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J. Biol. Chem., Vol. 280, Issue 43, 36141-36149, October 28, 2005

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The - and -subunits of the Human UDP-N-acetylglucosamine:Lysosomal Enzyme Phosphotransferase Are Encoded by a Single cDNA^*

Mariko Kudo, Ming Bao¶, Anil D'Souza||, Fu Ying**, Huaqin Pan, Bruce A. Roe**, and William M. Canfield1

From the Genzyme Corp., Oklahoma 73104, W. K. Warren Medical Research Institute and the Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, ^¶System Biology, Berlex Biosciences, Richmond, California 94804-0099, ^||Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, ^**Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, Oklahoma 73019, and Center for Integrated Fungal Research, North Carolina State University, Raleigh, North Carolina 27606

Lysosomal enzymes are targeted to the lysosome through binding to mannose 6-phosphate receptors because their glycans are modified with mannose 6-phosphate. This modification is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Bovine GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography, and an ₂₂₂-subunit structure was proposed. Although cDNA encoding the -subunit has been described, cDNAs for the - and -subunits have not. Using partial amino acid sequences from the bovine - and -subunits, we have isolated a human cDNA that encodes both the - and -subunits. Both subunits contain a single predicted membrane-spanning domain. The - and -subunits appear to be generated by a proteolytic cleavage at the Lys⁹²⁸-Asp⁹²⁹ bond. Transfection of 293T cells with the /-subunits-precursor cDNA with or without the -subunit cDNA results in a 3.6- or 17-fold increase in GlcNAc-phosphotransferase activity in cell lysates, suggesting that the precursor cDNA contains the catalytic domain. The sequence lacks significant similarity with any described vertebrate enzyme except for two Notch-like repeats in the -subunit. However, a 112-amino acid sequence is highly similar to a group of bacterial capsular polymerases (46% identity). A BAC clone containing the gene that spanned 85.3 kb and was composed of 21 exons was sequenced and localized to chromosome 12q23. We now report the cloning of both the cDNA and genomic DNA of the precursor of Glc-NAc-phosphotransferase. The completion of cloning all three subunits of GlcNAc-phosphotransferase allows expression of recombinant enzyme and dissection of lysosomal targeting disorders.

Received for publication, August 16, 2005 , and in revised form, August 24, 2005.

^* This work was supported in part by National Institutes of Health Grant HD31920, Oklahoma Center for the Advancement of Science and Technology Grant HS-024, Presbyterian Health Foundation Grant 916 (to W. M. C.), and National Institutes of Health Grant 5 U54 HG02152 (to B. A. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY687932.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.

¹ To whom correspondence should be addressed: Genzyme Corp., 800 Research Pkwy., Suite 200, Oklahoma City, OK 73104. Tel.: 405-271-8144; Fax: 405-271-8188; E-mail: William.Canfield{at}genzyme.com.

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