Mannose Analogue Research Articles

Targeted reprogramming of tumor-associated macrophages for overcoming glioblastoma resistance to chemotherapy and immunotherapy

The resistance of glioblastoma multiforme (GBM) to standard chemotherapy is primarily attributed to the existence of tumor-associated macrophages (TAMs) in the GBM microenvironment, particularly the anti-inflammatory M2 phenotype. Targeted modulation of M2-TAMs is emerging as a promising strategy to enhance chemotherapeutic efficacy. However, combination TAM-targeted therapy with chemotherapy faces substantial challenges, notably in terms of delivery efficiency and targeting specificity. In this study, we designed a pH-responsive hierarchical brain-targeting micelleplex loaded with temozolomide (TMZ) and resiquimod (R848) for combination chemo-immunotherapy against GBM. This delivery system, termed PCPA&PPM@TR, features a primary Angiopep-2 decoration on the outer layer via a pH-cleavable linker and a secondary mannose analogue (MAN) on the middle layer. This pH-responsive hierarchical targeting strategy enables effective BBB permeability while simultaneous GBM- and TAMs-targeting delivery. GBM-targeted delivery of TMZ induces alkylation and triggers an anti-GBM immune response. Concurrently, TAM-targeted delivery of R848 reprograms their phenotype from M2 to pro-inflammatory M1, thereby diminishing GBM resistance to TMZ and amplifying the immune response. In vivo studies demonstrated that targeted modulation of TAMs using PCPA&PPM@TR significantly enhanced anti-GBM efficacy. In summary, this study proposes a promising brain-targeting delivery system for the targeted modulation of TAMs to combat GBM.

An application of the Mitsunobu reaction in mannose series, using triethyl methanetricarboxylate as pronucleophile

Triethyl methanetricarboxylate, HC(CO2Et)3, is a pronucleophile used in order to achieve chain elongation and ramification. In the present paper, it has been coupled to the C6 position of mannose in a one-step synthesis, via the Mitsunobu reaction, in 71% yield. The synthesis provides a direct and rapid route to a product that might be further used to obtain mannose analogues with various functional groups (e. g. carboxylic acids) in C6 position.

Glucose and mannose analogs inhibit KSHV replication by blocking N-glycosylation and inducing the unfolded protein response.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent for Kaposi's sarcoma (KS), an HIV/AIDS-associated malignancy. Effective treatments against KS remain to be developed. The sugar analog 2-deoxy- d-glucose (2-DG) is an anticancer agent that is well-tolerated and safe in patients and was recently demonstrated to be a potent antiviral, including KSHV and severe acute respiratory syndrome coronavirus 2.Because 2-DG inhibits glycolysis and N-glycosylation, identifying its molecular targets is challenging. Here we compare the antiviral effect of 2-DG with2-fluoro-deoxy- d-glucose, a glycolysis inhibitor, and 2-deoxy-fluoro- d-mannose (2-DFM), a specific N-glycosylation inhibitor. At doses similar to those clinically achievable with 2-DG, the three drugs impair KSHV replication and virion production in iSLK.219 cells via downregulation of viral structural glycoprotein expression (K8.1 and gB), being 2-DFM the most potent KSHV inhibitor. Consistently with the higher potency of 2-DFM, we found that d-mannose rescues KSHV glycoprotein synthesis and virus production, indicating that inhibition of N-glycosylation is the main antiviral target using d-mannose competition experiments. Suppression of N-glycosylation by the sugar drugs triggers ER stress. It activates the host unfolded protein response (UPR), counteracting KSHV-induced inhibition of the protein kinase R-like endoplasmic reticulum kinasebranch, particularly activatingtranscription factor 4and C/EBP homologous protein expression. Finally, we demonstrate that sugar analogs induce autophagy (a prosurvival mechanism) and, thus, inhibit viral replication playing a protective role against KSHV-induced cell death, further supporting their direct antiviral effect and potential therapeutic use. Our work identifies inhibition of N-glycosylation leading to ER stress and UPR as an antienveloped virus target and sugar analogs such as 2-DG and the newly identified 2-DFM as antiviral drugs.

4′-Nitrobiphenyl thioglucoside as the Smallest, fluorescent photosensitizer with cancer targeting ligand

We have previously developed a glucose-linked biphenyl photosensitizer that can pass through glucose transporters, aiming for cancer-selective photodynamic therapy (PDT). Its small size (MW: 435) will allow oral administration and a fast clearance avoiding photosensitivity. However, its fluorescence efficiency was quite low, causing difficulty in monitoring cellular uptake. We thus synthesized a series of monosaccharide-linked biphenyl derivatives with a sulfur atom replacing an oxygen atom, in search of a photosensitizer with a brighter fluorescence. Among them, 4′-nitrobiphenyl thioglucoside showed a fluorescence emission extending to near infra-red region with a strength three times greater than that of the previous compound. This compound was found to have a higher 1O2-producing efficiency (ΦΔ: 0.75) than the previous compound (ΦΔ: 0.65). The thioglucoside indicated a significant photodamaging effect (IC50: 250 μM) against cancer cells. Although the galactose and mannose analogs exerted similar photodamaging effects, they were moderately toxic in the dark at a concentration of 300 μM. The thioglucoside and thiomannoside were at least partially uptaken through glucose transporters as demonstrated by inhibition with cytochalasin B, whereas no inhibition was observed for the galactoside. The behavior of d-glucose toward the cellular uptakes of these photosensitizers was bipolar: inhibitory at a low concentration and recovery or acceleratory at a higher concentration. These results indicate that 4′-nitrobiphenyl thioglucoside is the smallest (MW: 393) cancer-targeting photosensitizer with a trackable fluorescence property.

Sweet Drugs for Bad Bugs: A Glycomimetic Strategy against the DC-SIGN-Mediated Dissemination of SARS-CoV-2.

The C-type lectin receptor DC-SIGN is a pattern recognition receptor expressed on macrophages and dendritic cells. It has been identified as a promiscuous entry receptor for many pathogens, including epidemic and pandemic viruses such as SARS-CoV-2, Ebola virus, and HIV-1. In the context of the recent SARS-CoV-2 pandemic, DC-SIGN-mediated virus dissemination and stimulation of innate immune responses has been implicated as a potential factor in the development of severe COVID-19. Inhibition of virus binding to DC-SIGN, thus, represents an attractive host-directed strategy to attenuate overshooting innate immune responses and prevent the progression of the disease. In this study, we report on the discovery of a new class of potent glycomimetic DC-SIGN antagonists from a focused library of triazole-based mannose analogues. Structure-based optimization of an initial screening hit yielded a glycomimetic ligand with a more than 100-fold improved binding affinity compared to methyl α-d-mannopyranoside. Analysis of binding thermodynamics revealed an enthalpy-driven improvement of binding affinity that was enabled by hydrophobic interactions with a loop region adjacent to the binding site and displacement of a conserved water molecule. The identified ligand was employed for the synthesis of multivalent glycopolymers that were able to inhibit SARS-CoV-2 spike glycoprotein binding to DC-SIGN-expressing cells, as well as DC-SIGN-mediated trans-infection of ACE2+ cells by SARS-CoV-2 spike protein-expressing viruses, in nanomolar concentrations. The identified glycomimetic ligands reported here open promising perspectives for the development of highly potent and fully selective DC-SIGN-targeted therapeutics for a broad spectrum of viral infections.

The intrinsically disordered E-domains regulate the IGF-1 prohormones stability, subcellular localisation and secretion

Insulin-like growth factor-1 (IGF-1) is synthesised as a prohormone (proIGF-1) requiring enzymatic activity to yield the mature IGF-1. Three proIGF-1s are encoded by alternatively spliced IGF-1 mRNAs: proIGF-1Ea, proIGF-1Eb and proIGF-1Ec. These proIGF-1s have a common IGF-1 mature sequence but different E-domains. The structure of the E-domains has not been resolved, and their molecular functions are still unclear. Here, we show that E-domains are Intrinsically Disordered Regions that have distinct regulatory functions on proIGF-1s production. In particular, we identified a highly conserved N-glycosylation site in the Ea-domain, which regulated intracellular proIGF-1Ea level preventing its proteasome-mediated degradation. The inhibition of N-glycosylation by tunicamycin or glucose starvation markedly reduced proIGF-1Ea and mature IGF-1 production. Interestingly, 2-deoxyglucose, a glucose and mannose analogue, increased proIGF-1Ea and mature IGF-1 levels, probably leading to an accumulation of an under-glycosylated proIGF-1Ea that was still stable and efficiently secreted. The proIGF-1Eb and proIGF-1Ec were devoid of N-glycosylation sites, and hence their production was unaffected by N-glycosylation inhibitors. Moreover, we demonstrated that alternative Eb- and Ec-domains controlled the subcellular localisation of proIGF-1s, leading to the nuclear accumulation of both proIGF-1Eb and proIGF-1Ec. Our results demonstrated that E-domains are regulatory elements that control IGF-1 production and secretion.

A Diazido Mannose Analogue as a Chemoenzymatic Synthon for Synthesizing Di-N-acetyllegionaminic Acid-Containing Glycosides.

A chemoenzymatic synthon was designed to expand the scope of the chemoenzymatic synthesis of carbohydrates. The synthon was enzymatically converted into carbohydrate analogues, which were readily derivatized chemically to produce the desired targets. The strategy is demonstrated for the synthesis of glycosides containing 7,9-di-N-acetyllegionaminic acid (Leg5,7Ac2 ), a bacterial nonulosonic acid (NulO) analogue of sialic acid. A versatile library of α2-3/6-linked Leg5,7Ac2 -glycosides was built by using chemically synthesized 2,4-diazido-2,4,6-trideoxymannose as a chemoenzymatic synthon for highly efficient one-pot multienzyme (OPME) sialylation followed by downstream chemical conversion of the azido groups into acetamido groups. The syntheses required 10 steps from commercially available d-fucose and had an overall yield of 34-52 %, thus representing a significant improvement over previous methods. Free Leg5,7Ac2 monosaccharide was also synthesized by a sialic acid aldolase-catalyzed reaction.

A Diazido Mannose Analogue as a Chemoenzymatic Synthon for Synthesizing Di‐N‐acetyllegionaminic Acid‐Containing Glycosides

AbstractA chemoenzymatic synthon was designed to expand the scope of the chemoenzymatic synthesis of carbohydrates. The synthon was enzymatically converted into carbohydrate analogues, which were readily derivatized chemically to produce the desired targets. The strategy is demonstrated for the synthesis of glycosides containing 7,9‐di‐N‐acetyllegionaminic acid (Leg5,7Ac2), a bacterial nonulosonic acid (NulO) analogue of sialic acid. A versatile library of α2‐3/6‐linked Leg5,7Ac2‐glycosides was built by using chemically synthesized 2,4‐diazido‐2,4,6‐trideoxymannose as a chemoenzymatic synthon for highly efficient one‐pot multienzyme (OPME) sialylation followed by downstream chemical conversion of the azido groups into acetamido groups. The syntheses required 10 steps from commercially available d‐fucose and had an overall yield of 34–52 %, thus representing a significant improvement over previous methods. Free Leg5,7Ac2 monosaccharide was also synthesized by a sialic acid aldolase‐catalyzed reaction.

Dispersal and inhibitory roles of mannose, 2-deoxy-d-glucose and N-acetylgalactosaminidase on the biofilm of Desulfovibrio vulgaris.

Biofilms of sulfate-reducing bacteria (SRB) are often the major cause of microbiologically influenced corrosion. The representative SRB Desulfovibrio vulgaris has previously been shown to have a biofilm that consists primarily of protein. In this study, by utilizing lectin staining, we identified that the biofilm of D. vulgaris also consists of the matrix components mannose, fucose and N-acetylgalactosamine (GalNAc), with mannose predominating. Based on these results, we found that the addition of mannose and the nonmetabolizable mannose analog 2-deoxy-d-glucose inhibits the biofilm formation of D. vulgaris as well as that of D. desulfuricans; both compounds also dispersed the SRB biofilms. In addition, the enzyme N-acetylgalactosaminidase, which degrades GalNAc, was effective in dispersing D. vulgaris biofilms. Therefore, by determining composition of the SRB biofilm, effective biofilm control methods may be devised.

Bacterial pathogens: A spoonful of sugar could be the medicine.

Pili are filamentous bacterial structures that promote adhesion to host cells. It emerges that a small molecule that inhibits this adhesion can prevent colonization of the mouse gut by a pathogenic bacterium. See Letter p.528 Uropathogenic E. coli (UPEC) are responsible for 80% of community-acquired and 65% of nosocomial urinary tract infections (UTI), which together affect 150 million people annually. UPEC establishes reservoirs in the gut, but the factors involved in this process have remained unknown. Here, Scott Hultgren and colleagues show that both F17-like and type 1 pili promote intestinal colonization and bind to distinct glycans on epithelial cells distributed along colonic crypts. Using the high-affinity mannose analogue, mannoside M4284, which inhibits the adhesive function of type 1 pili, the authors demonstrate that it effectively reduces intestinal colonization of UPEC, while simultaneously treating UTI without significantly disrupting the composition of the gut microbiota. The authors suggest that this selective depletion of intestinal UPEC by mannosides could be used to reduce the occurrence of UTIs.

Small‐molecule inhibitors against type 1 pili selectively target uropathogenic E. coli in the gut and bladder

Urinary tract infections (UTI) affect over 150 million individuals worldwide every year. These infections are associated with significant morbidity and have a sizeable economic impact, with $5 billion being spent on UTI treatment in the USA annually. Uropathogenic E. coli (UPEC) are responsible for 80% of community aquired UTIs and 65% of nosocomial UTI. The current standard of care for UTI is antibiotic therapy. However, 30–50% of women experience recurrent UTI (rUTI) despite receiving antibiotic therapy. The prevalence of single and multi‐drug resistant UPEC strains has led to increased reliance on carbepenems, which are primarily reserved for multi‐drug resistant infections, to treat an increasing number of patients. Consequently E. coli strains resistant to colistin, a drug of last resort for treating carbapenem‐resistant Enterobacteriaceae, have now been isolated in over 30 counties. The rate at which drug‐resistant E. coli are spreading across the globe emphasizes the urgent nature of the antibiotic‐resistance crisis we currently face and the dire need for new, antibiotic‐sparing therapies that target UPEC virulence factors. To colonize host tissue, E. coli encode chaperone usher pathway (CUP) pili and single UPEC strains can encode up to 16 distinct CUP pilus operons. Although CUP pilus types that promote UPEC colonization of the urinary tract have been identified, no studies investigating a role for CUP pilus types in UPEC intestinal colonization have been published. Still, leading models of infection posit that UPEC in the gut seed UTI by being shed in the feces, and then colonizing the peri‐urethral or vaginal tissue and subsequently ascending through the urethra to access the bladder. Using a new mouse model of UPEC intestinal colonization we found that two (CUP) pili, F17‐like pili and type 1 pili, provide a fitness advantages for UPEC in the gut. We found that high‐affinity mannose analogues (mannosides) that target the function of type 1 pili and effectively treat UTI reduce intestinal colonization by clinical UPEC isolates, including a multi‐drug resistant strain, without disrupting gut microbiota structure. By decreasing the intestinal UPEC reservoir that seeds bladder infection, mannosides could significantly reduce the rate of UTI and rUTI. Further, mannosides act like a molecular scalpel, specifically targeting intestinal UPEC with minimal effects on the overall gut microbiota structure. Therapies like mannosides, which selectively target colonization by a specific pathogen, have potential to revolutionize treatment and prevention of rUTI.Support or Funding InformationThis work was supported by grants from the NIH [K08AI113184 (to ALK), AI029549 (SJH), DK051406 (SJH), and 1F31DK107057‐01A1 (CNS)]. CNS, JWJ, SJH are inventors on patent application US8,937,167 B2 that covers mannoside compounds. JWJ and SJH have ownership interest in Fimbrion Therapeutics, and may benefit if the company is successful in marketing mannosides.

2-Deoxy-d-glucose is a potent inhibitor of biofilm growth in Escherichia coli.

Escherichia coli strain 15 (ATCC 9723), which forms robust biofilms, was grown under optimal biofilm conditions in NaCl-free Luria-Bertani broth (LB*) or in LB* supplemented with one of the non-metabolizable analogues 2-deoxy-d-glucose (2DG), methyl α-d-mannopyranoside (αMM), or methyl α-d-glucopyranoside (αMG). Biofilm growth was inhibited by mannose analogue 2DG even at very low concentration in unbuffered medium, and the maximal inhibition was enhanced in the presence of either 100 mM KPO4 or 100 mM MOPS, pH 7.5; in buffered medium, concentrations of 0.02 % (1.2 mM) or more inhibited growth nearly completely. In contrast, mannose analogue αMM, which should not be able to enter the cells but has been reported to inhibit biofilm growth by binding to FimH, did not exhibit strong inhibition even at concentrations up to 1.8 % (108 mM). The glucose analogue αMG inhibited biofilm growth, but much less strongly than did 2DG. None of the analogues inhibited planktonic growth or caused a change in pH of the unbuffered medium. Similar inhibitory effects of the analogues were observed in minimal medium. The effects were not strain-specific, as 2DG and αMG also inhibited the weak biofilm growth of E. coli K12.

Unified picture for the conformation and stabilization of the O-glycosidic linkage in glycopeptide model structures

Glycoproteins play a central role in the immune response. In this study, we focus on the core of the common O-linked mucin-type glycopeptides. It has been observed that glycosylation stabilizes the protein in a stiffened, extended structure. We provide a unified picture for the conformation and stabilization of the O-glycosidic linkage using O-(2-acetylamino-2-deoxy-α- or -β-d-galacto- or -mannopyranosyl)-N-acetyl-l-serinamide model structures. We have calculated equilibrium geometries of the model structures with the B3LYP/6-31G(2df,p) method suggested in the Gaussian-4 theory. According to the relative energies, we confirm that the GalNAc-Ser linkage is more stable than its mannose analogues. The natural preference for the α-GalNAc-Ser over the β-GalNAc-Ser anomers can be explained by entropic effects. We explored the hydrogen bonding patterns on the carbohydrate unit calculating highly accurate dRPA@PBE0.25 and dRPA75 energies and found that in some cases, the acetamido group can be fixed by hydrogen bonding from the (O3Carb)H atom, but in most of the cases, it can rotate more freely. The torsion angles in the glycosidic linkage show that the linkage is stiffened more in the α-anomers and the most in the α-GalNAc-Ser structure because of the steric strains in the axial position and by two or three intramolecular hydrogen bonds. We also found that although, in our gas-phase model geometries, the peptide backbone prefers to be in a γ L-turn, a structural water molecule can stabilize a polyproline II helix of a proline-rich sequence, a β-sheet, or more likely random coils.

P-Aminophenyl-α-d-mannopyranoside engineered lipidic nanoparticles for effective delivery of docetaxel to brain

Lipidic systems are considered to be the most promising carrier for drug delivery to brain. Metabolic substrates like carbohydrates and amino acids are able to traverse the blood–brain barrier (BBB) by specific carrier-mediated transport systems like glucose transporters present on the both luminal and abluminal side of the BBB. With this objective, the docetaxel (DTX) loaded solid lipidic nanoparticles were formulated and surface modified with a mannose derived ligand p-aminophenyl-α-d-mannopyranoside (MAN) to develop MAN conjugated lipidic nanoparticles for targeting DTX to brain. Lipidic nanoparticles were prepared using emulsification and solvent evaporation method using stearic acid as charge modifying lipid and conjugated with MAN using carbodimide coupling. These lipidic nanoparticles were successfully characterized using various techniques like DLS, TEM, DSC and FTIR spectroscopy. Cytotoxicity and cell uptake unveiled enhanced efficacy of conjugated lipidic nanoparticles. Pharmacokinetic and brain distribution studies demonstrated increased DTX concentrations using lipidic nanoparticles in brain and conjugating MAN on surface of lipidic nanoparticles further augmented the inflow of the drug to brain. Present study revealed the prospective of mannose analog, MAN-conjugated lipidic nanoparticles as efficient vehicle for anticancer drug delivery to brain.

The role of glucose transporters in the distribution of p-aminophenyl-α-d-mannopyranoside modified liposomes within mice brain

The effective treatment of central nervous system diseases is a major challenge due to the presence of the blood–brain barrier (BBB). P-aminophenyl-α-d-mannopyranoside (MAN), a kind of mannose analog, was conjugated onto the surface of liposomes (MAN–LIP) to enhance the brain delivery. In this study, we investigated the brain distribution of MAN–LIP based on our previous studies and tried to explore the relationship between the distribution of MAN–LIP and glucose transporters (GLUTs) on the cells. In vivo optical imaging was used to assess the distribution of liposomes in mice brain. The mice administered with MAN–LIP had significantly higher brain fluorescence intensity and MAN–LIP relatively concentrated in the cerebellum and cerebral cortex. Fluorescent microscope and Western blot were used to evaluate the results of lentiviral vector-mediated hSLC2A1 and hSLC2A3 gene transfection into C6, PC12 and vessels of endothelial cell line, bEND.3. The results from live cell station and flow cytometry showed that the cellular uptake of MAN–LIP was significantly improved by GLUT1 and GLUT3 overexpression cells. The transport experiments also demonstrated that the transendothelial ability of MAN–LIP was much stronger when crossing LV-GLUT1/bEND.3 cell monolayers or LV-GLUT3/ bEND.3 cell monolayers, of which GLUT1 and GLUT3 were overexpressed. The combined data indicated that the transcytosis by GLUT1 and GLUT3 was a pathway of MAN–LIP into brain, and the special brain distribution of MAN–LIP was closely related to the non-homogeneous distribution of GLUT1 and GLUT3 in the brain.

Novel Monodisperse PEGtide Dendrons: Design, Fabrication, and Evaluation of Mannose Receptor-Mediated Macrophage Targeting

Novel PEGtide dendrons of generations 1 through 5 (G1.0–5.0) containing alternating discrete poly(ethylene glycol) (dPEG) and amino acid/peptide moieties were designed and developed. To demonstrate their targeting utility as nanocarriers, PEGtide dendrons functionalized with mannose residues were developed and evaluated for macrophage targeting. PEGtide dendrons were synthesized using 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS) protocols. The N-α-Fmoc-N-ε-(5-carboxyfluorescein)-l-lysine (Fmoc-Lys(5-FAM)-OH) and monodisperse Fmoc-dPEG6-OH were sequentially coupled to Fmoc-β-Ala-resin to obtain the resin-bound intermediate Fmoc-dPEG6-Lys(5-FAM)-β-Ala (1). G1.0 dendrons were obtained by sequentially coupling Fmoc-Lys(Fmoc)-OH, Fmoc-β-Ala-OH, and Fmoc-dPEG6-OH to 1. Dendrons of higher generation, G2.0–5.0, were obtained by repeating the coupling cycles used for the synthesis of G1.0. Dendrons containing eight mannose residues (G3.0-mannose8) were developed for mannose receptor (MR) mediated macrophage targeting by conjugating α-d-mannopyranosylphenyl isothiocyanate to G3.0 dendrons. In the present study PEGtide dendrons up to G5.0 were synthesized. The molecular weights of the dendrons determined by MALDI-TOF were in agreement with calculated values. The hydrodynamic diameters measured using dynamic light scattering (DLS) ranged from 1 to 8 nm. Cell viability in the presence of G3.0 and G3.0-mannose8 was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and was found to be statistically indistinguishable from that of untreated cells. G3.0-mannose8 exhibited 12-fold higher uptake than unmodified G3.0 control dendrons in MR-expressing J774.E murine macrophage-like cells. Uptake was nearly completely inhibited in the presence of 10 mg/mL mannan, a mannose analogue and known MR substrate. Confocal microscopy studies demonstrated the presence of significant intracellular punctate fluorescence colocalized with a fluid endocytosis marker with little surface fluorescence in cells incubated with G3.0-mannose8. No significant cell-associated fluorescence was observed in cells incubated with G3.0 dendrons that did not contain the targeting ligand mannose. The current studies suggest that PEGtide dendrons could be useful as nanocarriers in drug delivery and imaging applications.

Characterizing Pilus-Mediated Adhesion of Biofilm-Forming E. coli to Chemically Diverse Surfaces Using Atomic Force Microscopy

Biofilms are complex communities of microorganisms living together at an interface. Because biofilms are often associated with contamination and infection, it is critical to understand how bacterial cells adhere to surfaces in the early stages of biofilm formation. Even harmless commensal Escherichia coli naturally forms biofilms in the human digestive tract by adhering to epithelial cells, a trait that presents major concerns in the case of pathogenic E. coli strains. The laboratory strain E. coli ZK1056 provides an intriguing model system for pathogenic E. coli strains because it forms biofilms robustly on a wide range of surfaces.E. coli ZK1056 cells spontaneously form living biofilms on polylysine-coated AFM cantilevers, allowing us to measure quantitatively by AFM the adhesion between native biofilm cells and substrates of our choice. We use these biofilm-covered cantilevers to probe E. coli ZK1056 adhesion to five substrates with distinct and well-characterized surface chemistries, including fluorinated, amine-terminated, and PEG-like monolayers, as well as unmodified silicon wafer and mica. Notably, after only 0–10 s of contact time, the biofilms adhere strongly to fluorinated and amine-terminated monolayers as well as to mica and weakly to “antifouling” PEG monolayers, despite the wide variation in hydrophobicity and charge of these substrates. In each case the AFM retraction curves display distinct adhesion profiles in terms of both force and distance, highlighting the cells’ ability to adapt their adhesive properties to disparate surfaces. Specific inhibition of the pilus protein FimH by a nonhydrolyzable mannose analogue leads to diminished adhesion in all cases, demonstrating the critical role of type I pili in adhesion by this strain to surfaces bearing widely different functional groups. The strong and adaptable binding of FimH to diverse surfaces has unexpected implications for the design of antifouling surfaces and antiadhesion therapies.

Targeted Identification of Metastasis-associated Cell-surface Sialoglycoproteins in Prostate Cancer

Covalent attachment of carbohydrates to proteins is one of the most common post-translational modifications. At the cell surface, sugar moieties of glycoproteins contribute to molecular recognition events involved in cancer metastasis. We have combined glycan metabolic labeling with mass spectrometry analysis to identify and characterize metastasis-associated cell surface sialoglycoproteins. Our model system used syngeneic prostate cancer cell lines derived from PC3 (N2, nonmetastatic, and ML2, highly metastatic). The metabolic incorporation of AC4ManNAz and subsequent specific labeling of cell surface sialylation was confirmed by flow cytometry and confocal microscopy. Affinity isolation of the modified sialic-acid containing cell surface proteins via click chemistry was followed by SDS-PAGE separation and liquid chromatography-tandem MS analysis. We identified 324 proteins from N2 and 372 proteins of ML2. Using conservative annotation, 64 proteins (26%) from N2 and 72 proteins (29%) from ML2 were classified as extracellular or membrane-associated glycoproteins. A selective enrichment of sialoglycoproteins was confirmed. When compared with global proteomic analysis of the same cells, the proportion of identified glycoprotein and cell-surface proteins were on average threefold higher using the selective capture approach. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the vast majority of glycoproteins overexpressed in the metastatic ML2 subline were involved in cell motility, migration, and invasion. Our approach effectively targeted surface sialoglycoproteins and efficiently identified proteins that underlie the metastatic potential of the ML2 cells.

Abstract 3307: Cell-surface glycoproteomics identifies CUB-domain containing protein 1 to be associated with prostate cancer metastasis

Abstract Prostate cancer is still the most common and second leading cause of cancer death in men in the United States and is overwhelmingly lethal when metastatic. Evidence from histochemical analysis and experimental tumor models indicates alterations in cell-surface sialylation as a general observation associated with the metastatic potential of tumor cells. Our preliminary data from lectin staining also demonstrates changes of cell-surface sialylation in a syngeneic prostate cancer cell model, which includes two PC3 sublines, N2 (non-metastatic) and ML2 (highly metastatic). Characterization of cell-surface sialylated glycoproteins in this model system could provide new information related to the metastatic phenotype in prostate cancer. Thus, cell-surface sialoglycoproteins in the N2 and ML2 cell lines were metabolicly labeled with with an unnatural mannose analogue (AC4ManNAz); This was followed by chemoselective conjugation of the azido sugars with a biotinylated alkyne capture reagent via Cu (I) catalyzed click chemistry and affinity detection and enrichment by streptavidin-conjugated beads (or fluorescein). Captured glycoproteins were separated by 1D gel electrophoresis, in-gel proteolytic digestion, and protein identification by means of tandem mass spectrometry (LC-MS/MS). The expression of the targeted sialoglycoproteins was verified in our prostate cancer cell lines and clinical samples by means of Western blot, confocal microscopy and fluorescent immunohistochemistry. Forty three cell-surface N-linked glycoproteins were identified as differentially expressed between the PC3 metastasis variants, including CUB-domain containing protein 1 (CDCP1). Western blot analysis of the cell-surface glycoprotein enriched fractions and total cell lysate confirmed that CDCP1 was over-expressed in the metastatic ML2 cells. Expression of CDCP1 is dysregulated in prostate cancer tissues, with malignant glands showing a more intense cell surface staining pattern, whereas normal glands demonstrated weak membrane and intracellular granular staining on the basal side. We conclude that glycan metabolic labeling technology combined with mass spectrometry analysis provides a powerful tool for the identification of specific cell-surface glycoproteins that may contribute to prostate cancer metastasis, like CDCP1. Further studies are warranted to evaluate the clinical value of CDCP1 and to understand its function in prostate cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3307.

Synthesis and Quantitative Evaluation of Glycero‐D‐manno‐heptose Binding to Concanavalin A by Fluorous‐Tag Assistance

Fooling conA? Fluorous microarrays allow not only qualitative, but also quantitative assessment of binding to show that conA can accept modifications to its usual mannose ligand at the C-6 position and bind to bacterial seven-carbon mannose analogues.