Daniel M. Ratner

Tools for Glycomics: Mapping Interactions of Carbohydrates in Biological Systems

The emerging field of glycomics has been challenged by difficulties associated with studying complex carbohydrates and glycoconjugates. Advances in the development of synthetic tools for glycobiology are poised to overcome some of these challenges and accelerate progress towards our understanding of the roles of carbohydrates in biology. Carbohydrate microarrays, fluorescent neoglycoconjugate probes, and aminoglycoside antibiotic microarrays are among the many new tools becoming available to glycobiologists.

Probing protein-carbohydrate interactions with microarrays of synthetic oligosaccharides.

Formerly a TMneglected dimension∫ of biochemistry, recent years have seen growing interest in studying the biological function of carbohydrates and glycoconjugates. An emerging understanding of the physiological role of these biomolecules has uncovered their vital participation in a host of fundamental cellular processes. In the form of glycopeptides, glycolipids, glycosaminoglycans, and proteoglyans, glycoconjugates are known to be involved in inflammation, cell ± cell interactions, signal transduction, fertility, and development. 5] Unfortunately, current methods for elucidating the biochemical roles of glycoconjugates are often cumbersome. This demonstrates the need to develop techniques that will satisfy this growing field of study by enabling rapid and facile exploration of biochemical events involving carbohydrates. Inspired by the success of DNA and protein microarrays, 7] the chip-based approach has been put forward as a useful tool in the emerging field of glycomics. Nitrocellulose-coated slides have been employed for the noncovalent immobilization of microbial polysaccharides and neoglycolipid-modified oligosaccharides. 12] Hydrophobic interactions have been utilized to anchor lipid-bearing carbohydrates on polystyrene microtiter plates. Self-assembled monolayers presenting benzoquinone groups enabled the Diels ±Alder-mediated immobilization of cyclopentadiene-derivatized monosaccharides on a gold surface. Another covalent immobilization chemistry involved treating maleimide-functionalized monoand disaccharide glycosylamines with a thiol-derivatized glass slide, or, alternatively, thiol-functionalized carbohydrates with a self-assembled monolayer presenting maleimide groups. Our motivation for developing a system for arraying carbohydrates is based on the need to have microarrays that are fully phosphate buffer (0.1M) and Perfect Hyb hybridization buffer (Sigma, 1:1 v/v) for 5 h to give double-stranded DNA assembly on the surface. The resulting surfaces were rinsed with the hybridization buffer and immersed in a solution of hemin (1.2 M) in buffer (25 mM HEPES, 20 mM KCl, 200 mM NaCl, 0.05% Triton X-100, 1% DMSO; pH 7.4) for 12 h at room temperature. The resulting system was further treated with doxorubicin (5, 5 M) in phosphate buffer (0.1M, pH 7.4) for 1 h at room temperature.

Multisite and Multivalent Binding between Cyanovirin-N and Branched Oligomannosides: Calorimetric and NMR Characterization

Binding of the protein cyanovirin-N to oligomannose-8 and oligomannose-9 of gp120 is crucially involved in its potent virucidal activity against the human immunodeficiency virus (HIV). The interaction between cyanovirin-N and these oligosaccharides has not been thoroughly characterized due to aggregation of the oligosaccharide-protein complexes. Here, cyanovirin-Ns interaction with a nonamannoside, a structural analog of oligomannose-9, has been studied by nuclear magnetic resonance and isothermal titration calorimetry. The nonamannoside interacts with cyanovirin-N in a multivalent fashion, resulting in tight complexes with an average 1:1 stoichiometry. Like the nonamannoside, an alpha1-->2-linked trimannoside substructure interacts with cyanovirin-N at two distinct protein subsites. The chitobiose and internal core trimannoside substructures of oligomannose-9 are not recognized by cyanovirin-N, and binding of the core hexamannoside occurs at only one of the sites on the protein. This is the first detailed analysis of a biologically relevant interaction between cyanovirin-N and high-mannose oligosaccharides of HIV-1 gp120.

XPS and SPR Analysis of Glycoarray Surface Density

Despite the fact that the carbohydrate microarray has seen increasing use within the field of glycobiology, the surface chemistry of the glycoarray remains largely unexplored. Motivated by the need to develop surface analytical techniques to characterize carbohydrate-modified surfaces, we developed a quantitative X-ray photoelectron spectroscopy (XPS) and surface plasmon resonance imaging (SPR imaging) method to study glycan biosensors. We performed a comparative analysis on the relative coverage of mixed self-assembled monolayers (SAMs) on gold, consisting of a thiol-functionalized trimannoside (Manalpha1,2Manalpha1,2Manalpha-OEG-SH) at varying concentrations (0-100%) mixed separately with two thiol-containing polyethylene glycol oligomers. XPS C1s core level analysis was used to identify the O-C-O functionality unique to the carbohydrate acetal moiety and to separate and quantify the relative coverage of sugar in carbohydrate/OEG mixed SAMs. XPS spectra of the mixed SAMs demonstrated a proportional increase in the acetal signature of the glycan with increasing sugar concentration. To relate surface glycan density with biological function, we carried out a kinetic analysis of Concanavalin A (ConA) binding to SAMs of varying densities of carbohydrate using SPR imaging. We observed protein binding that was highly dependent on both glycan density and the nature of the OEG-thiol used in the mixed self-assembly. These results illustrate the utility of surface analytical techniques such as XPS and SPR in carbohydrate biosensor characterization and optimization.

A linear synthesis of branched high-mannose oligosaccharides from the HIV-1 viral surface envelope glycoprotein gp120

Described is a linear solution-phase synthesis of the HIV-1 viral surface envelope glycoprotein gp120 high-mannose nonasaccharide pentyl glycoside. Envisioning the automated solid-phase assembly of complex carbohydrates, the synthesis of the nonasaccharide and the related tri- and hexamannosides demonstrates the facile assembly of highly branched structures in a stepwise fashion incorporating monosaccharide building blocks. A differentially protected core trisaccharide was prepared and further elongated in two high-yielding tri-mannosylations to furnish the triantennary structure. The tri-, hexa-, and nonamannoside n-pentyl glycosides obtained via the described synthesis are currently being used for detailed study of the carbohydrate protein interactions responsible for binding of the anti-HIV protein cyanovirin-N to the glycoprotein gp120. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

Silicon photonic micro-disk resonators for label-free biosensing.

Silicon photonic biosensors are highly attractive for multiplexed Lab-on-Chip systems. Here, we characterize the sensing performance of 3 µm TE-mode and 10 µm dual TE/TM-mode silicon photonic micro-disk resonators and demonstrate their ability to detect the specific capture of biomolecules. Our experimental results show sensitivities of 26 nm/RIU and 142 nm/RIU, and quality factors of 3.3x10(4) and 1.6x10(4) for the TE and TM modes, respectively. Additionally, we show that the large disks contain both TE and TM modes with differing sensing characteristics. Finally, by serializing multiple disks on a single waveguide bus in a CMOS compatible process, we demonstrate a biosensor capable of multiplexed interrogation of biological samples.

Multiplexed inkjet functionalization of silicon photonic biosensors

The transformative potential of silicon photonics for chip-scale biosensing is limited primarily by the inability to selectively functionalize and exploit the extraordinary density of integrated optical devices on this platform. Silicon biosensors, such as the microring resonator, can be routinely fabricated to occupy a footprint of less than 50 × 50 µm; however, chemically addressing individual devices has proven to be a significant challenge due to their small size and alignment requirements. Herein, we describe a non-contact piezoelectric (inkjet) method for the rapid and efficient printing of bioactive proteins, glycoproteins and neoglycoconjugates onto a high-density silicon microring resonator biosensor array. This approach demonstrates the scalable fabrication of multiplexed silicon photonic biosensors for lab-on-a-chip applications, and is further applicable to the functionalization of any semiconductor-based biosensor chip.

Carbohydrate-Mediated Targeting of Antigen to Dendritic Cells Leads to Enhanced Presentation of Antigen to T Cells

The unique therapeutic value of dendritic cells (DCs) for the treatment of allergy, autoimmunity and transplant rejection is predicated upon our ability to selectively deliver antigens, drugs or nucleic acids to DCs in vivo. Here we describe a method for delivering whole protein antigens to DCs based on carbohydrate‐mediated targeting of DC‐expressed lectins. A series of synthetic carbohydrates was chemically‐coupled to a model antigen, ovalbumin (OVA), and each conjugate was evaluated for its ability to increase the efficiency of antigen presentation by murine DCs to OVA‐specific T cells (CD4+ and CD8+). In vitro data are presented that demonstrate that carbohydrate modification of OVA leads to a 50‐fold enhancement of presentation of antigenic peptide to CD4+ T cells. A tenfold enhancement is observed for CD8+ T cells; this indicates that the targeted lectin(s) can mediate cross‐presentation of antigens on MHC class I. Our data indicate that the observed enhancements in antigen presentation are unique to OVA that is conjugated to complex oligosaccharides, such as a high‐mannose nonasaccharide, but not to monosaccharides. Taken together, our data suggest that a DC targeting strategy that is based upon carbohydrate‐lectin interactions is a promising approach for enhancing antigen presentation via class I and class II molecules.

Suggestive Evidence for Darwinian Selection against Asparagine-Linked Glycans of Plasmodium falciparum and Toxoplasma gondii†

ABSTRACT We are interested in asparagine-linked glycans (N-glycans) of Plasmodium falciparum and Toxoplasma gondii, because their N-glycan structures have been controversial and because we hypothesize that there might be selection against N-glycans in nucleus-encoded proteins that must pass through the endoplasmic reticulum (ER) prior to threading into the apicoplast. In support of our hypothesis, we observed the following. First, in protists with apicoplasts, there is extensive secondary loss of Alg enzymes that make lipid-linked precursors to N-glycans. Theileria makes no N-glycans, and Plasmodium makes a severely truncated N-glycan precursor composed of one or two GlcNAc residues. Second, secreted proteins of Toxoplasma, which uses its own 10-sugar precursor (Glc3Man5GlcNAc2) and the host 14-sugar precursor (Glc3Man9GlcNAc2) to make N-glycans, have very few sites for N glycosylation, and there is additional selection against N-glycan sites in its apicoplast-targeted proteins. Third, while the GlcNAc-binding Griffonia simplicifolia lectin II labels ER, rhoptries, and surface of plasmodia, there is no apicoplast labeling. Similarly, the antiretroviral lectin cyanovirin-N, which binds to N-glycans of Toxoplasma, labels ER and rhoptries, but there is no apicoplast labeling. We conclude that possible selection against N-glycans in protists with apicoplasts occurs by eliminating N-glycans (Theileria), reducing their length (Plasmodium), or reducing the number of N-glycan sites (Toxoplasma). In addition, occupation of N-glycan sites is markedly reduced in apicoplast proteins versus some secretory proteins in both Plasmodium and Toxoplasma.

Biofunctional Paper via the Covalent Modification of Cellulose

Paper-based analytical devices are the subject of growing interest for the development of low-cost point-of-care diagnostics, environmental monitoring technologies, and research tools for limited-resource settings. However, there are limited chemistries available for the conjugation of biomolecules to cellulose for use in biomedical applications. Herein, divinyl sulfone (DVS) chemistry was demonstrated to immobilize small molecules, proteins, and DNA covalently onto the hydroxyl groups of cellulose membranes through nucleophilic addition. Assays on modified cellulose using protein-carbohydrate and protein-glycoprotein interactions as well as oligonucleotide hybridization showed that the membranes bioactivity was specific, dose-dependent, and stable over a long period of time. The use of an inkjet printer to form patterns of biomolecules on DVS-activated cellulose illustrates the adaptability of the DVS functionalization technique to pattern sophisticated designs, with potential applications in cellulose-based lateral flow devices.