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Design and fabrication of multivalent gal-containing quantum dots and study of its interactions with asialoglycoprotein receptor (ASGP-R)

Yang Yang, Yue-Tao Zhao, Ting-Ting Yan, Min Yu, Yin-Lin Sha, Zhi-Hui Zhao and Zhong-Jun Li

Tetrahedron Letters. (in press, 2010 doi:10.1016/j.tetlet.2010.06.002)

 

Multivalent lactose (Lac-QDs) and galactose (Gal-QDs) coated CdSeS-ZnS core-shell quantum dots (QDs) were prepared. The formula of the glyco-QDs was determined by Nuclear Magnetic Resonance (NMR) and Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). The uptake of the Gal-containing glyco-QDs by HepG2 cells was investigated. Flow Cytometry (FCM) and fluorescence microscopy analysis indicated that the uptake is receptor-mediated and selective. The prepared multivanlent glyco-QDs could be used to mimic the oligosaccharides in the study of hepatic endocytosis. Furthermore, this type of glyco-QDs can be used as a useful fluorescent probe in cell imaging and analysis of carbohydrate-protein interactions.


►►Characterization of multivalent lactose quantum dots and its application in carbohydrate-protein interactions study and cell imaging

Yang Yang,$ Min Yu,$ Ting-Ting Yan, Zhi-Hui Zhao, Yin-Lin Sha,* Zhong-Jun Li* ($ contribute equally)

Bioorganic & Medicinal Chemistry. (in press, 2010. doi: 10.1016/j.bmc.2010.05.046)

We have previously reported a facile and convenient method for the preparation of a new type of lactose-CdSeS/ZnS quantum dots conjugates (Lac-QDs) that exhibit biocompatibility, noncytotoxicity and specificity to leukocytes. In order to further study the carbohydrate-protein interactions, a series of Lac-QDs with different lactose densities and a PEGylated (n=3) lactose-QDs conjugate (LacPEG-QDs) with more flexible sugar ligands were prepared. The amount of the sugar molecules on QDs can be determined by NMR, which was in agreement with the results from TGA determination. The formula of the conjugates was determined with ICP-OES. The interactions between the conjugated QDs and the PNA protein were measured using SPR, which revealed that higher lactose density favored binding affinity under the same concentration, and Lac-QDs exhibit higher affinity than LacPEG-QDs. We further used a solid phase assay to assess the anti-adhesion activity of Lac-QDs and LacPEG-QDs on the cell level. The results showed that Lac-QDs had stronger activity in preventing THP1 from adhering to HUVEC than LacPEG-QDs, which was consistent with the SPR results. We reasoned that decrease in the conformational entropy induced by appropriate restriction of sugar flexibility could enhance the binding affinity of glyco-QDs, which implies that entropy change may be the main contributor to the interaction between high valent glyco-QDs and protein. The fabrication of lactose on QDs provides a fluorescent multivalent carbohydrate probe that can be used as mimics of glycoprotein for the study of carbohydrate-protein interactions and cell imaging.


►►Polyvalent lactose-quantum dot conjugate for fluorescent labeling of live leukocytes

Min Yu,$ Yang Yang,$ Rongcheng Han, Qiang Zheng, Lijun Wang, Yuankai Hong, Zhongjun Li,* and Yinlin Sha*($ contribute equally)

Langmuir. 26(11): 8534-8539, 2010

 

Oligosaccharides play crucial roles in many biorecognition processes by the so called “cluster glycosidic effect”. We here report a facile synthesis of lactose-CdSeS/ZnS quantum dot conjugate (Lac-QDs) by use of 1-thiol-β-D-lactose via ligand exchange, which exhibit significantly high affinity and specificity to leukocytes in contrast to the monovalent lactose. Structural analyses indicate that there are about 132 lactosyl molecules assembled on single QDs and the hydrodynamic diameter is small, close to 8.2 nm. Further, Lac-QDs display good fluorescence and physicochemical stability in physiological conditions, as well as extremely low cytotoxicity. These properties facilitate the use of Lac-QDs in fluorescent labeling of live leukocytes.


►►The role of calcium ions in the interactions of PrP106-126 amide with model membranes

Jian Zhong, Chunhui Yang, Wenfu Zheng, Lixin Huang, Yuankai Hong, Lijun Wang, Yinlin Sha*

Colloids and Surfaces B: Biointerfaces. 77: 40-46, 2010

In this work, we investigated the interactions of PrP106-126 amide with 1-palmitoyl-2-oleoyl-3-phosphocholine (POPC) and POPC/bovine brain sphingomyelin (BSM) membranes in the presence of calcium ions by in situ time-lapse atomic force microscopy (AFM) and circular dichroism (CD). The CD results show that Ca2+ has no obvious effects on the random coil conformation of PrP106-126 amide. The tapping mode AFM results demonstrate that electrostatic interaction decreases the measured heights of supported lipid bilayers (SLBs) in HBS-Ca2+ solution. Electrostatic interaction analysis also can be used to determine the applied force in liquid tapping mode AFM. The interactions of PrP106-126 amide with membranes by AFM demonstrate the following: (i) Ca2+ inhibits the interaction of PrP106-126 amide with POPC lipid and (ii) the co-interaction between Ca2+ and BSM increases the poration ability of PrP106-126 amide. These results imply that the main role of Ca2+ in the interactions of PrP106-126 amide with membranes is changing the surface properties of the membranes.


►►A designed β-hairpin forming peptide undergoes a consecutive process for self-assembly into nanofibrils

Chong Wang, Yinlin Sha*

Protein and Peptide Letters. 17(4): 410-415, 2010

We used a de novo designed, beta-hairpin forming T1 peptide as a model to investigate the kinetics of peptide fibrogenesis by a combination of light scattering (LS), circular dichroism (CD), fluorescence, and atomic force microscopy (AFM). The results demonstrated that the T1 fibrogenesis underwent a consecutive stepwise process, with a high degree of cooperation, presenting sigmoidal time-courses of the peptide aggregation, the subsequent conformational conversion of the backbone, and the peptide sidechains' rearrangement. We suggested that the conformational conversion was initiated after the peptide aggregates reach a dimensional size threshold, which could be a key step in the formation of beta-structural nuclei that catalyze the subsequent reactions. Furthermore, besides triggering the peptide aggregation, the interactions between the peptide sidechains predominately facilitate the regular alignment of the peptide molecules and the formation of a well-defined suprastructure. This work provides an insight of the hierarchical self-assembly of beta-hairpin forming peptides. It is helpful for designing beta-structural peptides for self-assembly into nanowires, which would have potential applications in the construction of nano-materials.


►►A facile synthesis of small-sized, highly photoluminescent and monodisperse CdSeS/SiO2 for live cell imaging

Rongcheng Han, Min Yu, Qiang Zheng, Lijun Wang, Yuankai Hong, Yinlin Sha*

Langmuir. 25 (20): 12250-5, 2009

In recent years, silica coating has been extensively investigated to fabricate the biocompatible interface of quantum dots (QDs) for biomedical applications. We here describe a facile and efficient method of synthesizing high-quality silica-coated CdSeS QDs (CdSeS QD/SiO2), where an immediate photoluminescence-favorable microenvironment is first created by assembling amphiphilic molecules around the CdSeS core, and a thin silica shell is further introduced to protect this hydrophobic interlayer. The prepared CdSeS QD/SiO2 exhibits excellent properties such as good water solubility, low cytotoxicity, and high quantum yield (QY, up to 0.49) as well as the resistance of photobleaching in aqueous solution. Also, the CdSeS QD/SiO2 nanoparticles homogeneously comprise single CdSeS cores and hold a comparatively small size up to about 11 nm in diameter. Particularly, this method leads to a significant increase in QY as compared to the uncoated CdSeS QDs (~109% of the initial QY), though only thin silica shells formed in the CdSeS QD/SiO2 structure. By coupling with folic acids, the CdSeS QD/SiO2 conjugates were successfully used for tumor cell labeling. Our results demonstrated a robust hydrophobic QDs-based approach for preparing highly photoluminescent, biocompatible QD/SiO2 through creation of a stable hydrophobic interlayer surrounding the QD cores, which could be also suitable for silica coating of other kinds of hydrophobic nanoparticles.


►►Ganglioside GM1 binding the N-terminus of amyloid precursor protein

Handi Zhang, Jixin Ding, Wenqiang Tian, Lijun Wang, Lixin Huang, Yan Ruan, Tianlan Lu, Yinlin Sha*, Dai Zhang*

Neurobiology of Aging. 30 (8): 1245-53, 2009

Secreted amyloid precursor protein (APPs) plays a role in several neuronal functions, including the promotion of synaptogenesis, neurite outgrowth and neuroprotection. Previous study has demonstrated that ganglioside GM1 inhibits the secretion of APPs; however the underlying mechanism remains unknown. Here we reported that GM1 can bind cellular full length APP and APPs secreted from APP695 stably-transfected SH-SY5Y cells. To characterize the GM1–APP interaction further, we expressed and purified recombinant fragments of the N-terminal APP. Immunoprecipitation experiments revealed that GM1 was able to bind the recombinant APP18–81 fragment. Moreover, the synthetic peptide APP52–81 could inhibit the binding. Therefore, the binding site for GM1 appears to be located within residues 52–81 of APP. Furthermore, we found that only GM1, but not GD1a, GT1b and ceramide, binds APP-N-terminus, indicating that the specific binding depends on the sugar moiety of GM1. Fluorescent studies revealed a decrease in the intrinsic fluorescence intensity of the APP52–81 peptide in phosphatidylcholine (PC)/GM1 vesicles. By using FTIR techniques, we found that the major secondary structure of the APP52–81 peptide was altered in PC/GM1 vesicles. Our results demonstrate that GM1 binds the N-terminus of APP and induces a conformational change. These findings suggest that secreted APP is decreased by membrane GM1 binding to its precursor protein and provide a possible molecular mechanism to explain the involvement of GM1 in APP proteolysis and pathogenesis of Alzheimer's disease.


►►PrP106-126 peptide disrupts lipid membranes: influence of C-terminal amidation

Wenfu Zheng, Lijun Wang, Yuankai Hong, Yinlin Sha*

Biochemical and Biophysical Research Communications. 379 (2): 298-303, 2009

PrP106–126 is located within the important domain concerning membrane related conformational conversion of human Prion protein (from cellular isoform PrPC to scrapie isoform PrPSc). Recent advances reveal that the pathological and physicochemical properties of PrP106–126 peptide are very sensitive to its N-terminal amidation, however, the detailed mechanism remains unclear. In this work, we studied the interactions of the PrP106–126 isoforms (PrP106–126CONH2 and PrP106–126COOH) with the neutral lipid bilayers by atomic force microscopy, surface plasmon resonance and fluorescence spectroscopy. The membrane structures were disturbed by the two isoforms in a similarly stepwise process. The distinct morphological changes of the membrane were characterized by formation of semi-penetrated defects and sigmoidal growth of flat high-rise domains on the supported lipid bilayers. However, PrP106–126COOH displayed a higher peptide–lipid binding affinity than PrP106–126CONH2 (~2.9 times) and facilitated the peptide–lipid interactions by shortening the lag time. These results indicate that the C-terminal amidation may influence the pathological actions of PrP106–126 by lowering the interaction potentials with lipid membranes.


►►Effects of lipid composition and phase on the membrane interaction of the Prion peptide 106–126 amide

Jian Zhong, Wenfu Zheng, Lixin Huang, Yuankai Hong, Lijun Wang, Yinlin Sha*

Biophysical Journal. 96(11): 4610-4621, 2009

Lipid rafts are specialized liquid-ordered (Lo) phases of the cell membrane that are enriched in sphingolipids and cholesterol (Chl), and surrounded by a liquid-disordered (Ld) phase enriched in glycerophospholipids. Lipid rafts are involved in the generation of pathological forms of proteins that are associated with neurodegenerative diseases. To investigate the effects of lipid composition and phase on the generation of pathological forms of proteins, we constructed an Ld-gel phase-separated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/sphingomyelin (from bovine brain (BSM))-supported lipid bilayer (SLB) and an Ld-Lo phase-separated POPC/BSM/Chl SLB. We used in situ time-lapse atomic force microscopy to study the interactions between these SLBs and the prion peptide K106TNMKHMAGAAAAGAVVGGLG126 (PrP106–126) amide, numbered according to the human prion-peptide sequence. Our results show that: 1), with the presence of BSM in the Ld phase, the PrP106–126 amide induces fully penetrated porations in the Ld phase of POPC/BSM SLB and POPC/BSM/Chl SLB; 2), with the presence of both BSM and Chl in the Ld phase, the PrP106–126 amide induces the disintegration of the Ld phase of POPC/BSM/Chl SLB; and 3), with the presence of both BSM and Chl in the Lo phase, PrP106–126 amide induces membrane thinning in the Lo phase of POPC/BSM/Chl SLB. These results provide comprehensive insight into the process by which the PrP106–126 amide interacts with lipid membranes.


Recent Presentation

  • 沙印林 第30次双清论坛,软物质材料科学前沿与交叉问题研讨会,2008 北京

  • 沙印林 第309次香山科学会议,软物质科学问题,2007 北京

  • 沙印林 量子点生物探针技术 北京大学生物医学跨学科讲座第149讲 2007年3月28日

  • 沙印林 量子点生物探针 南开大学元素有机化学国家重点实验室 2006年12月27日


北京大学 单分子与纳米生物学实验室

Single-molecule & Nanobiology Laboratory,Peking University