Ching-Ching Yu

A chemically functionalized magnetic nanoplatform for rapid and specific biomolecular recognition and separation

We have developed a target-molecule-functionalized magnetic nanoparticle (MNP)-based method to facilitate the study of biomolecular recognition and separation. The superparamagnetic property of MNPs allows the corresponding biomolecules to be rapidly separated from crude biofluids with a significant improvement in recovery yield and specificity. Various MNPs functionalized with tag molecules (chitin, heparin, and amylose) were synthesized for recombinant protein purification, and several probe-functionalized MNPs, such as nitrilotriacetic acid (NTA)@MNP and P(k)@MNP, exhibited excellent extraction efficiency for proteins. In a cell recognition study, mannose-functionalized MNPs allowed specific purification of Escherichia coli with FimH adhesin on the surface. In an immunoprecipitation assay, the antibody-conjugated MNPs reduced the incubation time from 12 to 1 h while maintaining a comparable efficiency. The functionalized MNPs were also used in a membrane proteomic study that utilized the interaction between streptavidin-functionalized MNPs and biotinylated cell membrane proteins. Overall, the functionalized MNPs were demonstrated to be promising probes for the specific separation of targets from proteins to cells and proteomics.

Site-specific immobilization of CMP-sialic acid synthetase on magnetic nanoparticles and its use in the synthesis of CMP-sialic acid

Through the native chemical ligation, CMP-sialic acid synthetase (CSS) was site-specifically immobilized on magnetic nanoparticles and presented excellent enzymatic performance.

Structure of human ST8SiaIII sialyltransferase provides insight into cell-surface polysialylation

Sialyltransferases of the mammalian ST8Sia family catalyze oligo- and polysialylation of surface-localized glycoproteins and glycolipids through transfer of sialic acids from CMP–sialic acid to the nonreducing ends of sialic acid acceptors. The crystal structure of human ST8SiaIII at 1.85-Å resolution presented here is, to our knowledge, the first solved structure of a polysialyltransferase from any species, and it reveals a cluster of polysialyltransferase-specific structural motifs that collectively provide an extended electropositive surface groove for binding of oligo–polysialic acid chain products. The ternary complex of ST8SiaIII with a donor sugar analog and a sulfated glycan acceptor identified with a sialyltransferase glycan array provides insight into the residues involved in substrate binding, specificity and sialyl transfer.

Fabrication of Highly Stable Glyco‐Gold Nanoparticles and Development of a Glyco‐Gold Nanoparticle‐Based Oriented Immobilized Antibody Microarray for Lectin (GOAL) Assay

The design of high-affinity lectin ligands is critical for enhancing the inherently weak binding affinities of monomeric carbohydrates to their binding proteins. Glyco-gold nanoparticles (glyco-AuNPs) are promising multivalent glycan displays that can confer significantly improved functional affinity of glyco-AuNPs to proteins. Here, AuNPs are functionalized with several different carbohydrates to profile lectin affinities. We demonstrate that AuNPs functionalized with mixed thiolated ligands comprising glycan (70 mol %) and an amphiphilic linker (30 mol %) provide long-term stability in solutions containing high concentrations of salts and proteins, with no evidence of nonspecific protein adsorption. These highly stable glyco-AuNPs enable the detection of model plant lectins such as Concanavalin A, wheat germ agglutinin, and Ricinus communis Agglutinin 120, at subnanomolar and low picomolar levels through UV/Vis spectrophotometry and dynamic light scattering, respectively. Moreover, we develop in situ glyco-AuNPs-based agglutination on an oriented immobilized antibody microarray, which permits highly sensitive lectin sensing with the naked eye. In addition, this microarray is capable of detecting lectins presented individually, in other environmental settings, or in a mixture of samples. These results indicate that glyconanoparticles represent a versatile and highly sensitive method for detecting and probing the binding of glycan to proteins, with significant implications for the construction of a variety of platforms for the development of glyconanoparticle-based biosensors.

Synthesis of sialic acid-containing saccharides.

Sialic acids are a diverse family of negatively charged monosaccharides with a shared nine-carbon carboxylated backbone, and they often serve as the terminal positions of cell surface glycoproteins and glycolipids. Sialic acids play essential roles in mediating or modulating numerous pathological, biological, and immunological recognition events. Advances in synthesis have provided chemically well-defined and structurally homogeneous sialic acid-containing carbohydrates that are crucial for studying glycobiology. This review highlights recent innovations in the chemical and chemoenzymatic synthesis of difficult α-sialosides, with a particular focus on methods developed for α-selective sialylation in the synthesis of O-linked and S-linked oligosialic acids.

Site-selective protein immobilization through 2-cyanobenzothiazole-cysteine condensation.

We described a rapid site‐selective protein immobilization strategy on glass slides and magnetic nanoparticles, at either the N or C terminus, by a 2‐cyanobenzothiazole (CBT)‐cysteine (Cys) condensation reaction. A terminal cysteine was generated at either terminus of a target protein by a combination of expressed protein ligation (EPL) and tobacco etch virus protease (TEVp) digestion, and was reacted with the CBT‐solid support to immobilize the protein. According to microarray analysis, we found that glutathione S‐transferase immobilized at the N terminus allowed higher substrate binding than for immobilization at the C terminus, whereas there were no differences in the activities of N‐ and C‐terminally immobilized maltose‐binding proteins. Moreover, immobilization of TEVp at the N terminus preserved higher activity than immobilization at the C terminus. The success of utilizing CBT‐Cys condensation and the ease of constructing a terminal cysteine using EPL and TEVp digestion demonstrate that this method is feasible for site‐selective protein immobilization on glass slides and nanoparticles. The orientation of a protein is crucial for its activity after immobilization, and this strategy provides a simple means to evaluate the preferred protein immobilization orientation on solid supports in the absence of clear structural information.

Sequential one-pot enzymatic synthesis of oligo-N-acetyllactosamine and its multi-sialylated extensions

A simple and efficient protocol for the preparative-scale synthesis of various lengths of oligo-N-acetyllactosamine (oligo-LacNAc) and its multi-sialylated extensions is described. The strategy utilizes one thermophilic bacterial thymidylyltransferase (RmlA) coupled with corresponding sugar-1-phosphate kinases to generate two uridine diphosphate sugars, UDP-galactose and UDP-N-acetylglucosamine. By incorporating glycosyltransferases, oligo-LacNAcs and their sialylated analogs were synthesized.