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

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. 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. INTRODUCTIONA key step in the targeting of newly synthesized acid hydrolases to lysosomes is the recognition by the enzyme UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine 1-phosphotransferase (abbreviated phosphotransferase) ( 1The abbreviations used are: phosphotransferaseN-acetylglucosamine 1-phosphotransferaseCD-MCD8membrane-anchored cathepsin DIGF-IIinsulin-like growth factor IIFITCfluorescein isothiocyanatemAbmonoclonal antibodyCPchimeric proteinPBSphosphate-buffered salineEndo Hendo-β-N-acetylglucosaminidase HERendoplasmic reticulum.)of a protein determinant shared among at least 40 different lysosomal hydrolases(1.von Figura K. Hasilik A. Annu. Rev. Biochem. 1986; 55: 167-193Crossref PubMed Scopus (635) Google Scholar, 2.Kornfeld S. Mellman I. Annu. Rev. Cell Biol. 1989; 5: 483-525Crossref PubMed Scopus (1232) Google Scholar). This interaction results in the phosphorylation of mannose residues on the Asn-linked high mannose oligosaccharides of the lysosomal hydrolases. 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The high the of the targeting of the acid hydrolases to D with to generate a high and the of on with the on which is the of the amino lobe in a to generate a high targeting to INTRODUCTIONA key step in the targeting of newly synthesized acid hydrolases to lysosomes is the recognition by the enzyme UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine 1-phosphotransferase (abbreviated phosphotransferase) ( 1The abbreviations used are: phosphotransferaseN-acetylglucosamine 1-phosphotransferaseCD-MCD8membrane-anchored cathepsin DIGF-IIinsulin-like growth factor IIFITCfluorescein isothiocyanatemAbmonoclonal antibodyCPchimeric proteinPBSphosphate-buffered salineEndo Hendo-β-N-acetylglucosaminidase HERendoplasmic reticulum.)of a protein determinant shared among at least 40 different lysosomal hydrolases(1.von Figura K. Hasilik A. Annu. Rev. Biochem. 1986; 55: 167-193Crossref PubMed Scopus (635) Google Scholar, 2.Kornfeld S. Mellman I. Annu. Rev. Cell Biol. 1989; 5: 483-525Crossref PubMed Scopus (1232) Google Scholar). This interaction results in the phosphorylation of mannose residues on the Asn-linked high mannose oligosaccharides of the lysosomal hydrolases. These phosphomannosyl residues serve high for of the hydrolases to mannose 6-phosphate in the and to the S. Annu. Rev. Biochem. PubMed Scopus Google of the recognition on lysosomal hydrolases was in a of the lysosomal cathepsin D and the protein for chimeric were for to S. PubMed Scopus Google Scholar). cathepsin D and are in amino acid and and PubMed Scopus Google Scholar, PubMed Scopus Google in cathepsin D is by a of is These to the of a recognition in the carboxyl lobe of cathepsin D by sequences and amino residues were to recognition by into in was the of regions of the amino lobe of cathepsin D phosphorylation of the chimeric was of an carboxyl lobe recognition or a recognition was the of a recognition on lobe of a cathepsin D/glycopepsinogen chimeric is to allow phosphorylation of oligosaccharides on both the oligosaccharide on the lobe the recognition was the S. Biol. PubMed Google Scholar, S. Biol. PubMed Google of sequences in the amino lobe of cathepsin D which contribute to recognition was by chimeric shared restriction in the amino lobe to generate S. Biol. PubMed Google Scholar). a cathepsin D amino lobe were with the were to This regions are for of the recognition or are for proper the cathepsin D amino lobe contribute to an and the of the chimeric in original a of and to generate a large library of chimeric and the library by a of cathepsin D and by oligonucleotide cassettes were shuffled together a strategy high and The library was in and chimeric and were were selected and This the of of in regions of the amino lobe of cathepsin D which contribute to proper and surface expression and regions phosphorylation of the amino lobe oligosaccharide Three models are to for