Structural basis for the substrate specificity of endo-beta-N-acetylglucosaminidase F(3).
Endo-beta-N-acetylglucosaminidase F(3) cleaves the beta(1-4) link between the core GlcNAc's of asparagine-linked oligosaccharides, with specificity for biantennary and triantennary complex glycans. The crystal structures of Endo F(3) and the complex with its reaction product, the biantennary octasaccharide, Gal-beta(1-4)-GlcNAc-beta(1-2)-Man-alpha(1-3)[Gal-beta(1-4)-GlcNAc-be ta(1-2)-Man-alpha(1-6)]-Man-beta(1-4)-GlcNAc, have been determined to 1.8 and 2.1 A resolution, respectively. Comparison of the structure of Endo F(3) with that of Endo F(1), which is specific for high-mannose oligosaccharides, reveals highly distinct folds and amino acid compositions at the oligosaccharide recognition sites. Binding of the oligosaccharide to the protein does not affect the protein conformation. The conformation of the oligosaccharide is similar to that seen for other biantennary oligosaccharides, with the exception of two links: the Gal-beta(1-4)-GlcNAc link of the alpha(1-3) branch and the GlcNAc-beta(1-2)-Man link of the alpha(1-6) branch. Especially the latter link is highly distorted and energetically unfavorable. Only the reducing-end GlcNAc and two Man's of the trimannose core are in direct contact with the protein. This is in contrast with biochemical data for Endo F(1) that shows that activity depends on the presence and identity of sugar residues beyond the trimannose core. The substrate specificity of Endo F(3) is based on steric exclusion of incompatible oligosaccharides rather than on protein-carbohydrate interactions that are unique to complexes with biantennary or triantennary complex glycans.
Waddling
C A
CA
Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Albany, New York 12201, USA.
Plummer
T H
TH
Tarentino
A L
AL
Van Roey
P
P
D002240:Carbohydrate Sequence; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D009844:Oligosaccharides; D011994:Recombinant Proteins; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase