Extensive Mannose Phosphorylation on Leukemia Inhibitory Factor (LIF) Controls Its Extracellular Levels by Multiple Mechanisms

Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2011; 286(28):24855-64. DOI: 10.1074/jbc.M111.221432
Source: PubMed


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.

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    • "LIF is also known as differentiation-stimulating factor (D-factor) (2), hepatocyte-stimulating factor III (HSF-III) (3), differentiation inhibitory activity (DIA) (4), human interleukin for DA cells (HILDA) (5, 6), melanomaderived lipoprotein lipase inhibitor (MLPLI) (7) and differentiation retarding factor (DRF) (8). Murine and human LIF are highly glycosylated and it has been shown that extensive mannose phosphorylation on LIF plays role in controlling extracellular levels of LIF (9). Its molecular weight varies between 32 to 62 kDa (10, 11), depending on the source. "
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    ABSTRACT: Leukemia inhibitory factor (LIF) plays important roles in cellular proliferation, growth promotion and differentiation of various types of target cells. In addition, LIF influences bone metabolism, cachexia, neural development, embryogenesis and inflammation. Human LIF (hLIF) is an essential growth factor for the maintenance of mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in a pluripotent, undifferentiated state. In this experimental study, we cloned hLIF into the pENTR-D/ TOPO entry vector by a TOPO reaction. Next, hLIF was subcloned into the pDEST17 destination vector by the LR reaction, which resulted in the production of a construct that was transferred into E. coli strain Rosetta-gami™ 2(DE3) pLacI competent cells to produce the His6-hLIF fusion protein. This straightforward method produced a biologically active recombinant hLIF protein in E. coli that has long-term storage ability. This procedure has provided rapid, cost effective purification of a soluble hLIF protein that is biologically active and functional as measured in mouse ESCs and iPSCs in vitro. Our results showed no significant differences in function between laboratory produced and commercialized hLIF.
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    • "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. "
    Tandem Mass Spectrometry - Applications and Principles, 02/2012; , ISBN: 978-953-51-0141-3
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    ABSTRACT: Carbohydrates play a central role in a wide range of biological processes. As with nucleic acids and proteins, modifications of specific sites within the glycan chain can modulate a carbohydrate's overall biological function. For example, acylation, methylation, sulfation, epimerization, and phosphorylation can occur at various positions within a carbohydrate to modulate bioactivity. Therefore, there is significant interest in identifying discrete carbohydrate modifications and understanding their biological effects. Additionally, enzymes that catalyze those modifications and proteins that bind modified glycans provide numerous targets for therapeutic intervention. This review will focus on modifications of glycans that occur after the oligomer/polymer has been assembled, generally referred to as post-glycosylational modifications.
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