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J. Biol. Chem., Vol. 280, Issue 39, 33318-33323, September 30, 2005

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Identification of the Minimal Lysosomal Enzyme Recognition Domain in Cathepsin D^*

From the Department of Internal Medicine, Washington University School of Medicine, Saint Louis, Missouri 63110

Specific recognition of lysosomal hydrolases by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose 6-phosphate residues, is governed by a common protein determinant. Previously, we generated a lysosomal enzyme recognition domain in the secretory protein glycopepsinogen by substituting in two regions (lysine 203 and amino acids 265–293 of the loop) from cathepsin D, a highly related lysosomal protease. Here we show that substitution of just two lysines (Lys-203 and Lys-267) stimulates mannose phosphorylation 116-fold. Substitution of additional residues in the loop, particularly lysines, increased phosphorylation 4-fold further, approaching the level obtained with intact cathepsin D. All the phosphorylation occurred at the carboxyl lobe glycan, indicating that additional elements are required for phosphorylation of the amino lobe glycan. These data support the proposal that as few as two lysines in the correct orientation to each other and to the glycan can serve as the minimal elements of the lysosomal enzyme recognition domain. However, our findings show that the spacing between lysines is flexible and other residues contribute to the recognition marker.

Received for publication, June 1, 2005 , and in revised form, July 19, 2005.

^* This work was supported by National Institutes of Health Grant CA08759 (to S. K.) and National Institutes of Health Training Grant 5T32-HL07088–30 (to R. S.). 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.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 314-362-8803; Fax: 314-362-8826; E-mail: skornfel{at}im.wustl.edu.

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