We describe a simple method for efficiently labeling cell surface glycans on virtually any living animal cell. The method employs mild Periodate oxidation to generate an aldehyde on sialic acids, followed by Aniline-catalyzed oxime Ligation with a suitable tag (PAL). Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of aminooxy-biotin at neutral pH to label the majority of cell surface glycoproteins while maintaining high cell viability. Keywordsglycoprotein; sialic acid; oxime ligation; aniline; periodate oxidation; metabolic labeling; live cell labelingThe expanding interest in glycoproteomics and the biological roles of glycoconjugates has increased efforts to develop efficient tools to label cell surface glycoproteins. Several elegant approaches have exploited metabolic labeling of cells, and even whole model organisms, using analogs of glycan precursors that carry bio-orthogonal groups (e.g. azide, alkyne, ketone or aldehyde), allowing the chemical ligation of reporter groups onto cell surface glycoconjugates 1, 2.The chemistries used for conjugation with these functional groups each have both advantages and disadvantages for use with living cells. The conjugation of azides with substituted triphenylphosphines using the Staudinger-Bertozzi ligation can be performed on living cells, but suffers from slow reaction kinetics 3 . Conjugation of azides with substituted-alkynes (or vice versa) with the Huisgen cycloaddition, or 'click chemistry', has rapid reaction kinetics, but requires a copper catalyst that is toxic to living cells4 , 5. The newly described ligation of azides with ring-strained alkynes is compatible with living cells and has rapid reaction kinetics, but requires reagents that are not currently commercially available 2 . Finally, imine (oxime or A limitation of all these methods is the need for culturing cells with a glycan precursor containing a bio-orthogonal group prior to labeling. As an alternative, aldehydes can be readily introduced into cell surface glycans by mild periodate oxidation, known for nearly 40 years to selectively oxidize the polyhydroxy side chain of sialic acids 7,8 . The recent demonstration that oxime ligation on complex biomolecules is dramatically accelerated using aniline as a nucleophilic catalyst 9-12 inspired us to explore the efficiency of this reaction with aldehydes on living cells introduced by metabolic labeling or periodate oxidation. While aniline can be efficiently used as a nucleophilic catalyst for labeling biomolecules in solution by oxime and hydrazone ligations, we chose to employ the oxime ligation, which gives a more stable product than the hydrazone ligation 13 . NIH Public AccessThe two approaches used for the introduction of aldehydes onto cell surface sialic acids for subsequent ligation with aminooxy-biotin are illustrated in Fig. 1a. Cells were subjected to mild periodate oxidation (1 mM NaIO 4 at 4 °C for 30 min; Supplementary Fig. 1) to selectively introduce an aldehyde at C-7 of sialic acid...
The projector augmented-wave ͑PAW͒ method was developed by Blöchl as a method to accurately and efficiently calculate the electronic structure of materials within the framework of density-functional theory. It contains the numerical advantages of pseudopotential calculations while retaining the physics of all-electron calculations, including the correct nodal behavior of the valence-electron wave functions and the ability to include upper core states in addition to valence states in the self-consistent iterations. It uses many of the same ideas developed by Vanderbilt in his ''soft pseudopotential'' formalism and in earlier work by Blöchl in his ''generalized separable potentials,'' and has been successfully demonstrated for several interesting materials. We have developed a version of the PAW formalism for general use in structural and dynamical studies of materials. In the present paper, we investigate the accuracy of this implementation in comparison with corresponding results obtained using pseudopotential and linearized augmented-plane-wave ͑LAPW͒ codes. We present results of calculations for the cohesive energy, equilibrium lattice constant, and bulk modulus for several representative covalent, ionic, and metallic materials including diamond, silicon, SiC, CaF 2 , fcc Ca, and bcc V. With the exception of CaF 2 , for which core-electron polarization effects are important, the structural properties of these materials are represented equally well by the PAW, LAPW, and pseudopotential formalisms. ͓S0163-1829͑97͒00404-9͔
The siglec family of sialic acid binding proteins participates in diverse cell surface biology that includes regulation of immune cell signaling and the interaction of neuronal cells with glial cells. The weak intrinsic affinity of the natural sialoside ligands has hampered the development of synthetic ligand based probes needed to elucidate their roles in siglec function. In this report, we describe a glycan microarray comprising a library of 9-acyl-substituted sialic acids incorporated into sialosides containing the Neu5Acalpha2-3Gal and Neu5Acalpha-6Gal linkages commonly recognized by the siglecs. The array is demonstrated to exhibit utility for detecting 9-acyl substituents that increase the affinity of siglecs for their ligands. Substituents that increase affinity are anticipated to be useful for the design of high affinity ligand based probes of siglec function.
CD22, a regulator of B-cell signaling, is a siglec that recognizes the sequence NeuAc␣2-6Gal on glycoprotein glycans as ligands. CD22 interactions with glycoproteins on the same cell (in cis) and apposing cells (in trans) modulate its activity in B-cell receptor signaling. Although CD22 predominantly recognizes neighboring CD22 molecules as cis ligands on B-cells, little is known about the trans ligands on apposing cells. We conducted a proteomics scale study to identify candidate trans ligands of CD22 on B-cells by UV photocross-linking CD22-Fc chimera bound to B-cell glycoproteins engineered to carry sialic acids with a 9-aryl azide moiety. Using mass spectrometrybased quantitative proteomics to analyze the cross-linked products, 27 glycoproteins were identified as candidate trans ligands. Next, CD22 expressed on the surface of one cell was photocross-linked to glycoproteins on apposing B-cells followed by immunochemical analysis of the products with antibodies to the candidate ligands. Of the many candidate ligands, only the B-cell receptor IgM was found to be a major in situ trans ligand of CD22 that is selectively redistributed to the site of cell contact upon interaction with CD22 on the apposing cell. Molecular & Cellular Proteomics 9:1339 -1351, 2010. Glycan-binding proteins (GBPs)1 mediate diverse aspects of cell communication through their interactions with their counter-receptors comprising glycan ligands carried on cell surface glycoproteins and glycolipids. Identification of the in situ counter-receptors of glycan-binding proteins is problematic due to the fact that the vast majority of the glycoproteins of a cell will carry highly related glycan structures because they share the same secretory pathway that elaborates their glycans post-translationally en route to the cell surface. Thus, although many glycoproteins will carry the glycan structure recognized by a GBP, the challenge is to determine whether one, several, or all of these cell surface glycoproteins (and glycolipids) are recognized in situ as physiologically relevant counter-receptors (1-4). Standard in vitro methods, such as co-precipitation from cell lysates or Western blotting using binding protein probes, are useful for identifying glycoproteins that contain the glycan structure recognized by the GBP. However, these may not be relevant ligands in situ due to constraints imposed by their microdomain localization and the geometric arrangement of their glycans relative to the GBP presented on the apposing cell.In this report, we examine the in situ ligands of CD22 (Siglec-2), a member of the siglec family and a regulator of B-cell receptor (BCR) signaling that recognizes glycans containing the sequence NeuAc␣2-6Gal as ligands (2, 5, 6). Regulation of BCR signaling by CD22 is effected by its proximity to the BCR through recruitment of a tyrosine phosphatase, SHP-1, which is in turn influenced by CD22 binding to its glycan ligands (6). Glycoproteins bearing CD22 ligands are abundantly expressed on B-cells and bind to CD22 in cis (on the sa...
In this study, a novel Co 3 O 4 /Co(OH) 2 heterostructure is obtained via electrodeposition on nickel (Ni) foam, forming sandwich-like structure and freestanding electrode. The outer Co(OH) 2 with layered structure can provide sufficient absorption sites and enable facile ion intercalation, meanwhile the presence of a conductive and robust interfacial Co 3 O 4 layer between Ni foam and Co(OH) 2 is found effectively minimizes the charge transfer resistance and stabilizes the interface, thus improving the electrode's rate and cycling performance with high capacity preserved synergistically. Furthermore, the structural evolution of Co(OH) 2 and Co 3 O 4 upon cycling are elucidated systematically using a series of in situ and ex situ techniques. The Co(OH) 2 is found irreversibly changed to CoOOH upon first charge, which is then reversibly converted to CoO 2 during the subsequent charge-discharge cycles. The Co 3 O 4 exhibits negligible phase changes of the bulk upon cycling, indicating its good structural integrity that contributes to the significantly improved cyclability. In general, this work not only offers an ease and effective approach to optimize the charge storage properties of Co 3 O 4 /Co(OH) 2 heterostructure via interfacial layer control, but also provides valuable insights in understanding their charge storage mechanisms, which may inspire the development of more heterostructures or extend to other applications.
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