A Novel Mutagenesis Strategy Identifies Distantly Spaced Amino Acid Sequences That Are Required for the Phosphorylation of Both the Oligosaccharides of Procathepsin D by N-Acetylglucosamine 1-Phosphotransferase

doi:10.1074/jbc.270.1.170

A Novel Mutagenesis Strategy Identifies Distantly Spaced Amino Acid Sequences That Are Required for the Phosphorylation of Both the Oligosaccharides of Procathepsin D by N-Acetylglucosamine 1-Phosphotransferase (*)

From the Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

↵^§ Fellow of the Janes Coffin Childs Fund. Present address: Dept. of Pathology, Jewish Hospital of St. Louis and Washington University School of Medicine, St. Louis, MO 63110.
↵^¶ Medical Scientist from the National Institute of General Medical Science. Current address: University of California, School of Medicine, San Francisco, CA.

** To whom correspondence should be addressed:
Dept. of Medicine, Washington University School of Medicine, Box 8125, 660 S. Euclid Ave., St. Louis, MO 63110
. Fax: 314-362-8826.

Abstract

A novel combinatorial mutagenesis strategy (shuffle mutagenesis) was developed to identify sequences in the propiece and amino lobe of cathepsin D which direct oligosaccharide phosphorylation by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. Propiece restriction fragments and oligonucleotide cassettes corresponding to 13 regions of the cathepsin D and glycopepsinogen amino lobes were randomly shuffled together to generate a large library of chimeric molecules. The library was inserted into an expression vector encoding the carboxyl lobe of cathepsin D with a carboxyl-terminal myc epitope and a CD8 transmembrane extension. Transfected COS1 cells expressing the membrane-anchored forms of the cathepsin D/glycopepsinogen chimeras at the cell surface were selected with solid phase mannose 6-phosphate receptor or an antibody to the myc epitope. Plasmids were rescued in Escherichia coli and sequenced by hybridization to the original oligonucleotide cassettes. Two regions of the cathepsin D amino lobe (segments 7 and 12) were found to contribute to proper folding, surface expression, and selective phosphorylation of the carboxyl lobe oligosaccharide. Two different cathepsin D regions (the propiece and segment 5) cooperated with a previously identified recognition element in the carboxyl lobe to allow efficient phosphorylation of both the amino and carboxyl lobe oligosaccharides.

Three general models for extending the catalytic reach of N-acetylglucosamine 1-phosphotransferase to widely spaced oligosaccharides are presented.

Footnotes

↵* This investigation was supported in part by United States Public Health Service Grant CA 08759 and by National Institutes of Health Research Award GM-07200. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

phosphotransferase

N-acetylglucosamine 1-phosphotransferase

CD-MCD8

membrane-anchored cathepsin D

IGF-II

insulin-like growth factor II

FITC

fluorescein isothiocyanate

mAb

monoclonal antibody

CP

chimeric protein

PBS

phosphate-buffered saline

Endo H

endo-β-N-acetylglucosaminidase H

ER

endoplasmic reticulum.
↵²G. Koelsch and M. Fusek, personal communication.
- Received October 7, 1994.
- Revision received October 27, 1994.