Mucin Glycopeptide-Protein Conjugates – Promising Antitumor Vaccine Candidates

Recent efforts towards the development of synthetic glycopeptide vaccines, which aim at the active immunization of patients against their own tumor tissues, are outlined. To achieve sufficient tumor selectivity, glycopeptides of the tandem repeat region of tumor-associated mucin, MUC1, have been synthesized. Since the endogenous structures usually exert low immunogenicity, these glycopeptide antigens, as B-cell epitopes, were conjugated with immunostimulating components. In the present short review, work is outlined in which the MUC1 B-cell epitope peptides are conjugated with bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or tetanus toxoid (TTox). In particular, the synthetic vaccines based on tetanus toxoid induce very strong tolerance-breaking immune responses in mice. The induced antibodies of the IgG type indicate the installation of an immunological memory. In addition, these antibodies strongly bind to human breast tumor cells in culture, demonstrated by flow cytometry experiments, and also to the tumor cells in mammary carcinoma tissues.     

Antibody Induction Directed against the Tumor‐Associated MUC4 Glycoprotein

Mucin glycoproteins are important diagnostic and therapeutic targets for cancer treatment. Although several strategies have been developed to explore anti‐tumor vaccines based on MUC1 glycopeptides, only few studies have focused on vaccines directed against the tumor‐associated MUC4 glycoprotein. MUC4 is an important tumor marker overexpressed in lung cancer and uniquely expressed in pancreatic ductual adenocarcinoma. The aberrant glycosylation of MUC4 in tumor cells results in an exposure of its peptide backbone and the formation of tumor‐associated glycopeptide antigens. Due to the low immunogenicity of these endogenous structures, their conjugation with immune stimulating peptide or protein carriers are required. In this study, MUC4 tandem‐repeat glycopeptides were conjugated to the tetanus toxoid and used for vaccination of mice. Immunological evaluations showed that our MUC4‐based vaccines induced very strong antigen‐specific immune responses. In addition, antibody binding epitope analysis on glycopeptide microarrays, were demonstrating a clear glycosylation site dependence of the induced antibodies.     

纳豆菌糖肽对RAW264.7巨噬细胞的免疫调节作用

为考察纳豆菌糖肽(BNGP)对正常状态及脂多糖(LPS)激活状态巨噬细胞的免疫调节作用,本文用不同浓度的BNGP单独处理或LPS和不同浓度的BNGP共同处理RAW264.7巨噬细胞,采用噻唑蓝(MTT)比色法检测细胞增殖作用,中性红吞噬试验检测吞噬能力,Griess试剂检测细胞培养上清液中NO含量,酶联免疫分析法(ELISA)检测细胞培养上清液中细胞因子(IL-1β、TNF-α)、促炎介质(PGE2)含量,Western Blot检测COX-2和i NOS蛋白表达水平。试验结果表明:BNGP在62.5~500μg/m L浓度范围内能提高正常状态的RAW264.7巨噬细胞的代谢活力,增加NO、IL-1β、TNF-α、PGE2的分泌量,提高COX-2和i NOS的表达量,高浓度(125μg/m L)能增强巨噬细胞的吞噬能力;当细胞处于LPS激活状态时,BNGP在62.5~500μg/m L浓度范围内能抑制IL-1β、TNF-α、PGE2的分泌,浓度为500μg/m L时可抑制NO的产生及i NOS的表达,高浓度(125μg/m L)则能下调COX-2的表达。     

Dissecting the Molecular Basis of the Role of the O‐Mannosylation Pathway in Disease: α‐Dystroglycan and Forms of Muscular Dystrophy

Dystroglycanopathies form a subgroup of muscular dystrophies that arise from defects in enzymes that are implicated in the recently elucidated O‐mannosylation pathway, thereby resulting in underglycosylation of α‐dystroglycan. The emerging identification of additional brain proteins modified by O‐mannosylation provides a broader context for interpreting the range of neurological consequences associated with dystroglycanopathies. This form of glycosylation is associated with protein mucin‐like domains that present numerous serine and threonine residues as possible sites for modification. Furthermore, the O‐Man glycans coexist in this region with O‐GalNAc glycans (conventionally associated with such protein sequences), thus resulting in a complex glycoconjugate landscape. Sorting out the relationships between the various molecular defects in glycosylation and the modes of disease presentation, as well as the regulatory interplay among the O‐Man glycans and the effects on other modes of glycosylation in the same domain, is challenging. Here we provide a perspective on chemical biology approaches employing synthetic and analytical methods to address these questions.     

Synthetic MUC1 Antitumor Vaccine Candidates with Varied Glycosylation Pattern Bearing R/S‐configured Pam3CysSerLys4

The Toll‐like receptor 2 ligand Pam₃CysSer is of particular interest for the construction synthetic vaccines because of its ability to stimulate of the innate immune system. Such vaccines usually comprise Pam₃CysSer with the natural R‐configuration at the glycerol 2‐position. Pam₃CysSer peptide vaccines with natural configuration have been shown to be more efficient than the corresponding R/S diastereomers. In order to clarify whether the effect of the configuration of Pam₃Cys on the immune response also applies to glycopeptide vaccines, MUC1 glycopeptide–lipopeptide vaccines bearing either R‐ or R/S‐configured Pam₃CysSerLys₄ were compared for their immunological effects. In order to find out whether glycosylated MUC1 tandem repeat domains comprise not only B‐cell epitopes but also T‐cell epitopes, two‐component vaccines containing the Pam₃CysSerLys₄ lipopeptide and MUC1 glycopeptides with various glycosylation patterns were synthesized, and their immune reactions in mice were studied.     

β-伴大豆球蛋白水解产物中糖肽的分离和鉴定

本研究对大豆蛋白水解产物中含糖的肽组分进行了分离和鉴定。从大豆蛋白中分离出相对纯度为75．5％、含糖量为3．23％的B．伴大豆球蛋白后，以β-伴大豆球蛋白为底物，用Alcalase进行酶解调制大豆肽。所得大豆肽SephadexG-25凝胶过滤，得到两个组分P1和P2，分子量较大P1含糖量为8．23％。用薄板层析对这一含糖的肽组分进行了鉴定，结果表明此含糖的肽为糖结合性肽。     

Chemoenzymatic Synthesis of Glycopeptides to Explore the Role of Mucin 1 Glycosylation in Cell Adhesion

Post-translational modifications affect protein biology under physiological and pathological conditions. Efficient methods for the preparation of peptides and proteins carrying defined, homogeneous modifications are fundamental tools for investigating these functions. In the case of mucin 1 (MUC1), an altered glycosylation pattern is observed in carcinogenesis. To better understand the role of MUC1 glycosylation in the interactions and adhesion of cancer cells, we prepared a panel of homogeneously O-glycosylated MUC1 peptides by using a quantitative chemoenzymatic approach. Cell-adhesion experiments with MCF-7 cancer cells on surfaces carrying up to six differently glycosylated MUC1 peptides demonstrated that different glycans have a significant impact on adhesion. This finding suggests a distinct role for MUC1 glycosylation patterns in cancer cell migration and/or invasion. To decipher the molecular mechanism for the observed adhesion, we investigated the conformation of the glycosylated MUC1 peptides by NMR spectroscopy. These experiments revealed only minor differences in peptide structure, therefore clearly relating the adhesion behaviour to the type and number of glycans linked to MUC1.