Structural features of the lysosomal hydrolase mannose 6-phosphate uncovering enzyme |
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Authors: | Yuqiang Wei Ten-Yang Yen Jian Cai John O. Trent William M. Pierce William W. Young Jr. |
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Affiliation: | (1) Department of Molecular, Cellular & Craniofacial Biology, School of Dentistry, University of Louisville, 501 S. Preston St., Louisville, KY, 40292;(2) Department of Chemistry & Biochemistry, San Francisco State University, 1600 Holloway Ave., San Francisco, CA, 94132;(3) James G. Brown Cancer Center, University of Louisville, USA;(4) Department of Pharmacology and Toxicology, School of Medicine, University of Louisville, USA;(5) Department of Biochemistry and Molecular Biology, School of Medicine, University of Louisville, USA;(6) Department of Molecular, Cellular, & Craniofacial Biology, University of Louisville, School of Dentistry Room 326, 501 S. Preston St., Louisville, KY, 40292 |
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Abstract: | The uncovering enzyme (UCE) removes N-acetylglucosamine from lysosomal enzymes to uncover the mannose 6-phosphate (Man-6-P) determinant necessary for targeting these enzymes to lysosomes. Failure to create the Man-6-P determinant is one cause of lysosomal storage diseases. Despite its medical importance, little structural information about UCE is available. In this report we have developed a model for the membrane proximal portion of the lumenal domain of UCE based on the structure of the EFG-3 and -4 domains of the extracellular segment of the beta chain of integrin V 3. In this model the EGF-like domains of UCE (residues 285–345) are predicted to form a rod-shaped stalk region, similar to the stem region in Golgi glycosyltransferases. This stalk causes the proposed catalytic domain (residues 1–277) to be extended away from the Golgi membrane. A portion of the proposed catalytic domain (residues 85-256) resides in Cluster of Orthologous Group (COG) 4632 with four bacterial proteins but is not homologous to any known eukaryotic proteins. Thus, UCE may have evolved from the fusion of a unique catalytic domain with a common EGF-like stalk domain. We have determined by mass spectrometry that the four disulfide bonds of the proposed catalytic domain are located between Cys2–Cys172, Cys66–Cys99, Cys83–Cys274, and Cys258–Cys265. Finally, we determined that four of the six potential N-linked glycosylation sites are glycosylated (Asn 159, Asn 165, Asn 247, and Asn 317) in COS cells. Published in 2005. |
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Keywords: | disulfide bonds mannose 6-phosphate mass spectrometry uncovering enzyme |
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