Acid phosphatase 5 is responsible for removing the mannose 6-phosphate recognition marker from lysosomal proteins

Abstract

Most newly synthesized proteins destined for the lysosome reach this location via a specific intracellular pathway. In the Golgi, a phosphotransferase specifically labels lysosomal proteins with mannose 6-phosphate (Man-6-P). This modification is recognized by receptors that target the lysosomal proteins to the lysosome where, in most cell types, the Man-6-P recognition marker is rapidly removed. Despite extensive characterization of this pathway, the enzyme responsible for the removal of the targeting modification has remained elusive. In this study, we have identified this activity. Preliminary investigations using a cell-based bioassay were used to follow a dephosphorylation activity that was associated with the lysosomal fraction. This activity was high in the liver, where endogenous lysosomal proteins are efficiently dephosphorylated, but present at a much lower level in the brain, where the modification persists. This observation, combined with an analysis of the expression of lysosomal proteins in different tissues, led us to identify acid phosphatase 5 (ACP5) as a candidate for the enzyme that removes Man-6-P. Expression of ACP5 in N1E-115 neuroblastoma cells, which do not efficiently dephosphorylate lysosomal proteins, significantly decreased the steady state levels of Man6-P glycoproteins. Analysis of ACP5-deficient mice revealed that levels of Man-6-P glycoproteins were highly elevated in tissues that normally express ACP5, and this resulted from a failure to dephosphorylate lysosomal proteins. These results indicate a central role for ACP5 in removal of the Man-6-P recognition marker and open up new avenues to investigate the importance of this process in cell biology and medicine.

Fig. 1.

Expression of recombinant ACP5 in mouse neuroblastoma cells. N1E-115 cells were cotransfected with plasmids expressing ACP2 or ACP5 and GFP as a marker for transfected cells (also see arrows, Right). Images were obtained at 40× magnification.

Fig. 2.

Levels of Man-6-P glycoproteins in tissues from ACP5-deficient mice. Twenty microgram protein equivalents of each tissue extract were fractionated by SDS/PAGE, transferred to nitrocellulose, and probed with 2 nM ¹²⁵I-CI-MPR to specifically detect glycoproteins containing Man-6-P. Tissue extracts were prepared from Acp5^−/− and Acp5^+/+ 129SvEv mice.

Fig. 3.

Lysosomal enzyme activities in ACP5-deficient mice. Activities of 12 lysosomal enzymes were measured in brain and liver from Acp5^+/+ and Acp5^−/− mice. Results were obtained from 2 animals of each genotype and expressed as activity in Acp5 mutant tissue normalized to wild-type control tissue with the line at y = 1 representing control activity. Mean activities are shown with error bars representing the range.

Fig. 4.

Phosphorylation state of lysosomal proteins in control and ACP5-deficient mouse brain and liver. Tissue homogenates from Acp5^+/+ (open symbols) and Acp5^−/− (filled symbols) mice were fractionated by affinity chromatography on immobilized sCI-MPR (9). Enzyme activities attributable to the Man-6-P glycoforms were calculated as the activity in the Man-6-P eluate/total recovered activity (flow through/wash+glucose-6 phosphate eluate+Man-6-P eluate). Bars represent mean of measurements from 2 animals of each genotype and error bars represent the range.

Fig. 5.

Localization of Man-6-P glycoproteins in ACP5-deficient mice. TPPI and Man-6-P glycoproteins were detected in the brains (A) and livers (B) of wild-type and mutant mice. In the merged images, the signals from GFP and Man-6-P proteins are in green and red, respectively, with yellow indicating colocalization. Images were obtained at 63× magnification.