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Synthesis of granulocyte-macrophage colony-stimulating factor as homogeneous glycoforms and early comparisons with yeast cell-derived material.

ABSTRACT: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a medicinally important glycoprotein, used as an immunostimulant following bone-marrow transplant. On the basis of reports of its potential utility as an anticancer vaccine adjuvant, we undertook to develop a synthetic route toward single-glycoform GM-CSF. We describe herein a convergent total synthesis of GM-CSF aglycone and two homogeneous glycoforms. Analytical and biological studies confirm the structure and activity of these synthetic congeners.

SUBMITTER: Zhang Q

PROVIDER: S-EPMC3939888 | biostudies-other | 2014 Feb

REPOSITORIES: biostudies-other

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Synthesis of granulocyte-macrophage colony-stimulating factor as homogeneous glycoforms and early comparisons with yeast cell-derived material.

Zhang Qiang Q Johnston Eric V EV Shieh Jae-Hung JH Moore Malcolm A S MA Danishefsky Samuel J SJ

Proceedings of the National Academy of Sciences of the United States of America 20140210 8

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a medicinally important glycoprotein, used as an immunostimulant following bone-marrow transplant. On the basis of reports of its potential utility as an anticancer vaccine adjuvant, we undertook to develop a synthetic route toward single-glycoform GM-CSF. We describe herein a convergent total synthesis of GM-CSF aglycone and two homogeneous glycoforms. Analytical and biological studies confirm the structure and activity of these synth ...[more]

PMID: 24516138

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Semisynthesis of Homogeneous, Active Granulocyte Colony-Stimulating Factor Glycoforms.

Project description:Granulocyte colony stimulating factor (G-CSF) is a cytokine used to treat neutropenia. Different glycosylated and non-glycosylated variants of G-CSF for therapeutic application are currently generated by recombinant expression. Here, we describe our approaches to establish a first semisynthesis strategy to access the aglycone and O-glycoforms of G-CSF, thereby enabling the preparation of selectively and homogeneously post-translationally modified variants of this important cytokine. Eventually, we succeeded by combining selenocysteine ligation of a recombinantly produced N-terminal segment with a synthetic C-terminal part, transiently equipped with a side-chain-linked, photocleavable PEG moiety, at low concentration. The transient PEGylation enabled quantitative enzymatic elongation of the carbohydrate at Thr133. Overall, we were able to significantly reduce the problems related to the low solubility and the tendency to aggregate of the two protein segments, which allowed the preparation of four G-CSF variants that were successfully folded and demonstrated biological activity in cell proliferation assays.

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Patient-derived granulocyte/macrophage colony-stimulating factor autoantibodies reproduce pulmonary alveolar proteinosis in nonhuman primates.

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Granulocyte and granulocyte-macrophage colony stimulating factors for newly diagnosed patients with myelodysplastic syndromes.

Project description: Not available

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Tumour-derived CSF2/granulocyte macrophage colony stimulating factor controls myeloid cell accumulation and progression of gliomas.

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| S-EPMC7403321 | biostudies-literature

Granulocyte-macrophage colony-stimulating factor neuroprotective activities in Alzheimer's disease mice.

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| S-EPMC5916331 | biostudies-literature

Granulocyte-Macrophage Colony Stimulating Factor Receptor Contributes to Plexiform Neurofibroma Initiation.

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Transcriptional mechanisms underlying sensitization of peripheral sensory neurons by granulocyte-/granulocyte-macrophage colony stimulating factors.

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Characterization of human and mouse granulocyte-macrophage-colony-stimulating factors derived from Escherichia coli.

Project description:Human and mouse granulocyte-macrophage-colony-stimulating factors (hGM-CSF and mGM-CSF, respectively), isolated from Escherichia coli cells expressing the corresponding human and mouse genes, have been characterized. The observed properties of the proteins have been compared with those properties which can be deduced from the DNA sequence alone and the published properties of natural GM-CSFs. The purified E. coli-derived proteins were found to have the expected molecular masses, amino acid compositions and N- and C-terminal amino acid sequences. The finding of 70-90% unprocessed N-terminal methionine for both proteins is discussed. The four Cys residues were found to be involved in two intramolecular disulphide bonds, linking the first and third, and second and fourth Cys residues. This disulphide bond arrangement is probably the one existing in natural material, since, although not glycosylated, both E. coli-derived proteins showed biological activity (colony stimulating assay for hGM-CSF, and cell proliferation assay for mGM-CSF) comparable with that reported for the respective proteins purified from animal cells.

| S-EPMC1148387 | biostudies-other

Proteomic Analysis Reveals Distinct Metabolic Differences Between Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Macrophage Colony Stimulating Factor (M-CSF) Grown Macrophages Derived from Murine Bone Marrow Cells.

Project description:Macrophages are crucial in controlling infectious agents and tissue homeostasis. Macrophages require a wide range of functional capabilities in order to fulfill distinct roles in our body, one being rapid and robust immune responses. To gain insight into macrophage plasticity and the key regulatory protein networks governing their specific functions, we performed quantitative analyses of the proteome and phosphoproteome of murine primary GM-CSF and M-CSF grown bone marrow derived macrophages (GM-BMMs and M-BMMs, respectively) using the latest isobaric tag based tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Strikingly, metabolic processes emerged as a major difference between these macrophages. Specifically, GM-BMMs show significant enrichment of proteins involving glycolysis, the mevalonate pathway, and nitrogen compound biosynthesis. This evidence of enhanced glycolytic capability in GM-BMMs is particularly significant regarding their pro-inflammatory responses, because increased production of cytokines upon LPS stimulation in GM-BMMs depends on their acute glycolytic capacity. In contrast, M-BMMs up-regulate proteins involved in endocytosis, which correlates with a tendency toward homeostatic functions such as scavenging cellular debris. Together, our data describes a proteomic network that underlies the pro-inflammatory actions of GM-BMMs as well as the homeostatic functions of M-BMMs.

| S-EPMC4597147 | biostudies-literature

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