Mice Lacking Mannose 6-Phosphate Uncovering Enzyme Activity Have a Milder Phenotype than Mice Deficient forN-Acetylglucosamine-1-Phosphotransferase Activity

Abstract

The mannose 6-phosphate (Man-6-P) lysosomal targeting signal on acid hydrolases is synthesized by the sequential action of uridine 5-diphosphate-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) and GlcNAc-1-phosphodiester ␣-N-acetylglucosaminidase ("uncovering enzyme" or UCE). Mutations in the two genes that encode GlcNAc-1-phosphotransferase give rise to lysosomal storage diseases (mucolipidosis type II and III), whereas no pathological conditions have been associated with the loss of UCE activity. To analyze the consequences of UCE deficiency, the UCE gene was inactivated via insertional mutagenesis in mice. The UCE ؊/؊ mice were viable, grew normally and lacked detectable histologic abnormalities. However, the plasma levels of six acid hydrolases were elevated 1.6-to 5.4-fold over wild-type levels. These values underestimate the degree of hydrolase hypersecretion as these enzymes were rapidly cleared from the plasma by the mannose receptor. The secreted hydrolases contained GlcNAc-P-Man diesters, exhibited a decreased affinity for the cation-independent mannose 6-phosphate receptor and failed to bind to the cation-dependent mannose 6-phosphate receptor. These data demonstrate that UCE accounts for all the uncovering activity in the Golgi. We propose that in the absence of UCE, the weak binding of the acid hydrolases to the cation-independent mannose 6-phosphate receptor allows sufficient sorting to lysosomes to prevent the tissue abnormalities seen with GlcNAc-1-phosphotranferase deficiency.

Glycan Microarray Analysis of P-type Lectins Reveals Distinct Phosphomannose Glycan Recognition

“…It is interesting to speculate that the ability of the CI-MPR to bind Man-P-GlcNAc phosphodiesters may be important to overall efficiency of lysosomal enzyme targeting and uptake. This is consistent with recent studies in mice generated to lack the ␣-N-acetylglucosaminidase, where the phenotype is milder than seen for mice deficient in the GlcNAc phosphotransferase (47). Unlike the CD-MPR, a fraction of the CI-MPR is found in plasma membranes, where it can mediate the uptake of lysosomal enzymes into an early endocytic compartment, and after dissociation of ligands, the CI-MPR recycles through the endocytic recycling compartment back to the plasma membrane (3).…”

Structural basis for recognition of phosphodiester-containing lysosomal enzymes by the cation-independent mannose 6-phosphate receptor

“…The crystal structure of 7 (i.e., domains 1-3, domains [11][12][13][14] out of the 15 MRH domains of the CI-MPR and the single MRH domain of the CD-MPR have been determined to date, which shows that each of these MRH domains shares a similar fold (17)(18)(19). Our previous crystal structures of the CD-MPR (17) and domains 1-3 of the CI-MPR (18) bound to Man-6-P identified conserved residues that serve as a signature motif for Man-6-P binding: CD-MPR (Q66, R111, E133, and Y143) and domain 3 of CI-MPR (Q348, R391, E416, and Y421) bind the 2-, 3-, and 4-hydroxyl groups of the mannose ring in the same manner.…”

“…The importance of providing lysosomes with an entire repertoire of degradative enzymes for normal cellular processes is illustrated by the existence of over 35 different monogenic human lysosomal storage diseases that are caused by a deficiency of a catabolic enzyme (31) that together are estimated to affect 1 in 5,000 live births (32). The ability of the CI-MPR to recognize the product of GlcNAc-phosphotransferase ensures that deficiencies in the uncovering enzyme will not be lethal (11) and allows those acid hydrolases that may be poor substrates for the uncovering enzyme to be delivered in sufficient quantities to the lysosome to prevent the pathologies associated with lysosomal storage diseases. The CI-MPR, via its fifth MRH domain, is the only lectin known to recognize Man-P-GlcNAc, and in the current report we show the molecular basis for this interaction.…”

“…Furthermore, a recent genetic study has made the intriguing observation that mutations in GlcNAc-phosphotransferase and the uncovering enzyme are associated with a common speech disorder, stuttering (9). Studies using transgenic mice have shown that, unlike GlcNAcphosphotransferase-deficient mice (10), mice lacking the uncovering enzyme, which results in their acid hydrolases bearing phosphodiesters (Man-P-GlcNAc), grew normally and exhibit no detectable histologic abnormalities (11). These studies also showed that secreted hydrolases from these animals bound to the CI-MPR but not the CD-MPR (11), a finding consistent with previous reports which demonstrated that the CI-MPR, but not the CD-MPR, binds phosphodiester-containing glycans (12,13).…”

Cathepsin D and its newly identified transport receptor SEZ6L2 can modulate neurite outgrowth

“…The protein of interest was immunoprecipitated and resolved by SDS-PAGE as described previously (Boonen et al, 2009). Signals were detected with a Cyclone™ Phosphor Imager, quantified with the OptiQuant™ software and the results were analyzed for statistical relevance using non-paired, two-tailed Student's t-tests.…”