Pichia pastoris-produced mucin-type fusion proteins with multivalent O-glycan substitution as targeting molecules for mannose-specific receptors of the immune system

Gustafsson, Anki; Sjöblom, Magnus; Strindelius, Lena; Johansson, Thomas; Fleckenstein, Tilly; Chatzissavidou, Nathalie; Lindberg, Lene; Ångström, Jonas; Holgersson, Jan

doi:10.1093/glycob/cwr046

Cited by 24 publications

(32 citation statements)

References 47 publications

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“…Pooled fractions were then dialyzed as described above, frozen, lyophilized, and stored at Ϫ80°C. The concentrations of recombinant fusion protein in purified fractions were determined by a two-antibody sandwich enzyme-linked immunosorbent assay (ELISA) method as described previously (30).…”

Section: Methodsmentioning

confidence: 99%

Recombinant Mucin-Type Fusion Proteins with a Galα1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors

et al. 2016

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The capability of a recombinant mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying Gal␣1,3Gal␤1,4GlcNAc determinants to bind and inhibit Clostridium difficile toxin A (TcdA) was investigated. The fusion protein, produced by a glyco-engineered stable CHO-K1 cell line and designated C-PGC2, was purified by affinity and gel filtration chromatography from large-scale cultures. Liquid chromatography-mass spectrometry was used to characterize Oglycans released by reductive ␤-elimination, and new diagnostic ions to distinguish Gal␣1,3Gal-from Gal␣1,4Gal-terminated O-glycans were identified. The C-PGC2 cell line, which was 20-fold more sensitive to TcdA than the wild-type CHO-K1, is proposed as a novel cell-based model for TcdA cytotoxicity and neutralization assays. The C-PGC2-produced fusion protein could competitively inhibit TcdA binding to rabbit erythrocytes, making it a high-efficiency inhibitor of the hemagglutination property of TcdA. The fusion protein also exhibited a moderate capability for neutralization of TcdA cytotoxicity in both C-PGC2 and CHO-K1 cells, the former with and the latter without cell surface Gal␣1,3Gal␤1,4GlcNAc sequences. Future studies in animal models of C. difficile infection will reveal its TcdA-inhibitory effect and therapeutic potential in C. difficile-associated diseases.C lostridium difficile is an opportunistic Gram-positive pathogenic bacterium responsible for antibiotic-associated diarrhea and other gastrointestinal diseases. C. difficile infections, collectively known as C. difficile-associated disease (CDAD), range from mild cases of diarrhea to fatal pseudomembranous colitis (1, 2). Various treatment options for CDAD include antibiotics, fecal transplantation therapy, and, potentially, toxin-specific antibodies, vaccines, and replenishment of the patient microflora with oral probiotic therapy (3, 4). However, the emergence of strains with reduced susceptibility to antibiotics such as metronidazole and vancomycin and high rates of recurrent infection have limited the treatment options, necessitating more effective therapeutics that target the pathogenic mechanism of C. difficile (5-7).Toxin A (TcdA) and toxin B (TcdB), with molecular masses of 308 kDa and 270 kDa, respectively, are the two primary virulence factors secreted by C. difficile, and can inactivate the Rho/Ras superfamily of GTPases by glucosylation (8-10). As Rho GTPases regulate a number of vital cellular processes, their functional inactivation results in the inhibition of cell migration, morphogenesis, division, and membrane trafficking. Glucosylation of Rho family GTPases causes breakdown of the actin cytoskeleton and activation of caspase-3, leading to apoptosis of intoxicated cells. Diarrhea and inflammation ensue, and ultimately, the control of intestinal epithelial barrier function is lost (11-15). Much of the pathology seen during a C. difficile infection is believed to be due to toxin effects. TcdA exhibits many pathobiological functions, such as cell roundin...

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Section: Methodsmentioning

confidence: 99%

Recombinant Mucin-Type Fusion Proteins with a Galα1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors

et al. 2016

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“…Analyses were performed on a BIAcore® T200 instrument (Biacore, GE Healthcare) as previously described (17). Recombinant macrophage mannose receptor was immobilized on a CM5 chip using standard amine coupling to maximum~12,000 RU on BIAcore T200.…”

Section: Mannose Receptor Binding Studies Using Surface Plasmon Resonmentioning

confidence: 99%

“…Instrument running buffer is the same as described above. RNAse B is a high mannose containing protein (18) that served as a positive control for MR binding, much like mannan or mannosylated IgG have been used in a previous study (17). On-rate, offrate, and equilibrium dissociation constants were calculated using the 1:1 binding model on BIAevaluation® software (Biacore, GE Healthcare).…”

Section: Mannose Receptor Binding Studies Using Surface Plasmon Resonmentioning

confidence: 99%

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Impact of Glycation on Antibody Clearance

Yang

Primack²,

Frohn

et al. 2014

AAPS J

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Abstract. Glycation of therapeutic proteins occurs during mammalian cell culture expression and upon administration to patients. Since the chemical attachment of mannose or other sugars via a chemical linker has been shown to increase a protein's clearance rate in mice through the mannose receptor, we explored the effect of mannose glycation on the clearance of an IgG in mice. An IgG decorated with high levels of mannose (~18 mol/mol protein) through glycation did not clear faster in mice than the underivatized protein, whereas the same IgG decorated with mannose attached in a way to maintain the normal glycosidic bond (2-imino-2-methoxyethyl-1-thiomannoside, or IMT-mannose) at similar derivatization levels cleared significantly faster. Surface plasmon resonance studies revealed that the IgG derivatized with IMT-mannose bound tightly to the mannose receptor (K D =20 nM) but the IgG glycated with mannose did not bind. These results indicate that glycation, even at unnaturally elevated levels, does not appear to be a clearance concern for therapeutic proteins.

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“…[293]. In general, the pH of the immobilisation buffer should be low enough to ensure electrostatic adsorption of the ligand (here the MR) onto the chip's surface is achieved [294].…”

Section: Methods Developmentmentioning

confidence: 99%

Mannosylated lipopetides as model vaccines for targeting the mannose receptor

Sedaghat¹

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The mannose receptor (MR) is an important component of the immune system and understanding the structural and conformational characteristics of this receptor is a key aspect of targeted vaccine design. Improved understanding of the role of carbohydrate recognition domains (CRDs) 4-7 in recognising glycosylated ligands present on the surface of pathogens such as C. albicans, P. carinii, L. donovani, and M. tuberculosis has given new insight into MR vaccine development. Initial studies identified mannan and its derivatives to be important ligands in MR targeting, providing essential knowledge about structural properties of the MR. During the last decade many attempts have been made to target this receptor for applications including vaccine and drug development.In the present study, a library of vaccine constructs comprising fluorescently-labelled mannosylated lipid dendrimers that contained the ovalbumin CD4 + epitope, OVA323-339, as the model peptide antigen were synthesised using fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS). The vaccine constructs were designed with an alanine spacer between the O-linked mannose moieties to investigate the impact of distance between the mannose units on receptor-mediated uptake and/or binding in antigen presenting cells.Mannosylated building blocks were prepared with a Lewis acid reaction and the reaction was successfully modified and monitored for a better yield. This was followed by solid phase synthesis of mannosylated peptides with an azide functional group and a fluorescent tag. Considering the presence of multi-components on the designed mannosylated peptide, different methods of preparation was used and a high yield of the final mannosylated peptides with an azide functional group and a fluorescent tag were prepared. Further, OVA323-339 lipopeptides with an alkyne functional group were prepared using microwave assisted peptide synthesis. Final vaccine structures were prepared by attaching azide and alkyne functional groups using click chemistry. The same methods were applied for the preparation of a library of control vaccine structures.In vitro uptake studies performed on macrophage (F4/80 + ) and dendritic (CD11c + ) cells showed significant uptake and/or binding for vaccine constructs containing mannose and lipid, and also for the lipopeptide vaccine construct without mannose when compared to the controls (containing no lipid, no mannose and no mannose or lipid). Further, in vitro mannan competition assays demonstrated that uptake of the mannosylated and lipidated vaccine constructs was MR mediated. To address the specificity of receptor uptake, surface plasmon resonance (SPR) was performed usingBiacore technology which confirmed a high affinity of the mannosylated and lipidated vaccine constructs towards the human recombinant MR in vitro when compared with vaccine constructs without mannose. These studies also confirmed that both mannose and lipid moieties play significant II roles in receptor-mediated uptake on APCs, potentially faci...

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Pichia pastoris-produced mucin-type fusion proteins with multivalent O-glycan substitution as targeting molecules for mannose-specific receptors of the immune system

Cited by 24 publications

References 47 publications

Recombinant Mucin-Type Fusion Proteins with a Galα1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors

Recombinant Mucin-Type Fusion Proteins with a Galα1,3Gal Substitution as Clostridium difficile Toxin A Inhibitors

Impact of Glycation on Antibody Clearance

Mannosylated lipopetides as model vaccines for targeting the mannose receptor

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