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.

Tandem Mass Spectrometry of Tagged and Permethylated Polysaccharides

“…In summary, tandem MS produces the greatest structural detail on permethylated glycans. Negative ion tandem MS is effective for producing useful structural information on native and tagged glycans, such as sialic acid containing, sulfated and phosphorylated glycan and those classes are commonly not compatible with permethylated glycans (Wheeler & Harvey, 2000;Larsen et al, 2006;Miller et al, 2006;Mechref et al, 2006;Lei et al, 2009;Yu et al, 2009;Barnes et al, 2011). For tagged glycans the mass shift varies according to the glycan's derivatives.…”

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

“…Cell lysates were prepared in detergent-containing buffer, and total protein concentration was determined using a BCA protein assay kit (Pierce). Medium from transfected CHO and HeLa cells and PMA-treated erythroleukemia cells was collected, concentrated using Centricon 10 spin columns (Amicon), and fractionated using an immobilized CI-MPR affinity column as described previously (11). Column fractions were precipitated with ethanol and resolved by SDS-PAGE.…”

“…CI-MPR Lectin Chromatography and Western Blot AnalysisHeLa and CHO cells were transfected as described previously (11). Cell lysates were prepared in detergent-containing buffer, and total protein concentration was determined using a BCA protein assay kit (Pierce).…”

“…Cell transfections were performed as described (11). In some cases, swainsonine (an inhibitor of Golgi-mannosidase II) was added (10 M) to the cultures after transient transfection to limit N-glycan processing and serve as an Endo H f control.…”

“…In light of the fact that many Man-6-P-modified nonlysosomal proteins are secreted glycoproteins, mannose phosphorylation is thought to facilitate their uptake from the extracellular space by cell surface Man-6-P receptors. Recent work has also demonstrated that the addition of phosphomannosyl residues on leukemia-inhibitory factor can limit its extracellular levels by another mechanism: direct sorting and disposal within lysosomes (11).…”