Study on the Lysosomal Enzyme Selection System.

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In many cell types, newly synthesized acid hydrolases acquire mannose 6-phosphate residues which serve as high affinity ligands for binding to Man-6-P receptors and targeting to lysosomes. A key step in this pathway is the ability of the enzyme UDP-G1cNAc: Lysosomal enzyme acetylglucosaminyl phosphotransferase to selective recognize and phosphorylate acid hydrolases. We have previously demonstrated that phosphotransferase recognizes conformational dependent protein determinants present on the acid hydrolases and absent in most nonlysosomal glycoproteins. It was recently reported that the secretory glycoprotein DNase I contains Man-6-P residues and we found that the human, bovine and mouse forms of this enzyme are phosphorylated to a low extent when expressed in COS cells. The poor phosphorylation of human and bovine DNase I is due to the lack of two critical lysines (position 27 and 74) present in the mouse enzyme whereas the poor phosphorylation of mouse DNase I appears to be due to the presence of "inhibitory" amino acid (Val23) that block the phosphotransferase recognition domain and the disappear of two critical amino acids (Tyr54 and Ser190). Then, we also report the substrate specificity of GPT for the oligosaccharides using variety of fluorescence labeled high mannose type oligosaccharides. As a point to be noticed, GPT can transfer the G1cNAc-phosphate to the G1cMan9GlcNAc2 that still remains glucose.
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