Hsin-Kai Liao

Carbohydrate-encapsulated gold nanoparticles for rapid target- protein identification and binding- epitope mapping

The interactions of cell-surface glycoproteins and glycolipids play important roles in cell–cell communication, proliferation, and differentiation. Combinations of saccharides, orientations of glycosidic bonds, and branching patterns of linkages allow complex carbohydrates to have a vast diversity of structures for molecular recognition. Thus, studies of carbohydrate-related interactions might provide new insights into their biological roles and reveal new possibilities for drug development. 4] Disclosure of the carbohydrate-recognition sites by X-ray crystallography and NMR spectroscopy has been a challenge due to the difficulty of cocrystallization of targeting proteins and carbohydrates. At present, most of the binding-epitope analysis methodologies are time-consuming as they screen sets of overlapping peptides spanning a known protein sequence. 7] The advent of an efficient, sensitive, general strategy to identify new carbohydrate-binding lectins and map epitopes is awaited to unravel the complexities of carbohydrate recognition. Recent developments in mass spectrometry have greatly expanded the possibility of characterizing unknown proteins, including mapping of protein glycosylation sites. Despite the advantages, the simultaneous characterization of the hundreds to thousands of proteins present in a complex medium still remains a challenge. However, when mass spectrometry is combined with a biologically active probe to rapidly and specifically target proteins of interest, this targeted proteomic approach can accelerate research for class-specific proteins or biomarkers. Recently, metal nanoparticles have been used in biological separation and promise to be superior to microbeads. Furthermore, biomolecule-conjugated gold nanoparticles (AuNPs) are the most popular probes because of their readily assembling with thiolated molecules, their large area/ volume ratio for investigating three-dimensional interactions, and their ease of separation by centrifugation. 13] However, the use of functionalized nanoparticles as probes combined with mass spectrometry for carbohydrate–protein recognition studies has not been explored. We report here a new approach of using carbohydrate-encapsulated AuNP (c-AuNP) as an affinity probe for the efficient separation and enrichment of target proteins, and then protein identification and epitope mapping by MALDI-TOF MS. The analytical scheme of the approach, nanoprobe-based affinity mass spectrometry (NBAMS), is illustrated in Scheme 1. Unlike other mass spectrometry-based affinity capture approaches that make use of agarose beads or biochips, the core component of our scheme is a nanosized biologically active affinity probe. Target proteins can be affinity captured from a mixture by the nanoprobe and directly analyzed on-probe by MALDI-TOF MS. Most significantly, once target proteins have been captured, on-probe digestion followed by removal of unbound peptides allows rapid mapping of carbohydrate-recognition peptide sequences in the proteins. To demonstrate the general applicability of the NBAMS technique in tackling carbohydrate–protein interactions, proof-ofprinciple was performed for the specific capture and identification of the galactophilic lectin Pseudomonas aeruginosa lectin I (PA-IL) by using c-AuNP. The medium-range affinity (Ka~3.4 10 m ) of monomeric d-galactose for PA-IL was enhanced by assembling sugars on nanoparticles. The resulting multivalent interactions between c-AuNP and PA-IL facilitated highly specific and stable surface affinity separation. To probe the subtle variations in the carbohydrate-binding domain of PA-IL, two carbohydrates—galactose and P antigen (Gala1! [a] Dr. Y.-J. Chen, Y.-W. Chang, H.-K. Liao, C.-Y. Chang, M.-D. Jan, Dr. C.-C. Lin Institute of Chemistry and Genomic Research Center Academia Sinica Sec. 2 Academia Road, Taipei, 115 (Taiwan) Fax: (+ 886) 2-2783-1237 E-mail : yjchen@chem.sinica.edu.tw cclin@chem.sinica.edu.tw [b] S.-H. Chen, Y.-Y. Chien, Dr. K.-T. Wang Department of Chemistry, National Taiwan University Taipei 115 (Taiwan) Supporting information for this article is available on the WWW under http ://www.chembiochem.org or from the author.

Nuclear efflux of heterogeneous nuclear ribonucleoprotein C1/C2 in apoptotic cells: a novel nuclear export dependent on Rho-associated kinase activation

Using a proteomic approach, we searched for protein changes dependent on Rho-associated kinase (ROCK) during phorbol-12-myristate-13-acetate (PMA)-induced apoptosis. We found that heterogeneous nuclear ribonucleoprotein C1 and C2 (hnRNP C1/C2), two nuclear restricted pre-mRNA binding proteins, are translocated to the cytosolic compartment in a ROCK-dependent manner in PMA-induced pro-apoptotic cells, where nuclear envelopes remain intact. The subcellular localization change of hnRNP C1/C2 appears to be dependent on ROCK-mediated cytoskeletal change and independent of caspase execution and new protein synthesis. Such a ROCK-dependent translocation is also seen in TNFα-induced apoptotic NIH3T3 cells. By overexpressing the dominant active form of ROCK, we showed that a ROCK-mediated signal is sufficient to induce translocation of hnRNP C1/C2. Deletion experiments indicated that the C-terminal 40-amino-acid region of hnRNP C1/C2 is required for ROCK-responsive translocation. By using nuclear yellow fluorescent protein (YFP) fusion, we determined that the C-terminal 40-amino-acid region of hnRNP C1/C2 is a novel nuclear export signal responsive to ROCK-activation. We conclude that a novel nuclear export is activated by the ROCK signaling pathway to exclude hnRNP C1/C2 from nucleus, by which the compartmentalization of specific hnRNP components is disturbed in apoptotic cells.

Molecular Identification of Canine Podocalyxin-Like Protein 1 as a Renal Tubulogenic Regulator

GP135 is an apical membrane protein expressed in polarized MDCK epithelial cells. When cultured in three-dimensional collagen gel, MDCK cells form branching tubules in response to hepatocyte growth factor stimulation in a manner that simulates the embryonic renal development. During this process, GP135 displays transient loss of membranous localization but reappears at the cell surface when nascent lumen emerges from the developing tubules. Despite being used for decades as the canonical hallmark of apical surface, the molecular identity and the significance of the dynamic expression of GP135 during the tubulogenic process remain elusive. For exploring the function of GP135, the full-length cDNA encoding GP135 was obtained. Sequence alignments and features analysis confirm GP135 as a canine homolog of podocalyxin, confirming the finding of an earlier independent study. Immunohistochemical assays on canine kidney sections identified both glomerular and tubular distribution of GP135 along the nephron. Mutant MDCK cells expressing siRNA targeted at two regions of GP135 show defects in hepatocyte growth factor-induced tubulogenesis. Re-expression of full-length and an O-linked glycosylation abbreviated construct of GP135 could recapitulate the tubulogenesis process lacking in siRNA knockdown cells; however, a deletion construct devoid of the cytoplasmic domain failed to rescue the phenotype. In summary, the data identify the MDCK apical domain marker GP135 as a tubular form of podocalyxin and provide evidence for its importance in renal tubulogenesis.