Henning Stöckmann

The "sweet" side of the protein corona: effects of glycosylation on nanoparticle-cell interactions.

The significance of a protein corona on nanoparticles in modulating particle properties and their biological interactions has been widely acknowledged. The protein corona is derived from proteins in biological fluids, many of which are glycosylated. To date, the glycans on the proteins have been largely overlooked in studies of nanoparticle-cell interactions. In this study, we demonstrate that glycosylation of the protein corona plays an important role in maintaining the colloidal stability of nanoparticles and influences nanoparticle-cell interactions. The removal of glycans from the protein corona enhances cell membrane adhesion and cell uptake of nanoparticles in comparison with the fully glycosylated form, resulting in the generation of a pro-inflammatory milieu by macrophages. This study highlights that the post-translational modification of proteins can significantly impact nanoparticle-cell interactions by modulating the protein corona properties.

Automated, High-Throughput IgG-Antibody Glycoprofiling Platform

One of todays key challenges is the ability to decode the functions of complex carbohydrates in various biological contexts. To generate high-quality glycomics data in a high-throughput fashion, we developed a robotized and low-cost N-glycan analysis platform for glycoprofiling of immunoglobulin G antibodies (IgG), which are central players of the immune system and of vital importance in the biopharmaceutical industry. The key features include (a) rapid IgG affinity purification and sample concentration, (b) protein denaturation and glycan release on a multiwell filtration device, (c) glycan purification on solid-supported hydrazide, and (d) glycan quantification by ultra performance liquid chromatography. The sample preparation workflow was automated using a robotic liquid-handling workstation, allowing the preparation of 96 samples (or multiples thereof) in 22 h with excellent reproducibility and, thus, should greatly facilitate biomarker discovery and glycosylation monitoring of therapeutic IgGs.

Imaging sialylated tumor cell glycans in vivo

Cell surface glycans are involved in numerous physiological processes that involve cell‐cell interactions and migration, including lymphocyte trafficking and cancer metastasis. We have used a bioorthogonal metabolic labeling strategy to detect cell surface glycans and demonstrate, for the first time, fluorescence and radionuclide imaging of sialylated glycans in a murine tumor model in vivo. Peracetylated azido‐labeled N‐acetyl‐man‐nosamine, injected intraperitoneally, was used as the metabolic precursor for the biosynthesis of 5‐azidoneuraminic, or azidosialic acid. Azidosialic acid‐labeled cell surface glycans were then reacted, by Staudinger ligation, with a biotinylated phosphine injected intraperitoneally, and the biotin was detected by subsequent intravenous injection of a fluorescent or radiolabeled avidin derivative. At 24 h after administration of NeutrAvidin, labeled with either a far‐red fluorophore or 111In, there was a significant azido‐labeled N‐acetyl‐mannosamine‐dependent increase in tumor‐to‐tissue contrast, which was detected using optical imaging or single‐photon‐emission computed tomography (SPECT), respectively. The technique has the potential to translate to the clinic, where, given the prognostic relevance of altered sialic acid expression in cancer, it could be used to monitor disease progression.—Neves, A. A., Stöckmann, H., Harmston, R. R., Pryor, H. J., Alam, I. S., Ireland‐Zecchini, H., Lewis, D. Y., Lyons, S. K., Leeper, F. J., Brindle, K. M. Imaging sialylated tumor cell glycans in vivo. FASEB J. 25, 2528–2537 (2011). www.fasebj.org

Imaging cell surface glycosylation in vivo using "double click" chemistry.

Dynamic alterations in cell surface glycosylation occur in numerous biological processes that involve cell–cell communication and cell migration. We report here imaging of cell surface glycosylation in live mice using double click chemistry. Cell surface glycans were metabolically labeled using peracetylated azido-labeled N-acetylgalactosamine and then reacted, in the first click reaction, with either a cyclooctyne, in a Huisgen [3 + 2] cycloaddition, or with a Staudinger phosphine, via Staudinger ligation. The second click reaction was a [4 + 2] inverse electron demand Diels–Alder reaction between a trans-cyclooctene and a tetrazine, where the latter reagent had been fluorescently labeled with a far-red fluorophore. After administration of the fluorescent tetrazine, the bifunctional cyclooctyne-cyclooctene produced significant azido sugar-dependent fluorescence labeling of tumor, kidney, liver, spleen, and small intestine in vivo, where the kidney and tumor could be imaged noninvasively in the live mouse.

Development and evaluation of new cyclooctynes for cell surface glycan imaging in cancer cells

Two reagents have been synthesized for selective labeling of cell surface azidoglycans, an unusually stable version of a dibenzocyclooctyne (TMDIBO) and a third-generation difluorinated cyclooctyne (DIFO3). Both syntheses are efficient with minimal purification, and the dibenzocyclooctyne is stable under basic and acidic conditions. Flow cytometric measurements with azidosugar labeled cancer cells, in which these reagents were linked to the fluorophore Alexa Fluor 647, gave a signal-to-background ratio of up to 35 with TMDIBO as compared to ≈10 for DIFO3 and ≈5 for a phosphine reagent. TMDIBO-based probes should have applications in molecular imaging of cell surface glycans in vivo.

Bacterial Biosynthetic Gene Clusters Encoding the Anti-cancer Haterumalide Class of Molecules BIOGENESIS OF THE BROAD SPECTRUM ANTIFUNGAL AND ANTI-OOMYCETE COMPOUND, OOCYDIN A

Background: Oocydin A is an anticancer haterumalide with strong antimicrobial activity against agriculturally important plant pathogenic fungi and oomycetes. Results: The oocydin A gene cluster has been identified and characterized in four plant-associated enterobacteria. Conclusion: The ooc gene cluster is organized in three transcriptional units encoding enzymes that belong to a growing class of trans-acyltransferase polyketide synthases. Significance: Oocydin A has potential agricultural, pharmacological, and chemotherapeutic applications. Haterumalides are halogenated macrolides with strong antitumor properties, making them attractive targets for chemical synthesis. Unfortunately, current synthetic routes to these molecules are inefficient. The potent haterumalide, oocydin A, was previously identified from two plant-associated bacteria through its high bioactivity against plant pathogenic fungi and oomycetes. In this study, we describe oocydin A (ooc) biosynthetic gene clusters identified by genome sequencing, comparative genomics, and chemical analysis in four plant-associated enterobacteria of the Serratia and Dickeya genera. Disruption of the ooc gene cluster abolished oocydin A production and bioactivity against fungi and oomycetes. The ooc gene clusters span between 77 and 80 kb and encode five multimodular polyketide synthase (PKS) proteins, a hydroxymethylglutaryl-CoA synthase cassette and three flavin-dependent tailoring enzymes. The presence of two free-standing acyltransferase proteins classifies the oocydin A gene cluster within the growing family of trans-AT PKSs. The amino acid sequences and organization of the PKS domains are consistent with the chemical predictions and functional peculiarities associated with trans-acyltransferase PKS. Based on extensive in silico analysis of the gene cluster, we propose a biosynthetic model for the production of oocydin A and, by extension, for other members of the haterumalide family of halogenated macrolides exhibiting anti-cancer, anti-fungal, and other interesting biological properties.

Metabolic Glycan Imaging by Isonitrile–Tetrazine Click Chemistry

Seeing the sugar coating: N-Acetyl-glucosamine and mannosamine derivatives tagged with an isonitrile group are metabolically incorporated into cell-surface glycans and can be detected with a fluorescent tetrazine. This bioorthogonal isonitrile-tetrazine ligation is also orthogonal to the commonly used azide-cyclooctyne ligation, and so will allow simultaneous detection of the incorporation of two different sugars.

Ultrahigh Throughput, Ultrafiltration-Based N-Glycomics Platform for Ultraperformance Liquid Chromatography (ULTRA3)

Accurate, reproducible, and fast quantification of N-glycans is crucial not only for the development and quality control of modern glycosylated biopharmaceuticals, but also in clinical biomarker discovery. Several methods exist for fluorescent labeling of N-glycans and subsequent chromatographic separation and quantification. However, the methods can be complex, lengthy, and expensive. Here we report an automated ultrafiltration-based N-glycoanalytical workflow combined with a glycan labeling strategy that is based on the reaction of glycosylamines with fluorescent carbamate. The entire protocol is quick, simple, and cost-effective and requires less than 1 μg of protein per sample. As many as 768 affinity purified IgG glycoprotein samples can be prepared in a single run with a liquid handling platform.

N-Glycan Abnormalities in Children with Galactosemia

Galactose intoxication and over-restriction in galactosemia may affect glycosylation pathways and cause multisystem effects. In this study, we describe an applied hydrophilic interaction chromatography ultra-performance liquid chromatography high-throughput method to analyze whole serum and extracted IgG N-glycans with measurement of agalactosylated (G0), monogalactosylated (G1), and digalactosylated (G2) structures as a quantitative measure of galactose incorporation. This was applied to nine children with severe galactosemia (genotype Q188R/Q188R) and one child with a milder variant (genotype S135L/S135L). The profiles were also compared with those obtained from three age-matched children with PMM2-CDG (congenital disorder of glycosylation type Ia) and nine pediatric control samples. We have observed that severe N-glycan assembly defects correct in the neonate following dietary restriction of galactose. However, treated adult galactosemia patients continue to exhibit ongoing N-glycan processing defects. We have now applied informative galactose incorporation ratios as a method of studying the presence of N-glycan processing defects in children with galactosemia. We identified N-glycan processing defects present in galactosemia children from an early age. For G0/G1, G0/G2, and (G0/G1)/G2 ratios, the difference noted between galactosemia patients and controls was found to be statistically significant (p = 0.002, 0.01, and 0.006, respectively).

Residual ligand entropy in the binding of p-substituted benzenesulfonamide ligands to bovine carbonic anhydrase II.

In studies on the thermodynamics of ligand-protein interactions, it is often assumed that the configurational and conformational entropy of the ligand is zero in the bound state (i.e., the ligand is rigidly fixed in the binding pocket). However, there is little direct experimental evidence for this assumption, and in the case of binding of p-substituted benzenesulfonamide inhibitors to bovine carbonic anhydrase II (BCA II), the observed thermodynamic binding signature derived from isothermal titration calorimetry experiments leads indirectly to the conclusion that a considerable degree of residual entropy remains in the bound ligand. Specifically, the entropy of binding increases with glycine chain length n, and strong evidence exists that this thermodynamic signature is not driven by solvent reorganization. By use of heteronuclear (15)N NMR relaxation measurements in a series (n = 1-6) of (15)N-glycine-enriched ligands, we find that the observed thermodynamic binding signature cannot be explained by residual ligand dynamics in the bound state, but rather results from the indirect influence of ligand chain length on protein dynamics.