Mucin-type O-glycosylation and its potential use in drug and vaccine development

Mucin-type O-glycans are found on mucins as well as many other glycoproteins. The initiation step in synthesis is catalyzed by a large family of polypeptide GalNAc-transferases attaching the first carbohydrate residue, GalNAc, to selected serine and threonine residues in proteins. During the last decade an increasing number of GalNAc-transferase isoforms have been cloned and their substrate-specificities partly characterized. These differences in substrate specificities have been exploited for in vitro site-directed O-glycosylation. In GlycoPEGylation™, polyehylene glycol (PEG) is transferred to recombinant therapeutics to specific acceptor sites directed by GalNAc-transferases. GalNAc-transferases have also been used to control density of glycosylation in the development of glycopeptide-based cancer vaccines. The membrane-associated mucin-1 (MUC1) has long been considered a target for immunotherapeutic and immunodiagnostic measures, since it is highly overexpressed and aberrantly O-glycosylated in most adenocarcinomas, including breast, ovarian, and pancreatic cancers. By using vaccines mimicking the glycosylation pattern of cancer-cells, it is possible to overcome tolerance in transgenic animals expressing the human MUC1 protein as a self-antigen providing important clues for an improved MUC1 vaccine design. The present review will highlight some of the potential applications of site-directed O-glycosylation.     

Mucin-type O-glycosylation in Mesocestoides vogae (syn. corti)

Protein glycosylation is an important post-translational modification underlying host-parasite interactions, which may determine the outcome of infection. Although Mesocestoides vogae represents an important model for investigating the various aspects of cestode biology, virtually no information is available about the structure and synthesis of glycans in this parasite. In this work, focused on the initiation pathway of mucin-type O-glycosylation in M. vogae, we characterized O-glycoproteins bearing the simple mucin-type cancer-associated Tn and sialyl-Tn antigens, and the expression and activity of ppGalNAc-T, the key enzyme responsible for the first step of mucin-type O-glycosylation. Using immunohistochemistry, Tn and sialyl-Tn antigens were detected mainly in the tegument (microtriches) and in parenchymal cells. Tn expression was also observed in lateral nerve cords. Both Tn and sialyl-Tn antigens were detected in in vitro cultured parasites. Based on their electrophoretic mobility, Tn- and sialyl-Tn-bearing glycoproteins from M. vogae were separated into several components of 22 to 60 kDa. The observation that Tn and sialyl-Tn glycoproteins remained in the 0.6N perchloric acid-soluble fraction suggested that they could be good candidates for characterizing mucin-type glycosylation in this parasite. O-glycoproteins were purified and initially characterized using a proteomic approach. Immunohistochemical analysis of the tissue distribution of ppGalNAc-T revealed that this enzyme is expressed in the sub-tegumental region and in the parenchyma of the parasite. In M. vogae cultured in vitro, ppGalNAc-T was mainly detected in the suckers. Using a panel of 8 acceptor substrate synthetic peptides, we found that M. vogae ppGalNAc-T preferentially glycosylate threonine residues, the best substrates being peptides derived from human mucin MUC1 and from Trypanosoma cruzi mucin. These results suggest that M. vogae might represent a useful model to study O-glycosylation, and provide new research avenues for future studies on the glycopathobiology of helminth parasites.     

Parasite annexins--new molecules with potential for drug and vaccine development

In the last few years, annexins have been discovered in several nematodes and other parasites, and distinct differences between the parasite annexins and those of the hosts make them potentially attractive targets for anti-parasite therapeutics. Annexins are ubiquitous proteins found in almost all organisms across all kingdoms.Here, we present an overview of novel annexins from parasitic organisms, and summarize their phylogenetic and biochemical properties, with a view to using them as drug or vaccine targets. Building on structural and biological information that has been accumulated for mammalian and plant annexins, we describe a predicted additional secondary structure element found in many parasite annexins that may confer unique functional properties, and present a specific antigenic epitope for use as a vaccine.     

Liposome-mediated cytosolic delivery of macromolecules and its possible use in vaccine development.

In the majority of bacterial and viral infections the generation of cytotoxic T cells is of particular interest because such pathogens are able to escape the host defence mechanisms by surviving intracellularly within the phagocytic cells. To generate a CD8+ T lymphocyte response against exogenous antigens, the prerequisite is their delivery into the cytosol followed by processing and presentation along with class I major histocompatibility complex (MHC-I) molecules. In the present study we describe the method of liposome-based delivery of antigens and other macromolecules into the cytosol of target cells. To develop safe and effective methods for generating CD8+ T lymphocytes, we exploited the fusogenic character of lipids derived from lower organisms, that is baker's yeast (Saccharomyces cerevisiae). The degree of fusion with model membrane systems using yeast lipid liposomes varied from 40-70%, as opposed to 1-8% observed with egg PtdCho liposomes, depending on the assay system used. The fusion of yeast lipid liposomes with macrophages resulted in effective delivery of the entrapped solutes into the cytoplasmic compartment. This was further supported by the inhibition of cellular protein synthesis in J774 A1 cells by ricin A, encapsulated in the yeast lipid liposomes. Interestingly, the model antigen ovalbumin, when entrapped in the yeast lipid liposomes, successfully elicited antigen reactive CD8+ T cell responses. It may be concluded that the liposomes made of lipids derived from S. cerevisiae can spontaneously fuse with macrophages, delivering a significant portion of their contents into the cytoplasmic compartment of the cells.     

Mucin-type O-glycosylation in Fasciola hepatica: characterisation of carcinoma-associated Tn and sialyl-Tn antigens and evaluation of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity

Simple mucin-type cancer-associated O-glycan structures, such as the Tn antigen (GalNAc-O-Ser/Thr), are expressed by certain helminth parasites. These antigens are involved in several types of receptor-ligand interactions, and they are potential targets for immunotherapy. The aim of this work was to study the initiation pathway of mucin-type O-glycosylation in Fasciola hepatica, performing a biochemical and immunohistochemical characterisation of Tn and sialyl-Tn antigens, and evaluating the ppGaNTase activity, which catalyses the first step in O-glycan biosynthesis. Using ELISA, both Tn and sialyl-Tn antigens were detected predominantly in the somatic and deoxycholate extracts. Immunofluorescence analysis revealed that Tn antigen is preferentially expressed in testis, while sialyl-Tn glycoproteins were more widely distributed, being present in parenchymal cells, basal membrane of the tegument, and apical surface of epithelial cells lining the caeca. On the basis of their electrophoretic mobility, Tn glycoproteins were resolved as six components of 10, 37, 76, 125, 170 and 205 kDa, and sialyl-Tn components showed an apparent molecular mass of 28 and 32 kDa, and two broad bands of 90-110 and 170-190 kDa. The observation that only the 76 kDa Tn-glycoprotein remained in the 0.6 N perchloric acid-soluble fraction suggests that it could be a good candidate for mucin characterisation in this parasite. The ppGaNTase activity showed its maximal activity at pH 7-7.5 and 37 degrees C, showing that Mn(2+) was the best divalent cation activator. Using a panel of nine synthetic peptides as acceptor substrates, we found that F. hepatica ppGaNTase was able to glycosylate both threonines and serines, the best substrates being the peptides derived from the tandem repeat region of human mucins (MUC2 and MUC6), and from Trypanosoma cruzi and Trypanosoma brucei glycoproteins. The results reported here constitute the first evidence on O-glycosylation pathways in F. hepatica, and may help to identify new biological characteristics of this parasite as well as of the host-parasite relationship.     

The E. coli alpha-hemolysin secretion system and its use in vaccine development.

Many Gram-negative bacteria use a type I secretion system to translocate proteins, including pore-forming toxins, proteases, lipases and S-layer proteins, across both the inner and outer membranes into the extracellular surroundings. The Escherichia coli alpha-hemolysin (HlyA) secretion system is the prototypical and best characterized type I secretion system. The structure and function of the components of the HlyA secretion apparatus, HlyB, HlyD and TolC, have been studied in great detail. The functional characteristics of this secretion system enable it to be used in a variety of different applications, including the presentation of heterologous antigens in live-attenuated bacterial vaccines. Such vaccines can be an effective delivery system for heterologous antigens, and the use of a type I secretion system allows the antigens to be actively exported from the cytoplasm of the bacterial carrier rather than only becoming accessible to the host immune system after bacterial disintegration.     

The development and use of vaccine adjuvants

Interest in vaccine adjuvants is intense and growing, because many of the new subunit vaccine candidates lack sufficient immunogenicity to be clinically useful. In this review, I have emphasized modern vaccine adjuvants injected parenterally, or administered orally, intranasally, or transcutaneously with licensed or experimental vaccines in humans. Every adjuvant has a complex and often multi-factorial immunological mechanism, usually poorly understood in vivo. Many determinants of adjuvanticity exist, and each adjuvanted vaccine is unique. Adjuvant safety is critical and can enhance, retard, or stop development of an adjuvanted vaccine. The choice of an adjuvant often depends upon expensive experimental trial and error, upon cost, and upon commercial availability. Extensive regulatory and administrative support is required to conduct clinical trials of adjuvanted vaccines. Finally, comparative adjuvant trials where one antigen is formulated with different adjuvants and administered by a common protocol to animals and humans can accelerate vaccine development.     

Issues in tick vaccine development: identification and characterization of potential candidate vaccine antigens

It is well established that acquired immunity against tick infestation can be induced by repeated tick infestation or by active immunization with either crude or purified native as well as recombinant antigens. This review provides insights into the development of tick vaccines with reference to identification, purification and molecular cloning of candidate target antigens.     

The acceptor substrate specificity of human beta4-galactosyltransferase V indicates its potential function in O-glycosylation.

In order to assess the function of the different human UDP-Gal:GlcNAc beta4-galactosyltransferases, the cDNAs of two of them, beta4-GalT I and beta4-GalT V, were expressed in the baculovirus/insect cell expression system. The soluble recombinant enzymes produced were purified from the medium and used to determine their in vitro substrate specificities. The specific activity of the recombinant beta4-GalT V was more than 15 times lower than that of beta4-GalT I, using GlcNAc beta-S-pNP as an acceptor. Whereas beta4-GalT I efficiently acts on all substrates having a terminal beta-linked GlcNAc, beta4-GalT V appeared to be far more restricted in acceptor usage. Beta4-GalT V acts with high preference on acceptors that contain the GlcNAc beta1-->6GalNAc structural element, as found in O-linked core 2-, 4- and 6-based glycans, but not on substrates related to V-linked or blood group I-active oligosaccharides. These results suggest that beta4-GalT V may function in the synthesis of lacNAc units on O-linked chains, particularly in tissues which do not express beta4-GalT I, such as brain.     

昆虫几丁质酶及其在植物保护中的应用

本文从生理学、生物化学和分子生物学３个方面综述了昆虫几丁质酶的研究进展,并概述了它在害虫防治、主要是在抗虫育种中作为重要的基因源中的应用。e     

Gene expression profiling and its practice in drug development

The availability of sequenced genomes of human and many experimental animals necessitated the development of new technologies and powerful computational tools that are capable of exploiting these genomic data and ask intriguing questions about complex nature of biological processes. This gave impetus for developing whole genome approaches that can produce functional information of genes in the form of expression profiles and unscramble the relationships between variation in gene expression and the resulting physiological outcome. These profiles represent genetic fingerprints or catalogue of genes that characterize the cell or tissue being studied and provide a basis from which to begin an investigation of the underlying biology. Among the most powerful and versatile tools are high-density DNA microarrays to analyze the expression patterns of large numbers of genes across different tissues or within the same tissue under a variety of experimental conditions or even between species. The wide spread use of microarray technologies is generating large sets of data that is stimulating the development of better analytical tools so that functions can be predicted for novel genes. In this review, the authors discuss how these profiles are being used at various stages of the drug discovery process and help in the identification of new drug targets, predict the function of novel genes, and understand individual variability in response to drugs.     

The use of functional human tissues in drug development

Fresh, functional human tissues have long been considered the closest possible model of human in vivo function and can be used to measure a wide range of pharmacological responses. Despite this, relatively little drug development is conducted using fresh human tissue because of the logistical and ethical difficulties surrounding the availability of tissue and practicalities of experimental work. Most tests of drug activity require a living test system comprising cells, tissues or whole organisms. In some instances, “living” (fresh) human tissues have the potential to reduce or replace animal tests through superior prediction of drug safety and efficacy. Before functional human tissue tests become a routine part of drug development, two factors must co-exist. Firstly, organisations such as Biopta must continue to create compelling evidence that human tissues are more predictive than alternative models; such evidence will drive demand from the pharmaceutical industry for human tissue-based tests. Secondly, the vast number of tissues and organs residual to surgery or unsuitable for transplant must be routinely consented for medical research and made available to all researchers in an equitable and timely manner. This requires a concerted effort throughout the NHS and consistent demand as well as financial support from researchers, particularly within industry. It is our view that the next 5–10 years will generate compelling evidence of the value of functional human tissue-based tests and recognition that more efficient use of residual or non-transplantable tissues and organs is an urgent priority for the development of new medicines.     

The use of functional human tissues in drug development

Drug development currently depends on animal models to provide an accurate prediction of human physiology and pathophysiology. However, as is clear from clinical trial failures during phases II and III, such in vivo models do not always predict the effects that a drug can elicit in humans. Tests with human tissues, which are obviously considered to be the closest model of human in vivo function, could fill the gap between animal-based tests and trials in patients. Despite clear advantages, logistical and ethical barriers prevent fresh human tissues from being widely used during drug development. Biopta is aiming to make human tissue testing a regular element of drug development, and works to lower the barriers surrounding the availability of tissue and practicalities of experimental work.     

跨国土地利用及其生态影响

在全球食物价格不断上涨,粮食安全已经威胁到经济安全的环境下,跨国农用地投资成为国际浪潮。跨国农用地投资推动着各国的土地资源向全球化资源转变,对土地的跨国利用成为全球土地资源优化配置的必然。在对跨国土地利用的背景及现状进行阐述的基础上,指出"代理性农业耕作"方式对区域生态环境可能造成的影响。同时,新型农业科技与跨国土地利用的结合,也在推动着农业经济发展的同时,改变着传统的土地利用模式,进而改变着区域生态环境。最后,指出需要对跨国土地利用做出理性分析并建立相关的国际规则,从而维护可持续粮食安全及生态安全。