Mutation of amino acids in the alpha 1,3-fucosyltransferase motif affects enzyme activity and Km for donor and acceptor substrates.

Alpha 1,3-fucosyltransferases (FucT) share a conserved amino acid sequence designated the alpha 1,3 FucT motif that has been proposed to be important for nucleotide sugar binding. To evaluate the importance of the amino acids in this motif, each of the alpha 1,3 FucT motif amino acids was replaced with alanine (alanine scanning mutagenesis) in human FucT VI, and the resulting mutant proteins were analyzed for enzyme activity and kinetically characterized in those cases in which the mutant protein had sufficient activity. Two of the mutant proteins were inactive, six had less than 1% of wild-type activity, and four had approximately 10-50% of wild-type enzyme activity. Three of the mutant proteins with significant enzyme activity had substantially larger Km (5 to 15 times) for GDP-fucose than FucT VI wild-type enzyme. The fourth mutant protein with significant enzyme activity (S249A) had a Km at least 10 times larger than wild-type FucT VI for the acceptor substrate, with only a slightly larger (2-3 times) Km for GDP-fucose. Thus mutation of any of the amino acids within the alpha 1,3 FucT motif to Ala affects alpha 1,3-FucT activity, and substitution of Ala for some of the alpha 1,3 FucT motif amino acids results in proteins with altered kinetic constants for both the acceptor and donor substrates. Secondary structure prediction suggests a helix-loop-helix fold for the alpha 1,3 FucT motif, which can be used to rationalize the effects of mutations in terms of 3D structure.

High level of alpha1-acid glycoprotein in human seminal plasma is associated with high branching and expression of Lewis(a) groups on its glycans: supporting evidence for a prostatic origin.

BACKGROUND: Changes in concentration of seminal plasma alpha(1)-acid glycoprotein (AGP) have been studied in detail before. However, the source of high levels of AGP as well as the glycosylation of seminal plasma AGP has not been elucidated yet. METHODS: The glycosylation of AGP was studied by crossed affinity immunoelectrophoresis using fucose-specific lectins and immunostaining. Glycan structure and monosaccharide analyses were performed by high pH anion exchange chromatography with pulsed amperometric detection. Fucosyltransferases were analyzed for activity and their substrate specificity was determined. RESULTS: Two types of fucosylation were detected; Lewis(x) and Lewis(a). Lewis(a) groups were only present on AGP of individuals with a high concentration and were completely absent when the AGP concentration in seminal plasma was low. Lewis(a) expression coincides with a higher degree of branching of the glycans and a relative increased alpha4-fucosyltransferase activity. The molecular weight of all seminal plasma AGP was slightly higher than of blood plasma AGP (approx. 47 vs. 41-43 kDa). CONCLUSIONS: The results indicate that AGP in seminal fluid most likely originates from the prostate and that it is either alpha3- or alpha4-fucosylated.