Evaluation of human N-linked glycosylation sites in murine granulocyte-macrophage colony-stimulating factor

Altmann, Scott; Prystowsky, Michael B.

doi:10.1016/0003-9861(92)90405-l

Cited by 4 publications

(3 citation statements)

References 23 publications

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“…As demonstrated by earlier studies, other cytokines such as macrophage colony-stimulating factor also bear Man-6-P residues on their N-glycans (11). Thus, the mechanism we propose for LIF may help explain the relationship between glycosylation and activity that was observed by other investigators (60,61). Although the extent of mannose phosphorylation on secreted LIF was significant (30 -40%), these residues can be detected on proteins at much lower frequencies than what was observed for LIF.…”

Section: Discussionsupporting

confidence: 72%

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

Barnes

Lim

Godard

et al. 2011

Journal of Biological Chemistry

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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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Section: Discussionsupporting

confidence: 72%

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

Barnes

Lim

Godard

et al. 2011

Journal of Biological Chemistry

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“…Our results show that both anti-GMCSF and anti-IL9 antibodies recognized the same secreted protein at a molecular weight of ~50 kD (Figure 1B). The molecular weight spread of GIFT9 protein observed on SDS gel electrophoresis are likely due to the glycosylation of the fusion protein, as it has been previously reported that both GMCSF and IL9 can be glycosylated at multiple sites [22,23]. …”

Section: Resultsmentioning

confidence: 99%

A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity

Yuan

Galipeau

2013

PLoS ONE

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Cytokine receptors are randomly distributed on the cell surface membrane and are activated upon binding of their extracellular ligands to mediate downstream cellular activities. We hypothesized that pharmaceutical clustering of ligand-bound, activated receptors may lead to heretofore unrealized gain-of-function with therapeutically desirable properties. We here describe an engineered bifunctional cytokine borne of the fusion of Granulocyte Macrophage Colony Stimulating Factor (GMCSF) and Interleukin-9 (IL9) (hereafter GIFT9 fusokine) and demonstrate that it chaperones co-clustering of the functionally unrelated GMCSF receptor (GMCSFR) and IL9 receptor (IL9R) on cell surface of target cells. We demonstrate that GIFT9 treatment of MC/9 cells leads to transhyperphosphorylation of IL9R-associated STAT1 by GMCSFR-associated JAK2. We also show that IL9R-associated JAK1 and JAK3 augment phosphorylation of GMCSFR-linked STAT5. The functional relevance of these synergistic JAK/STAT transphosphorylation events translates to an increased mitogenic response by GMCSFR/IL9R-expressing primary marrow mast cells. The notion of inducing heterologous receptor clustering by engineered fusokines such as GIFT9 opens the door to a novel type of biopharmaceutical platform where designer fusokines modulate cell physiology through clustering of targeted receptor complexes.

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“…The size, number, branching and degree of sialylation of N-glycans can generate numerous glycoforms of a single polypeptide such as erythropoietin (629)(630)(631)(632)(633)(634)(635)(636)(637)(638)(639)(640) or GM-CSF (641)(642)(643)(644). It turns out that the nature of the glycosylation, extent of branching and level of sialylation modulate the activity of such cytokines over a range of function, by affecting its interaction with its cognate receptor, and also by altering the rate of clearance from the circulation.…”

Section: Tuning a Range Of Functionmentioning

confidence: 99%

Biological roles of glycans

2016

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Simple and complex carbohydrates (glycans) have long been known to play major metabolic, structural and physical roles in biological systems. Targeted microbial binding to host glycans has also been studied for decades. But such biological roles can only explain some of the remarkable complexity and organismal diversity of glycans in nature. Reviewing the subject about two decades ago, one could find very few clear-cut instances of glycan-recognition-specific biological roles of glycans that were of intrinsic value to the organism expressing them. In striking contrast there is now a profusion of examples, such that this updated review cannot be comprehensive. Instead, a historical overview is presented, broad principles outlined and a few examples cited, representing diverse types of roles, mediated by various glycan classes, in different evolutionary lineages. What remains unchanged is the fact that while all theories regarding biological roles of glycans are supported by compelling evidence, exceptions to each can be found. In retrospect, this is not surprising. Complex and diverse glycans appear to be ubiquitous to all cells in nature, and essential to all life forms. Thus, >3 billion years of evolution consistently generated organisms that use these molecules for many key biological roles, even while sometimes coopting them for minor functions. In this respect, glycans are no different from other major macromolecular building blocks of life (nucleic acids, proteins and lipids), simply more rapidly evolving and complex. It is time for the diverse functional roles of glycans to be fully incorporated into the mainstream of biological sciences.

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Evaluation of human N-linked glycosylation sites in murine granulocyte-macrophage colony-stimulating factor

Cited by 4 publications

References 23 publications

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

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

A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity

Biological roles of glycans

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