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The specificity of Golgi-membrane glycoprotein 6-alpha-[L]-fucosyltransferase [GDP-[L]-fucose: 2-acetamido-2-deoxy- beta-[D]-glucoside (Fuc-->Asn-linked GlcNAc) 6-alpha-[L]-fucosyltransferase; EC 2.4.1.68] has been assessed with regard to substrate covalent structures and the effect of a protein matrix on the conformational display of those covalent structures. Specificity was studied by direct comparison of the substrate quality of nine 6-biotinamidohexanoylAsn (= R) derivatives of intermediates and products in the pathway from Man5GlcNAc2-R to a fully sialylated biantennary complex-type glycan. The Man5 derivative and the sialic acid-containing glycans were completely inactive as substrates. The other glycans were all fucosylated; the best substrate was GlcNAcMan3GlcNAc2-R. The protein-matrix effect was studied by comparing the substrate quality of the same 6-biotinamidohexanoylAsn derivatives as well as the corresponding biotinylAsn derivatives free in solution and bound to streptavidin. On the basis of a model derived from the known 3D structure of biotin (biocytin)-saturated streptavidin, it was predicted that the fucosylation site in the substrates would be completely masked in the biotin-binding pocket in the biotinyl derivatives (proximal display), and at least partially masked in the 6-biotinamidohexanoyl derivatives (distal display). The activity measurements were in agreement with these predictions; the glycan structures GlcNAcMan5GlcNAc2-, GlcNAcMan3GlcNAc2-, and GlcNAc2-Man3GlcNAc2- were readily fucosylated as derivatives free in solution, but were totally inert in the proximal complex with streptavidin. In the distal complexes the latter two structures were found to be fucosylated very slowly while the former structure was inactive.

Sugar-binding characteristics of rat serum mannose-binding protein (MBP) were studied using the carbohydrate-recognition domain of this protein expressed from a cloned cDNA. To assess the binding affinity of various test compounds, they were added as inhibitors in a binding assay in which 125I-MBP was incubated with yeast cells and the extent of binding was estimated from the radioactivity associated with the pelleted cells. The results of such inhibition assays suggest that MBP has a small binding site which is probably of the trough-type. The 3- and 4-OH of the target sugar are indispensable, while the 6-OH is not required. These characteristics are shared by the rat hepatic lectin and chicken hepatic lectin, both of which are C-type lectins containing carbohydrate-recognition domains highly homologous to that of MBP. Apparently, the related primary structures of these lectins give rise to similar gross architecture of their binding sites, despite the fact that each exhibits different sugar binding specificities.

In order to assess the basis for the regulatory effects of the protein matrix on the processing of glycans in glycoproteins, we have used the avidin-biotinylglycan neoglycoprotein model system to compare the kinetic parameters for three rat liver Golgi enzymes acting on their free and protein-bound glycan substrates. Two modes of glycan display in the avidin complex were produced by the use of either the biotinyl- or the 6-biotinamidohexanoyl-group as ligands for the avidin binding. N-Acetylglucosaminyltransferase I gave a 100-fold decrease in Vmax/Km for the avidin complex of Man5GlcNAc2-(biotinyl)Asn as compared to the free glycan derivative; the rate difference reflects a large (25x) decrease in the Vmax and a relatively small increase (4x) in Km. When the substrate with the extension arm (Man5GlcNAc2-(6-biotinamidohexanoyl)Asn) was used, the difference between Vmax/Km for free and avidin-bound substrate was only 6-fold. The Vmax/Km ratio for N-acetylglucosaminyltransferase II also showed a 10-fold difference for free and avidin-bound GlcNAcMan3GlcNAc2-(biotinyl)Asn; the introduction of the extension arm in the complex reduced the difference to about 3-fold. The third enzyme, galactosyltransferase, acting on the substrate GlcNAcMan5-GlcNAc2-R in the presence of the mannosidase II-inhibitor swainsonine, showed a small, 2- to 3-fold, decrease in the Vmax for the bound substrates, both with and without the extension arm. The results suggest that the protein matrix affects the catalytic efficiency rather than the substrate affinity of the processing enzymes.