine.

The relationship between post-translational modifications of macromolecules and their intracellular routing is of fundamental importance. The availability of the indolizidine alkaloid, swainsonine, which interferes with glycoprotein processing, provides a new probe for studying relationships between glycosylation of proteins and their cellular routing. Using fibronectin as a model glycoprotein, we have explored the effect of swainsonine upon oligosaccharide structure, glycoprotein synthesis, and secretion. Confluent human fibroblasts were labeled with radioactive mannose or glucosamine in the presence or absence of swainsonine. Fibronectin was secreted into the medium of swainsonine-treated cultures and found to contain endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides, instead of the normal complex endo-beta-N-acetylglucosaminidase H-resistant oligosaccharides. Pronase-released glycopeptides and hydrazine-released oligosaccharides of isolated fibronectin from the culture media were analyzed using endo-beta-N-acetylglucosaminidase H and specific exoglycosidase digestions in conjunction with calibrated gel filtration chromatography. The structure of the swainsonine-modified endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide was found to be a hybrid type, Gal beta leads to GlcNAc beta leads to Man alpha leads to [Man alpha leads to (Man alpha leads to) Man alpha leads to]Man beta leads to GlcNAc beta leads to (+/- Fuc alpha leads to)-GlcNAc. Other experiments showed that the synthesis and secretion of fibronectin were not affected by its change in glycosylation. The results also infer that swainsonine inhibits Golgi mannosidase II in intact cells, and that removal of two mannose residues by that mannosidase is not a prerequisite for addition of galactose to the existing peripheral nonreducing GlcNAc or for the addition of fucose to the innermost reducing GlcNAc. The composite results indicate that significant changes in oligosaccharide structure had little effect upon the routing and cellular release of a typical N-asparagine-linked glycoprotein.

Glycosylation and secretion of an altered immunoglobulin heavy chain in mouse myeloma MOPC 315.

A variant line (LV-1) of mouse myeloma MOPC 315 (IgA, lambda 2) has lost the ability to synthesize L chain. It synthesizes an altered H chain (H' chain) that is turned over intracellularly and is not secreted. Rescue of H' chain secretion can be accomplished by fusion of LV-1 to a variant of another myeloma line, MPC 11 (IgG2b, kappa), which only synthesizes a light chain. The hybrid (X-2) secretes the H' chain in a four chain structure (kappa 2 alpha' 2). In wild-type MOPC 315 cells, it was reported previously that inhibition of core sugar addition blocks the secretion of the H chain polypeptide. We have studied glycosylation in MPOC 315 wild-type, LV-1 variant, and X-2 hybrid cell lines. The ability of all three lines to add the core sugars mannose and glucosamine to heavy chain was demonstrated. Due to the instability of the H' chain in LV-1, it is difficult to assess H' chain fucosylation directly. To study fucose addition in LV-1, the enveloped virus vesicular stomatitis (VSV), which can infect the three lines, was utilized. The fucosylation and secretion of VSV glycoprotein G was discernible in all three lines; however, only LV-1 cannot activate free fucose, and instead fucosylates through conversion of the mannose intermediate. Normal fucose addition to H chain in a wild-type cell occurred immediately before secretion. The fact that degradation of H' chain in LV-1 begins before fucosylation suggests that the rescue of H' chain secretion by formation of the X-2 hybrid is due to the acquired presence of a suitable L chain rather than complementation of a sugar defect. These observations indicate that proper assembly of the polypeptide components of some secretory proteins, e.g., Ig molecules, is required for the secretion of the individual chains.

Structural studies of the endoglycosidase H-resistant oligosaccharides present on human beta-glucuronidase.

Human beta-glucuronidase bears 3-4 oligosaccharide moieties/subunit of Mr = 75,000. We have previously characterized the endoglycosidase H-releasable oligosaccharides of this enzyme including those which are phosphorylated and involved in targeting to lysosomes. In this study, we report the characterization of the endoglycosidase H-resistant oligosaccharides which were released from beta-glucuronidase with anhydrous hydrazine. Approximately 65% of the hydrazine-released oligosaccharides are of the high mannose type, with the predominant species containing 9 mannose residues. The remaining oligosaccharides appear to originate from incomplete complex oligosaccharides. Their basic structures are Man alpha 1,6Man beta 1,4Glc-NAc beta 1,4GlcNAcol, and Man alpha 1,3[Man alpha 1,6]Man beta 1,4Glc-NAc beta 1,4GlcNAcol with roughly half of each species containing an additional fucose linked alpha 1,6 to the N-acetylglucosaminitol (GlcNAcol) residue. The small amount of complex oligosaccharide present bearing 1 sialic acid was heterogeneous in nature with incompletion of the nonsialylated branch. In addition, there was a minor specie of high mannose-type oligosaccharide bearing 5 mannose residues with an alpha 1,6-linked fucose on the GlcNAcol. This structure was not expected since high mannose-type oligosaccharides have been reported to not be substrates for the alpha 1,6-fucosyl transferase.

Conformational analysis of some mannose‐containing and milk oligosaccharides: Correlation of their shape with inhibitory properties in antigen/antibody interactions

AbstractPossible conformations of two mannotetraoses and several milk oligosaccharides have been studied by an energy minimization procedure using semi‐empirical potential functions. Changes in the terminal residue at the reducing end (cyclic to acyclic form) of these molecules do not affect the favored conformations of the remaining oligosaccharide moiety. However, differences in the overall shape of the native and reduced forms of the mannotetraose, mannose α(1–3) mannose α(1–2) mannose α(1–2) mannose are much less marked than between the native and reduced forms of lacto‐N‐tetraose. These differences are related to the effectiveness of the native forms as inhibitors of antibodies produced using synthetic antigens. Changes in the linkage of the residues at the nonreducing end of these molecules affect significantly the overall shape of these molecules. These differences also are related to their effectiveness as inhibitors. In the fucose‐containing milk oligosaccharides the additional fucose residues only restrict the orientation of the backbone tetrasaccharide and do not push it into a totally new conformation. The fucose residues come on a surface of the molecule which is away from the region which may be important for binding. The present studies show that it is the overall shape of the molecule which is important in determining its inhibitory properties and give information as to how best to use the immune method for identification of unknown oligosaccharide sequences by specially prepared antibodies.

A fucogalactoxyloglucan from rapeseed hulls

A polysaccharide, isolated from rapeseed hulls by extraction with aqueous sodium hydroxide—sodium tetraborate, contained residues of l-arabinose, l-fucose, d-xylose, d-galactose, and d-glucose in the proportions of 2:8:25:13:52. Acetolysis furnished cellobiose, 6- O-α- d-xylopyranosyl- d-xylopyranosyl- d-glucose, and 2- O-β- d-galactopyranosyl d-xylose. The cleavage products from the methylated polysaccharide were examined by g.l.c. of the methyl glycosides and g.l.c.—mass spectrometry of the partially methylated, alditol acetates. The results show that the polysaccharide is a member of the xyloglucan group in which additional fucose and galactose residues terminate some of the side-chains. For comparative purposes, aspects of the structures of xyloglucans from nasturtium seeds and suspension-cultured sycamore cells have been re-examined.

OLIGOSACCHARIDES IN INFANTS' FAECES.

Concentrated faecal extracts from breast and artificially fed infants during the first week of life were examined by paper chromatography. Besides lactose, glucose and galactose, five oligosaccharide spots were detected. Breast-fed infants showed an additional fucose spot. Complete hydrolysis of the individual oligosaccharides showed glucose and galactose always and fucose in two instances. The oligosaccharides are probably similar to those isolated by other workers from colostrum and human milk. This is a preliminary report.