Yan-Mei Li

Copper binding properties of a tau peptide associated with Alzheimer's disease studied by CD, NMR, and MALDI-TOF MS

We have previously reported the copper binding properties of R3 peptide (residues 318-335: VTSKCGSLGNIHHKPGGG, according to the longest tau protein) derived from the third repeat microtubule-binding domain of water-soluble tau protein. In this work, we have investigated copper binding properties of R2 peptide (residues 287-304: VQSKCGSKDNIKHVPGGG) derived from the second repeat region of tau protein. Similar to R3 peptide, R2 peptide also plays an important role in the formation of neurofibrillary tangles (NFTs) which is one of the two main biological characteristics of Alzheimers disease (AD). Based on the copper binding properties of R2 peptide, the possible influences of the binding on the formation of NFTs were investigated. Results from circular dichroism (CD) spectra, nuclear magnetic resonance (NMR) spectroscopy, and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) suggest that the binding is pH-dependent and stoichiometry-determined. In addition, these results also reveal that R2 peptide adopts a monomeric alpha-helical structure in aqueous solutions at physiological pH after the addition of 1 mol equiv. of Cu2+. Since alpha-helix structure is responsible for the formation of paired helical filaments (PHFs) which aggregate into NFTs, it is hypothesized that Cu2+ induces R2 peptide to self-assemble into a PHFs-like structure. Hence, it is postulated that Cu2+ plays an important role in the aggregation of R2 peptide and tau protein and that copper binding to R2 peptide may be another possible involvement in AD.

Self‐Adjuvanting Synthetic Antitumor Vaccines from MUC1 Glycopeptides Conjugated to T‐Cell Epitopes from Tetanus Toxoid

The T-helper epitope peptide P30 (green in the scheme) from tetanus toxoid was used as the immunostimulant in MUC1 glycopeptide antitumor vaccines and apparently also acts as a built-in adjuvant. P30-conjugated glycopeptide vaccines containing three glycans in the immunodominant motifs PDTRP and GSTAP induced much stronger immune responses and complement dependent cytotoxicity mediated killing of tumor cells when applied in plain PBS solution without complete Freunds adjuvant.

Fully Synthetic Self‐Adjuvanting Thioether‐Conjugated GlycopeptideLipopeptide Antitumor Vaccines for the Induction of Complement‐Dependent Cytotoxicity against Tumor Cells

Glycopeptides of tumor-associated mucin MUC1 are promising target structures for the development of antitumor vaccines. Because these endogenous structures were weakly immunogenic, they were coupled to immune-response-stimulating T-cell epitopes and the Pam(3)Cys lipopeptide to induce strong immune responses in mice. A new thioether-ligation method for the synthesis of two- and three-component vaccines that contain MUC1 glycopeptides as the B-cell epitopes, a T-cell epitope peptide, and the Pam(3)CSK(4) lipopeptide is described. The resulting fully synthetic vaccines were used for the vaccination of mice, either in a liposome with Freunds adjuvant or in aqueous PBS buffer. The three-component vaccines that contained the Tetanus Toxoid P2 T-cell epitope peptide induced strong immune responses, even when administered just in PBS. By activation of the complement-dependent cytotoxicity (CDC) complex, the antisera induced the killing of tumor cells.

Variation of the Glycosylation Pattern in MUC1 Glycopeptide BSA Vaccines and Its Influence on the Immune Response

Because of its overexpression on almost all types of epithelial tumor tissues, the tumor-associated mucin MUC1 is an attractive target antigen for cancer immunotherapy. A number of research groups are interested in the development of antitumor vaccines based on this mucin. Compared with MUC1 on normal cells, the glycan profile of tumor-associated MUC1 is characteristically changed, in particular, in the extracellular domain, which consists of a variable number of tandem repeat sequences HGVTSAPDTRPAPGSTAPPA containing five potential O-glycosylation sites (T4, S5, T9, S15, and T16). As a resulting of downregulation of a glucosaminyl transferase and concomitant overexpression of sialyl transferases, MUC1 on tumor cells carries short, often prematurely sialylated glycan side chains. The tumorassociated carbohydrate antigens comprise the Thomsen– Friedenreich antigen (T antigen), its precursor (Tn antigen), and their respective sialylated derivatives STn and 2,6-ST. Because of the truncated, short saccharide side chains, peptide epitopes within the protein backbone of tumorassociated MUC1 are uncovered. These aberrant glycopeptides are considered promising target structures for the development of antitumor vaccines. Owing to the biological microheterogeneity of glycoproteins, MUC1 isolated from tumor cell membranes is not suitable for vaccination. 2e] Synthetic glycopeptides of the MUC1 tandem repeat sequence which contain structurally defined saccharides should facilitate the induction of sufficiently tumor-selective immune responses. As recently discussed, the glycosylation of serine and/or threonine in the MUC1 tandem repeat sequence can distinctly influence the conformation of the peptide backbone and the immunogenicity of the tumor-associated glycopeptides. Therefore, the influence of the different saccharide attachment sites within the MUC1 glycopeptide vaccine on the immune responses is of particular interest. In a previous study, synthetic MUC1 glycopeptides bearing Tn and/or T antigens at serine 15 (S15) and threonine 9 (T9) of the tandem repeat sequence were conjugated to bovine serum albumin (BSA) as the carrier protein (Scheme 1). Immunization of Balb/c mice with these vaccines revealed that the glycosylation of the tandem repeat sequence with either the Tn or the T antigen at position T9, which belongs to the immunodominant PDTRP epitope, enhanced the immune response. These results prompted us to synthesize MUC1 tandem repeat peptides bearing the tumorassociated sialylated saccharide antigens STn or 2,6-ST at position S15, while threonine T9 is linked to Tn or T antigen or is not glycosylated. As sialyltransferases have been found to be strongly overexpressed on epithelial tumor cells, the investigation of MUC1 vaccines with sialylated saccharide antigens is considered of particular interest. The glycopeptides were covalently linked to BSA. The resulting vaccines were used for immunization of Balb/c mice. The antibody titers of the induced antisera were determined by enzyme-linked immunosorbent assay (ELISA). In addition, the isotypes of the induced antibodies were examined. The binding of the induced sera to the MUC1 expressed on human MCF-7 breast tumor cells was examined by flow cytometry (FACS analysis). The microwave-supported solid-phase synthesis of the MUC1 glycopeptides containing the STn or the 2,6-ST antigen at site S15 and Tn or T antigen at site T9 was performed starting from a 2-chlorotrityl resin preloaded with Fmoc-alanine (Scheme 2). The peptide coupling was carried out with Fmoc amino acids (6 equiv) using HBTU/HOBt. The more reactive HATU/HOAt was used for coupling of the [*] H. Cai, Z. H. Huang, L. Shi, Z. Y. Sun, Prof. Dr. Y. F. Zhao, Prof. Dr. Y. M. Li Key Lab of Bioorganic Phosphorus Chemistry & Chemical Biology Department of Chemistry, Tsinghua University Beijing 100084 (P.R. China) E-mail: liym@mail.tsinghua.edu.cn

Copper-induced cytotoxicity: reactive oxygen species or islet amyloid polypeptide oligomer formation

Copper enhances amyloid cytotoxicity and mediates human islet amyloid polypeptide (hIAPP) oligomerization; nickel, a redox inactive metal with similar protein binding affinity to copper, also mimics this effect, thereby demonstrating copper-mediated hIAPP cytotoxicity is due mainly to granular oligomer generation rather than ROS accumulation in type 2 diabetes.

Towards a Fully Synthetic MUC1‐Based Anticancer Vaccine: Efficient Conjugation of Glycopeptides with Mono‐, Di‐, and Tetravalent Lipopeptides Using Click Chemistry

The membrane-bound tumor-associated glycoprotein MUC1 is aberrantly glycosylated in cancer cells compared with normal cells, and is therefore considered an attractive target for cancer immunotherapy. However, tumor-associated glycopeptides from MUC1 do not elicit a sufficiently robust immune response. Therefore, antitumor vaccines were developed, which consist of MUC1 glycopeptides as the B epitopes and immune-stimulating toll-like receptoru20092 (TLRu20092) lipopeptide ligands. These fully synthetic vaccine candidates were prepared by solid-phase synthesis of the MUC1 glycopeptides. The Pam(3) Cys lipopeptide, also synthesized on solid-phase, was C-terminally coupled to oligovalent lysine cores, which N-terminally incorporate O-propargyl oligoethylene glycol acyl side chains. The MUC1 glycopeptides and lipopeptide lysine constructs were then conjugated by click chemistry to give oligovalent synthetic vaccines. Oligovalent glycopeptide-lipopeptide conjugates are considered more immunogenic than their monovalent analogues.

Influence of hydrophobicity on the surface-catalyzed assembly of the islet amyloid polypeptide.

The islet amyloid polypeptide (IAPP) is a hormonal factor secreted by the β-cells in the pancreas. Aggregation of misfolded IAPP molecules and subsequent assembly of amyloid nanofibrils are critical for the development of type 2 diabetes mellitus. In the physiological environment, amyloid aggregation is affected by the presence of interfaces such as cell membranes. The physicochemical properties of the interface dictates the interaction of the peptide with the surface, i.e., electrostatic and hydrophobic interactions on hydrophilic and hydrophobic surfaces, respectively. We have studied the influence of hydrophobicity on the surface-catalyzed assembly of IAPP on ultrasmooth hydrocarbon films grown on ion-beam-modified mica surfaces by atomic force microscopy. The contact angle θ of these surfaces can be tuned continuously in the range from ≤20° to ∼90° by aging the samples without significant changes of the chemical composition or the topography of the surface. On hydrophilic surfaces with a θ of ∼20°, electrostatic interactions induce the assembly of IAPP nanofibrils, whereas aggregation of large (∼2.6 nm) oligomers is observed at hydrophobic surfaces with a θ of ∼90°. At intermediate contact angles, the interplay between electrostatic and hydrophobic substrate interactions dictates the pathway of aggregation with fibrillation getting continuously delayed when the contact angle is increased. In addition, the morphology of the formed protofibrils and mature fibrils at intermediate contact angles differs from those observed at more hydrophilic surfaces. These results might contribute to the understanding of the surface-catalyzed assembly of different amyloid aggregates and may also have implications for the technologically relevant controlled synthesis of amyloid nanofibrils of desired morphology.

N-Phosphoryl Amino Acids and Biomolecular Origins. Review Paper in Honor of the 50th Anniversary of the Publication of ``A Production of Amino Acids under Possible Primitive Earth Conditions'' (Miller, 1953)

The possible role of phosphoryl amino acids for biomolecular origins is briefly reviewed. Peptide formation, ester formation, ester exchange on phosphorus and N to O migration occurred when the N-phosphoryl amino acid was incubated at room temperature. Short nucleotides and peptides were formed when nucleoside was reacted with N-phosphoryl amino acid at room temperature. Serine and threonine residues in their conjugate with different nucleosides (mediated with phosphorus) showed different self-cleavage activities. N-phosphoryl Histine and Ser-His dipeptide could cleave nucleic acids, proteins and esters in neutral medium. Based on a simple model, a pathway of `co-evolution of protein and nucleic acid was proposed.

O-GlcNAcylation modulates the self-aggregation ability of the fourth microtubule-binding repeat of tau

In Alzheimers disease (AD), tau protein is abnormally hyperphosphorylated and aggregated into paired helical filaments (PHFs). It was discovered recently that tau is also O-GlcNAcylated in human brains. And O-GlcNAcylation may regulate phosphorylation of tau in a site-specific manner. In this work, we focused on the fourth microtubule-binding repeat (R4) of tau, which has an O-GlcNAcylation site-Ser356. The aggregation behavior of this repeat and its O-GlcNAcylated form was investigated by turbidity, precipitation assay and electron microscopy. In addition, conformations of these two peptides were analyzed with circular dichroism (CD). Our results revealed that O-GlcNAcylation at Ser356 could greatly slow down the aggregation speed of R4 peptide. This modulation of O-GlcNAcylation on tau aggregation implies a new perspective of tau pathology.

DNA cleavage function of seryl-histidine dipeptide and its application

Summary.The double-stranded DNA or circular plasmid DNA can be cleaved by the Ser-His dipeptide by hydrolysis of the DNA-phospho-diester bond. The proper sequential order of the amino acids serine and histidine is apparently crucial in its unique cleavage activity as compared to the other di- or tri-peptides containing one of these amino acids. An inverted sequence of this dipeptide to a His-Ser linkage renders the peptide ineffective in the cleavage of DNA. In addition to the DNA cleavage function, Ser-His is also capable of cleaving other molecules, e.g., proteins, esters and RNAs. The cooperative actions of the hydroxyl group and the basic groups in the serine and histidine or related amino acids can be found in contemporary enzymes, such as DNase, serine proteases, lipases, esterases, chymotrypsin, trypsin, and elastase, etc. The Ser-His and related oligopeptides might have played important roles in the evolution of enzyme functions.