title: Structural features of the lysosomal hydrolase mannose 6-phosphate uncovering enzyme.
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 alpha Vbeta 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 Cys(2)-Cys(172), Cys(66)-Cys(99), Cys(83)-Cys(274), and Cys(258)-Cys(265). 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.
authors.affiliation: Department of Molecular, Cellular & Craniofacial Biology, School of Dentistry, University of Louisville, KY 40292, USA.
authors.forename: Yuqiang
authors.initials: Y
authors.lastname: Wei
authors.forename: Ten-Yang
authors.initials: TY
authors.lastname: Yen
authors.forename: Jian
authors.initials: J
authors.lastname: Cai
authors.forename: John O
authors.initials: JO
authors.lastname: Trent
authors.forename: William M
authors.initials: WM
authors.lastname: Pierce
authors.forename: William W
authors.initials: WW
authors.lastname: Young
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002853:Chromatography, Liquid; D006031:Glycosylation; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000737:chemistry; D008247:Lysosomes / Q000201:enzymology*; D008360:Mannosephosphates / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D039302:Integrin alphaVbeta3; D008360:Mannosephosphates; C027693:mannose-6-phosphate; D010727:Phosphoric Diester Hydrolases; C026645:N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase