Enzyme-independent, orientation-selective conjugation of whole human complement C3 to protein surfaces

Mitchell, Daniel A.; Ilyas, Rebecca; Dodds, Alister W.; Sim, Robert B.

doi:10.1016/j.jim.2008.05.011

Cited by 5 publications

(6 citation statements)

References 28 publications

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“…This mode of immobilization emulates more closely the natural orientation of C3b on surfaces (52). Figure 3 C shows representative equilibrium binding single-site saturation curves for rH19-20 wildtype as well as examples of a high-affinity (L1189R) and a low-affinity (R1215G) mutant binding to C3b.…”

Section: Resultssupporting

confidence: 53%

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The Binding of Factor H to a Complex of Physiological Polyanions and C3b on Cells Is Impaired in Atypical Hemolytic Uremic Syndrome

Ferreira

Herbert

Cortés

et al. 2009

The Journal of Immunology

159

215

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Factor H (fH) is essential for complement homeostasis in fluid-phase and on surfaces. Its two C-terminal domains (CCP 19-20) anchor fH to self surfaces where it prevents C3b amplification in a process requiring its N-terminal four domains. In atypical hemolytic uremic syndrome (aHUS), mutations clustering towards the C-terminus of fH may disrupt interactions with surface-associated C3b or polyanions and thereby diminish the ability of fH to regulate complement. To test this we compared a recombinant protein encompassing CCP 19-20 with sixteen mutants. The mutations had only very limited and localized effects on protein structure. While we found four aHUS-linked fH mutations that decreased binding to C3b and/or to heparin (a model compound for cell-surface polyanionic carbohydrates), we identified five aHUS-associated mutants with increased affinity for either or both ligands. Strikingly, these variable affinities for the individual ligands did not correlate with the extent to which all the aHUS-associated mutants were found to be impaired in a more physiological assay that measured their ability to inhibit cell surface complement functions of full-length fH. Taken together, our data suggest that disruption of a complex fH-self surface recognition process, involving a balance of affinities for protein and physiological carbohydrate ligands, predisposes to aHUS.

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Section: Resultssupporting

confidence: 53%

“…Human C3b (800-2000 response units (RU)) was amine coupled to a CM5 sensor chip (GE Healthcare). In addition, a streptavidin (SA) sensor chip (GE Healthcare) was coated with biotinylated C3b, generated as recently described (52). For each type of sensor chip, three flow cells (Fc2, Fc3 and Fc4) were independently coupled with C3b.…”

Section: Methodsmentioning

confidence: 99%

The Binding of Factor H to a Complex of Physiological Polyanions and C3b on Cells Is Impaired in Atypical Hemolytic Uremic Syndrome

Ferreira

Herbert

Cortés

et al. 2009

The Journal of Immunology

159

215

View full text Add to dashboard Cite

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“…Recently, an ingenious method has been used to label C3 efficiently with larger molecules, C3 is treated with methylamine, and as the thioester is aminolysed the cysteine side-chain becomes transiently available for derivatization. This promising approach has been used to conjugate C3 to harness its immunological properties (Mitchell et al 2008). We have previously demonstrated, that purified C3 thioester can be directly reacted via the thioester bond with much larger biomolecules than were used before.…”

Section: Introductionmentioning

confidence: 99%

Chemical labelling of active serum thioester proteins for quantification

Holm¹,

Ackland

Edwards

et al. 2012

Immunobiology

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The complement serum proteins C3 and C4 and the protease inhibitor α-2 macroglobulin are all members of the C3/α-2M thioester protein family, an evolutionarily ancient and conserved family that contains an intrachain thioester bond. The chemistry of the thioester bond is a key to the function of the thioester proteins. All these proteins function by covalently linking to their target by acyl transfer of the protein via the thioester moiety. We show that the signature thioester bond can be targeted with nucleophiles linked to a bioreporter molecule, site-specifically modifying the whole, intact thioester protein. Conditions were optimised to label selectively and efficiently pull-down unprocessed thioester-containing proteins from serum. We demonstrated pull-down of full-length C3, α-2M and C4 from sera in high salt, using a biotinylated nucleophile and streptavidin-coated resin, confirmed by MALDI-TOF MS identification of the gel bands. The potential for the development of a quantitative method for measuring active C3 in serum was investigated in patient sera pre and post operation. Quantifying active C3 in clinical assays using current methods is difficult. Methods based on antibody detection (e.g. nephelometry) do not distinguish between active C3 and inactive breakdown products. C3-specific haemolytic assays can be used, but these require use of relatively unstable reagents. The current work represents a promising robust, enzyme- and antibody-free chemical method for detecting active thioester proteins in blood, plasma or serum.

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“…The OPSS group readily forms disulfide bonds with available sulfhydryl groups 16 . Since activated C3 contains an exposed sulfhydryl group, we hypothesized that OPSS-liposomes would form a disulfide bond with C3, allowing for their uptake by MDSCs which express the receptor for activated C3 17 . Liposomes with the OPSS group had a diameter of 167 ± 92 nm which was not significantly different from control liposomes with no OPSS group (135 ± 55 nm) (Figure 1A).…”

Section: Resultsmentioning

confidence: 99%

Complement C3 mediated targeting of liposomes to granulocytic myeloid derived suppressor cells

Kullberg

Martinson

Mann

et al. 2015

Nanomedicine: Nanotechnology, Biology and Medicine

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In cancer patients, granulocytic myeloid derived suppressor cells (G-MDSCs) expand in number, infiltrating tumor and lymphatic tissues where they suppress an anti-tumor immune response. We report here the development of a liposomal drug delivery system that selectively targets GMDSCs. The liposomes form a disulfide bond with activated complement C3 after intravenous injection and are taken up by G-MDSCs, which express the receptor for activated C3. In vitro experiments utilizing serum from a C3 knockout mouse demonstrate that G-MDSCs take up these liposomes in a C3-dependent manner. After systemic administration to tumor bearing mice, liposomes were incorporated by 22% of G-MDSCs in the blood and were also present in a percentage of G-MDSCs in the tumor (11%), spleen (22%), liver (35%) and lungs (26%). This liposomal system offers a versatile means of targeted drug delivery to G-MDSCs and could be an important tool for restoring anti-tumor immunity in cancer patients.

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Enzyme-independent, orientation-selective conjugation of whole human complement C3 to protein surfaces

Cited by 5 publications

References 28 publications

The Binding of Factor H to a Complex of Physiological Polyanions and C3b on Cells Is Impaired in Atypical Hemolytic Uremic Syndrome

The Binding of Factor H to a Complex of Physiological Polyanions and C3b on Cells Is Impaired in Atypical Hemolytic Uremic Syndrome

Chemical labelling of active serum thioester proteins for quantification

Complement C3 mediated targeting of liposomes to granulocytic myeloid derived suppressor cells

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