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Extensive mannose phosphorylation on leukemia inhibitory factor (LIF) controls its extracellular levels by multiple mechanisms.

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.

SUBMITTER: Barnes J

PROVIDER: S-EPMC3137060 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Extensive mannose phosphorylation on leukemia inhibitory factor (LIF) controls its extracellular levels by multiple mechanisms.

Barnes Jarrod J Lim Jae-Min JM Godard Anne A Blanchard Frédéric F Wells Lance L Steet Richard R

The Journal of biological chemistry 20110525 28

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 ef ...[more]

PMID: 21613225

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Project description:Parkinson's disease (PD) is characterized neuropathologically by intracellular aggregates of fibrillar α-synuclein, termed Lewy bodies (LBs). Approximately 90% of α-synuclein deposited as LBs is phosphorylated at Ser129 in brains with PD. In contrast, only 4% of total α-synuclein is phosphorylated at Ser129 in brains with normal individuals. It is unclear why extensive phosphorylation occurs in the pathological process of PD. To address this issue, we investigated a mechanism and role of Ser129-phosphorylation in regulating accumulation of α-synuclein. In CHO cells, the levels of Ser129-phosphorylated soluble α-synuclein were maintained constantly to those of total α-synuclein in intracellular and extracellular spaces. In SH-SY5Y cells and rat primary cortical neurons, mitochondrial impairment by rotenone or MPP+ enhanced Ser129-phosphorylation through increased influx of extracellular Ca2+. This elevation was suppressively controlled by targeting Ser129-phosphorylated α-synuclein to the proteasome pathway. Rotenone-induced insoluble α-synuclein was also targeted by Ser129-phosphoryation to the proteasome pathway. Experiments with epoxomicin and chloroquine showed that proteasomal targeting of insoluble Ser129-phosphorylated α-synuclein was enhanced under lysosome inhibition and it reduced accumulation of insoluble total α-synuclein. However, in a rat AAV-mediated α-synuclein overexpression model, there was no difference in the number of total α-synuclein aggregates between A53T mutant and A53T plus S129A double mutant α-synuclein, although Ser129-phosphorylated α-synuclein-positive aggregates were increased in rats expressing A53T α-synuclein. These findings suggest that Ser129-phosphorylation occurs against stress conditions, which increases influx of extracellular Ca2+, and it prevents accumulation of insoluble α-synuclein by evoking proteasomal clearance complementary to lysosomal one. However, Ser129-phosphorylation may provide an ineffective signal for degradation-resistant aggregates, causing extensive phosphorylation in aggregates.

Dataset Information

Extensive mannose phosphorylation on leukemia inhibitory factor (LIF) controls its extracellular levels by multiple mechanisms.

Publications

Extensive mannose phosphorylation on leukemia inhibitory factor (LIF) controls its extracellular levels by multiple mechanisms.

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