Specificity studies of the GDP-[L]-fucose: 2-acetamido-2-deoxy-beta-[D]-glucoside (Fuc-->Asn-linked GlcNAc) 6-alpha-[L]-fucosyltransferase from rat-liver Golgi membranes.
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.
Shao
M C
MC
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
Sokolik
C W
CW
Wold
F
F
D000818:Animals; D001216:Asparagine / Q000032:analysis; D001426:Bacterial Proteins / Q000378:metabolism / Q000494:pharmacology; D001710:Biotin / Q000032:analysis; D002240:Carbohydrate Sequence; D005647:Fucosyltransferases / Q000187:drug effects / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D008358:Mannose / Q000031:analogs & derivatives; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D016339:Spectrometry, Mass, Fast Atom Bombardment; D019809:Streptavidin; D013379:Substrate Specificity; D017026:Swainsonine / Q000494:pharmacology
D001426:Bacterial Proteins; D009844:Oligosaccharides; D001710:Biotin; D001216:Asparagine; D019809:Streptavidin; D005647:Fucosyltransferases; C020559:Glycoprotein 6-alpha-L-fucosyltransferase; D008358:Mannose; D017026:Swainsonine