We have generated a database of 639 glycosidic linkage structures by an exhaustive survey of the available crystallographic data for isolated oligosaccharides, glycoproteins, and glycan-binding proteins. For isolated oligosaccharides there is relatively little crystallographic data available. A much larger number of glycoprotein and glycan-binding protein structures have now been solved in which two or more linked monosaccharides can be resolved. In the majority of these cases, only a few residues can be seen. Using the 639 glycosidic linkage structures, we have identified one or more distinct conformers for all the linkages. The O5-C1-O-C(x)' torsion angles for all these distinct conformers appear to be determined chiefly by the exo-anomeric effect. The Manalpha1-6Man linkage appears to be less restrained than the others, showing a wide degree of dispersion outside the ranges of the defined conformers. The identification of distinct conformers for glyco-sidic linkages allows "average" glycan structures to be modeled and also allows the easy identification of distorted glycosidic linkages. Such an analysis shows that the interactions between IgG Fc and its own N-linked glycan result in severe distortion of the terminal Galbeta1-4GlcNAc linkage only, indicating the strong interactions that must be present between the Gal residue and the protein surface. The applicability of this crystallographic based analysis to glycan structures in solution is discussed. This database of linkagestructures should be a very useful reference tool in three-dimensional structure determinations.
Tyrosinase is a copper-containing enzyme that regulates melanin biosynthesis in mammals. Mutations at a single N-glycosylation sequon of tyrosinase have been reported to be responsible for oculocutaneous albinism type IA in humans, characterized by inactive tyrosinase and the total absence of pigmentation. To probe the role that each N-glycosylation site plays in the synthesis of biologically active tyrosinase, we analyzed the calnexin mediated folding of tyrosinase N-glycosylation mutants. We have determined that four of the six potential Nglycosylation sites, including that associated with albinism, are occupied. Analysis of the folding pathway and activity of 15 tyrosinase mutants lacking one or more of the occupied N-glycosylation sites shows that glycans at any two N-glycosylation sites are sufficient to interact with calnexin and give partial activity, but a specific pair of sites (Asn 86 and Asn 371 ) is required for full activity. The mutants with less than two N-glycosylation sites do not interact with calnexin and show a complete absence of enzyme activity. Copper analysis of selected mutants suggests that the observed partial activity is due to two populations with differential copper content. By correlating the degree of folding with the activity of tyrosinase, we propose a local folding mechanism for tyrosinase that can explain the mechanism of inactivation of tyrosinase N-glycosylation mutants found in certain pigmentation disorders.
Contents 1. Introduction 4697 2. N-Glycosylation of Glycoproteins 4698 2.1. N-Glycan Processing in the ER and Golgi 4698 2.2. Early Stages of N-Glycan Processing Involved in Protein Folding 4699 2.3. Glycosylation Inhibitors 4701 3. Tyrosinase and Tyrosinase-Related Proteinss The Regulating Enzymes of Melanogenesis 4701 4. Tyrosinase and TRP-1 as Probes of the Role of Calnexin/Calreticulin 4703 4.1. Tyrosinase Folding Is Calnexin Dependent 4703 4.2. Kinetics of TRP-1 Folding Is Unchanged in the Presence of Calnexin 4704 5. Tyrosinase and TRP-1 Glycosylation Is Protein Specific 4706 5.1. N-Glycan Composition 4706 5.2. TRP-1 Is a Substrate for Endomannosidase 4706 6. Individual Glycans in Tyrosinase Folding 4707 7. Tyrosinase Folding and Copper Loading 4708 8. Malignant Melanomas and Tyrosinase 4708 9. Concluding Remarks 4709 10. Acknowledgments 4710 11. References 4710
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