Abstract
In addition to soluble acid hydrolases, many nonlysosomal proteins have been shown to bear mannose 6-phosphate (Man-6-P) residues. Quantification of the extent of mannose phosphorylation and the relevance to physiological function, however, remain poorly defined. In this study, we investigated the mannose phosphorylation status of leukemia inhibitory factor (LIF), a previously identified high affinity ligand for the cation-independent mannose 6-phosphate receptor (CI-MPR), and we analyzed the effects of this modification on its secretion and uptake in cultured cells. When media from LIF-overexpressing cells were fractionated using a CI-MPR affinity column, 35-45% of the total LIF molecules were bound and specifically eluted with free Man-6-P thus confirming LIF as a bona fide Man-6-P-modified protein. Surprisingly, mass spectrometric analysis of LIF glycopeptides enriched on the CI-MPR column revealed that all six N-glycan sites could be Man-6-P-modified. The relative utilization of these sites, however, was not uniform. Analysis of glycan-deleted LIF mutants demonstrated that loss of glycans bearing the majority of Man-6-P residues leads to higher steady-state levels of secreted LIF. Using mouse embryonic stem cells, we showed that the mannose phosphorylation of LIF mediates its internalization thereby reducing extracellular levels and stimulating embryonic stem cell differentiation. Finally, immunofluorescence experiments indicate that LIF is targeted directly to lysosomes following its biosynthesis, providing another mechanism whereby mannose phosphorylation serves to control extracellular levels of LIF. Failure to modify LIF in the context of mucolipidosis II and its subsequent accumulation in the extracellular space may have important implications for disease pathogenesis.
Highlights
JULY 15, 2011 VOLUME 286 NUMBER 28 studied functions for protein-bound glycans
Overexpression in CHO and HeLa Cells Results in the Secretion of leukemia inhibitory factor (LIF) That Is Highly mannose 6-phosphate (Man-6-P)-modified—In an effort to quantify the relative amount of secreted LIF protein that contains mannose-phosphorylated glycans, we subjected purified human LIF and culture media from CHO cells stably expressing mouse LIF (CHO-mLIF) to affinity chromatography using an immobilized cation-independent mannose 6-phosphate receptor (CI-MPR) column
The extent of mannose phosphorylation of secreted LIF in CHO and HeLa cells is comparable with the level seen on overexpressed cathepsin D in these cell lines indicating that LIF is a good substrate for the GlcNAc-1-phosphotransferase enzyme [33]
Summary
JULY 15, 2011 VOLUME 286 NUMBER 28 studied functions for protein-bound glycans The biosynthesis of these residues on lysosomal hydrolases and other glycoproteins proceeds via a two-step enzymatic process. Our data further demonstrated that the mannose phosphorylation of LIF serves as a mechanism to control its extracellular levels through direct intracellular degradation of Man-6-P-modified LIF within the lysosome as well as rapid Man-6-P-mediated uptake following secretion. The implications of these novel findings in the pathogenesis of ML-II and relevance of mannose phosphorylation on other nonlysosomal proteins are discussed
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