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
Tyrosinase and tyrosinase-related protein-1 (TRP-1) are two melanogenic enzymes that regulate melanin biosynthesis. Both are glycoproteins and belong to the TRP-1 gene family. They share a significant level of sequence similarity in several regions, including the catalytic domain and the potential N-glycosylation sites. We have recently shown that inhibition of the early steps of N-glycan processing in B16F1 cells dramatically affects tyrosinase activity and melanin synthesis. We present here results on N-glycan processing of TRP-1 and tyrosinase and compare the maturation process and activity of both glycoproteins in the presence of inhibitors of the endoplasmic reticulum stages of N-glycosylation. N-glycan analysis reveals that each of these two glycoproteins contains a mixture of high-mannose and sialylated complex N-glycans. However, in contrast to TRP-1, tyrosinase presents a homogeneous high-mannose glycoform, also. In the presence of alpha-glucosidases inhibitors, the maturation of tyrosinase N-glycans is completely inhibited, whereas TRP-1 is still able to acquire some complex glycans, indicating that endomannosidase acts preferentially on the later glycoprotein. In addition, the dopa-oxidase activity of tyrosinase is totally abolished, whereas for TRP-1 it is only partially affected. The results suggest that despite their structural similarity, tyrosinase is more sensitive than TRP-1 to perturbations of early N-glycan processing, in terms of maturation and catalytical activity.
Human CD1d molecules consist of a transmembrane CD1 (cluster of differentiation 1) heavy chain in association with  2 -microglobulin ( 2 m). Assembly occurs in the endoplasmic reticulum (ER) and involves the initial glycan-dependent association of the free heavy chain with calreticulin and calnexin and the thiol oxidoreductase ERp57. Folding and disulfide bond formation within the heavy chain occurs prior to  2 m binding. There are four N-linked glycans on the CD1d heavy chain, and we mutated them individually to ascertain their importance for the assembly and function of CD1d- 2 m heterodimers. None of the four were indispensable for assembly or the ability to bind ␣-galactosyl ceramide and to present it to human NKT cells. Nor were any required for the CD1d molecule to bind and present ␣-galactosyl ceramide after lysosomal processing of a precursor lipid, galactosyl-(␣1-2)-galactosyl ceramide. However, one glycan, glycan 2 at Asn-42, proved to be of particular importance for the stability of the CD1d- 2 m heterodimer. A mutant CD1d heavy chain lacking glycan 2 assembled with  2 m and transported from the ER more rapidly than wild-type CD1d and dissociated more readily from  2 m upon exposure to detergents. A mutant expressing only glycan 1 dissociated completely from  2 m upon exposure to the detergent Triton X-100, whereas a mutant expressing only glycan 2 at Asn-42 was more stable. In addition, glycan 2 was not processed efficiently to the complex form in mature wild-type CD1d molecules. Modeling the glycans on the published structure indicated that glycan 2 interacts significantly with both the CD1d heavy chain and  2 m, which may explain these unusual properties.The human CD1 5 (cluster of differentiation 1) family consists of five transmembrane glycoproteins encoded by linked genes (1). They are divided into two groups based on amino acid sequence homology; group 1 includes CD1a, -b, and -c, and group 2 consists of CD1d, the only isoform present in mice and rats. The fifth member of the family, CD1e, has an amino acid sequence intermediate between the two groups. CD1 heavy chains are structurally similar to MHC class I molecules and possess a short C-terminal cytosolic tail, a hydrophobic transmembrane region, and an extracellular region that interacts non-covalently with  2 -microglobulin ( 2 m). The role of CD1 molecules is to bind lipid antigens and present them to T cells, and the ␣1 and ␣2 domains of the extracellular region fold in a similar manner to the analogous domains in MHC class I molecules to generate the lipid binding site.CD1 heavy chain folding and association with  2 m occurs in the endoplasmic reticulum (ER). After exiting the ER, the assembled CD1 molecules pass through the secretory pathway and reach the plasma membrane. From there, with the exception of CD1a, they enter the endocytic system by adaptor protein (AP)-dependent internalization using tyrosine-based endocytic motifs (YXX⌽, X ϭ any amino acid and ⌽ ϭ bulky hydrophobic amino acid). Similar to MHC class II molecul...
Tyrosinase and tyrosinase-related protein-1 (TRP-1) are two melanogenic enzymes that regulate melanin biosynthesis. Both are glycoproteins and belong to the TRP-1 gene family. They share a significant level of sequence similarity in several regions, including the catalytic domain and the potential N-glycosylation sites. We have recently shown that inhibition of the early steps of N-glycan processing in B16F1 cells dramatically affects tyrosinase activity and melanin synthesis. We present here results on Nglycan processing of TRP-1 and tyrosinase and compare the maturation process and activity of both glycoproteins in the presence of inhibitors of the endoplasmic reticulum stages of Nglycosylation.N-glycan analysis reveals that each of these two glycoproteins contains a mixture of high-mannose and sialylated complex N-
Background V(D)J recombination is essential for adaptive immunity in jawed vertebrates and is initiated by the RAG1-RAG2 endonuclease. The RAG1 and RAG2 genes are thought to have evolved from a RAGL (RAG-like) transposon containing convergently-oriented RAG1-like (RAG1L) and RAG2-like (RAG2L) genes. Elements resembling this presumptive evolutionary precursor have thus far only been detected convincingly in deuterostomes, leading to the model that the RAGL transposon first appeared in an early deuterostome. Results We have identified numerous RAGL transposons in the genomes of protostomes, including oysters and mussels (phylum Mollusca) and a ribbon worm (phylum Nemertea), and in the genomes of several cnidarians. Phylogenetic analyses indicate that the RAGL transposon family evolved in a vertical manner within the Bilateria clade. Many of the RAGL transposons identified in protostomes are intact elements containing convergently oriented RAG1L and RAG2L genes flanked by terminal inverted repeats (TIRs) and target site duplications with striking similarities with the corresponding elements in deuterostomes. In addition, protostome genomes contain numerous intact RAG1L-RAG2L adjacent gene pairs that lack detectable flanking TIRs. Domains and critical active site and structural amino acids needed for endonuclease and transposase activity are present and conserved in many of the predicted RAG1L and RAG2L proteins encoded in protostome genomes. Conclusions Active RAGL transposons were present in multiple protostome lineages and were transmitted vertically during protostome evolution. It appears that the RAGL transposon family was broadly active during bilaterian evolution, undergoing multiple duplication and loss/fossilization events, with the RAGL genes that persist in present day protostomes perhaps constituting both active RAGL transposons and domesticated RAGL genes. Our findings indicate that the RAGL transposon arose earlier in evolution than previously thought, either in an early bilaterian or prior to the divergence of bilaterians and non-bilaterians, and alter our understanding of the evolutionary history of this important transposon family.
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