Neethling FA, Joziasse D, Bovin N, Cooper DK, Oriol R
Transplant international : official journal of the European Society for Organ Transplantation •
Synthetic galactosyl oligosaccharides were tested for their ability to inhibit the cytotoxic reaction of human and baboon natural antibodies on PK15 cells in culture. Methyl-alpha-Gal gave weak inhibition, Gal alpha 1-3Gal substantially inhibited the reaction (400muM), and Gal alpha 1-3Gal beta 1-4GLcNAc was ten times more efficient (30 muM). The modification from alpha to beta anomeric configuration of the nonreducing end resulted in a complete loss of activity, while substitutions at the reducing end induced only a partial loss of activity. These observations suggest that natural anti-alphaGal antibodies recognize the epitope from its nonreducing end, but that substitutions at the reducing terminus can modify the antibody-binding capacity. Modified tri- and tetrasaccharides are better inhibitors than the disaccharide but not as good as Gal alpha 1-3Gal beta 1-4GlcNAc. The reducing terminus therefore contributes some energy to the reaction, indicating that certain oligosaccharides will be of more potential clinical use than others.
Harmsen MM, Langedijk AC, van Tuinen E, Geerse RH, Raué HA, et al.
Gene •
We fused the yeast-derived sequences encoding the invertase, acid phosphatase and alpha-factor pre- and prepro-signal peptides (SP) to the Cyamopsis tetragonoloba (guar plant) alpha-galactosidase(alpha Gal)-encoding gene and expressed these gene fusions in yeast. Whereas the amount of fusion protein produced by each of the constructs did not vary significantly, the secretion efficiency of the fusion protein that carried the SP of the prepro-alpha-factor (MF alpha 1) was consistently found to be about 10% higher than that of the other fusions (99% vs. 90%). Furthermore, when the secretion of alpha Gal was directed by the invertase (SUC2) SP, the intracellular enzyme localized to the endoplasmic reticulum (ER), whereas use of the MF alpha 1 SP caused the intracellular enzyme to be outer-chain-glycosylated and processed by the KEX2 endoproteinase, implying that it had passed the ER. These results suggest that the pro-peptide of MF alpha 1 stimulates the efflux of the heterologous protein from the ER. Null mutants of PMR1 (encoding a Ca(2+)-dependent ATPase) are known to give higher secretion efficiencies for a number of different heterologous proteins. Therefore, we also studied the secretion of alpha Gal in a pmr 1 disruption mutant. Structural analysis of the enzyme secreted by the mutant cells showed that it was completely processed by KEX2 and outer-chain-glycosylated, although the length of the outer-chain carbohydrate moiety was reduced when compared with the enzyme secreted by wild-type cells. These results contradict the hypothesis advanced by Rudolph et al. [Cell 58 (1989) 133-145] that disruption of PMR1 causes the secretory pathway to bypass the Golgi apparatus.
The porcine small intestine contains several polypeptides that could function as receptors for K88-positive Escherichia coli. The mucus fraction contained three proteins with molecular weights of 25, 35 and 60 kDa respectively, which showed a high affinity for K88-positive E. coli cells, whereas brush borders contained a 16 kDa protein and a set of proteins ranging from 40-70 kDa. Depending on the K88 serotype tested, differences in binding to these proteins were observed. In particular, E. coli cells carrying K88ad fimbriae exhibited only a rather weak binding to mucus proteins. The influence of age of the pig on the presence of K88 receptors was also investigated. One-week-old and 35-days-old post-weaning piglets were shown to contain K88 receptors in their mucus while these receptors were hardly detectable in the mucus of 6-month-old pigs. The presence of receptors in the brush border fraction was shown to be independent of age. The binding of K88 fimbriae to mucus proteins was blocked using a lectin of Euonymus europeaus which specifically recognizes the Gal alpha(1-3)Gal sequence, indicating that this disaccharide forms a significant part of the receptor structure.
