Identification of the Minimal Lysosomal Enzyme Recognition Domain in Cathepsin D

Abstract

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.The enzyme UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (abbreviated "phosphotransferase") 3 has a central role in the targeting of newly synthesized lysosomal hydrolases to lysosomes. This enzyme recognizes a conformation-dependent protein determinant shared by the many different lysosomal hydrolases and then transfers N-acetylglucosamine 1-phosphate from UDP-GlcNAc to selected mannose residues of the high mannose oligosaccharides of the hydrolases. The N-acetylglucosamine residues are subsequently excised by "uncovering enzyme" generating mannose 6-phosphate monoesters that allow high affinity binding to mannose 6-phosphate receptors in the Golgi and translocation to lysosomes (1).The nature of the common protein recognition determinant has received considerable attention because of its role in this targeting pathway. Our laboratory undertook a molecular dissection of the phosphotransferase recognition marker using a pair of aspartyl proteases, the lysosomal hydrolase cathepsin D and the secretory protein pepsinogen (2, 3). These proteins are 45% identical in amino acid sequence and share similar secondary and tertiary structure but differ in that cathepsin D is highly phosphorylated by phosphotransferase, whereas glycopepsinogen, the glycosylated form of pepsinogen, is not. By analyzing numerous chimeric proteins, we were able to define a phosphotransferase recognition patch in the carboxyl lobe of cathepsin D formed by two noncontinuous primary sequences, specifically Lys-203 and amino acids 265-293 that form a loop structure directed toward Lys-203 (2). Lysine residues 267 and 293 at the base of the loop made significant contributions to the degree of phosphorylation, implicating lysines as important elements to the recognition patch. It was further observed tha...

Murine UDP-GlcNAc:Lysosomal Enzyme N-Acetylglucosamine-1-phosphotransferase Lacking the γ-Subunit Retains Substantial Activity toward Acid Hydrolases

“…The minimum number of substitutions needed to generate a recognition motif was two lysines at the correct positions (22). However, the binding signal was substantially enhanced by the substitution of additional residues.…”

“…In prior studies we have generated a GlcNAc-1 phosphotransferase protein recognition motif in the secretory protein pepsinogen by substituting elements from the related aspartic protease cathepsin D into equivalent positions of pepsinogen (22,26,35,40). The minimum number of substitutions needed to generate a recognition motif was two lysines at the correct positions (22).…”

“…Cathepsin D in the cells and media was immunoprecipitated with a polyclonal anti-cathepsin D antibody and analyzed by SDS-PAGE as previously described (22).…”

The DMAP interaction domain of UDP-GlcNAc:lysosomal enzyme N -acetylglucosamine-1-phosphotransferase is a substrate recognition module

“…A number of studies from our laboratory and others have shown that the protein recognition domain of acid hydrolases is a complex conformation-dependent determinant that includes a number of critical lysine residues (16)(17)(18)(19)(20)(21)(22)(23). On this basis, it seems likely that the interaction of an acid hydrolase with GlcNAc-1-phosphotransferase would involve multiple contacts between the surfaces of the two proteins.…”

Latency-associated Peptide of Transforming Growth Factor-β1 Is Not Subject to Physiological Mannose Phosphorylation

“…The determination of the percentage of latent TGF-␤1 glycans that are Man-6-Pmodified was performed as described earlier but without ammonium chloride in the culture medium (33)(34)(35). The percentage phosphorylation was calculated as the radioactivity (cpm) recovered in Endo H f -released glycans with one or two phosphates/total cpm in Endo H f -released glycans ϩ 2 Endo H f -resistant complex glycans.…”