Glycomics: an integrated systems approach to structure-function relationships of glycans

In comparison with genomics and proteomics, the advancement of glycomics has faced unique challenges in the pursuit of developing analytical and biochemical tools and biological readouts to investigate glycan structure-function relationships. Glycans are more diverse in terms of chemical structure and information density than are DNA and proteins. This diversity arises from glycans' complex nontemplate-based biosynthesis, which involves several enzymes and isoforms of these enzymes. Consequently, glycans are expressed as an 'ensemble' of structures that mediate function. Moreover, unlike protein-protein interactions, which can be generally viewed as 'digital' in regulating function, glycan-protein interactions impinge on biological functions in a more 'analog' fashion that can in turn 'fine-tune' a biological response. This fine-tuning by glycans is achieved through the graded affinity, avidity and multivalency of their interactions. Given the importance of glycomics, this review focuses on areas of technologies and the importance of developing a bioinformatics platform to integrate the diverse datasets generated using the different technologies to allow a systems approach to glycan structure-function relationships.     

Characterization of new G protein-coupled adenine receptors in mouse and hamster

The nucleobase adenine has previously been reported to activate G protein-coupled receptors in rat and mouse. Adenine receptors (AdeR) thus constitute a new family of purine receptors, for which the designation “P0-receptors” has been suggested. We now describe the cloning and characterization of two new members of the AdeR family from mouse (MrgA10, termed mAde1R) and hamster (cAdeR). Both receptors were expressed in Sf9 insect cells, and radioligand binding studies were performed using [³H]adenine. Specific binding of the radioligand was detected in transfected, but not in untransfected cells, and K _D values of 286 nM (mAde1R, B _max 1.18 pmol/mg protein) and 301 nM (cAdeR, B _max 17.7 pmol/mg protein), respectively, were determined. A series of adenine derivatives was investigated in competition binding assays. Minor structural modifications generally led to a reduction or loss of affinity, with one exception: 2-fluoroadenine was at least as potent as adenine itself at the cAdeR. Structure–activity relationships at all AdeR orthologs and subtypes investigated so far were similar, but not identical. For functional analyses, the cAdeR was homologously expressed in Chinese hamster ovary (CHO) cells, while the mAde1R was heterologously expressed in 1321N1 astrocytoma cells. Like the previously described AdeRs from rat (rAdeR) and mouse (mAde2R), the mAde1R (EC₅₀ 9.77 nM) and the cAdeR (EC₅₀ 51.6 nM) were coupled to inhibition of adenylate cyclase. In addition, the cAdeR from hamster expressed in CHO cells produced an increase in intracellular calcium concentrations (EC₅₀ 6.24 nM) and was found to be additionally coupled to G_q proteins.     

JosD1, a Membrane-targeted Deubiquitinating Enzyme,Is Activated by Ubiquitination and Regulates Membrane Dynamics,Cell Motility,and Endocytosis

The functional diversity of deubiquitinating enzymes (DUBs) is not well understood. The MJD family of DUBs consists of four cysteine proteases that share a catalytic “Josephin” domain. The family is named after the DUB ATXN3, which causes the neurodegenerative disease Machado-Joseph disease. The two closely related Josephin domain-containing (JosD) proteins 1 and 2 consist of little more than the Josephin domain. To gain insight into the properties of Josephin domains, we investigated JosD1 and JosD2. JosD1 and JosD2 were found to differ fundamentally in many respects. In vitro, only JosD2 can cleave ubiquitin chains. In contrast, JosD1 cleaves ubiquitin chains only after it is monoubiquitinated, a form of posttranslational-dependent regulation shared with ATXN3. A significant fraction of JosD1 is monoubiquitinated in diverse mouse tissues. In cell-based studies, JosD2 localizes to the cytoplasm whereas JosD1 preferentially localizes to the plasma membrane, particularly when ubiquitinated. The membrane occupancy by JosD1 suggests that it could participate in membrane-dependent events such as cell motility and endocytosis. Indeed, time-lapse imaging revealed that JosD1 enhances membrane dynamics and cell motility. JosD1 also influences endocytosis in cultured cells by increasing the uptake of endocytic markers of macropinocytosis while decreasing those for clathrin- and caveolae-mediated endocytosis. Our results establish that two closely related DUBs differ markedly in activity and function and that JosD1, a membrane-associated DUB whose activity is regulated by ubiquitination, helps regulate membrane dynamics, cell motility, and endocytosis.     

Clinical mass spectrometry in neuroscience. Proteomics and peptidomics.

In this review we discuss the merits and drawbacks with the use of proteomic and peptidomic strategies for identification of proteins and peptides in their multidimensional interactions in complex biological processes. The progress in proteomics and peptidomics during the last years offer us new challenges to study changes in the protein and peptide synthesis. These strategies also offer new tools to follow post-translational modifications and other disturbed chemical processes that may be indicative of pathophysiological alteration(s). Furthermore these techniques can contribute to improvements in the diagnosis and therapy of neurodegenerative diseases, such as Alzheimer's disease, and psychiatric diseases, as depression and post traumatic stress disorders. We also consider different practical aspects of the applications of mass spectrometry in clinical neuroscience, illustrated by example from our laboratories. The new proteomic and peptidomic strategies will further enable the progress for clinical neuroscience research.     

Tropical Secondary Forest Management Influences Frugivorous Bat Composition,Abundance and Fruit Consumption in Chiapas,Mexico

Most studies on frugivorous bat assemblages in secondary forests have concentrated on differences among successional stages, and have disregarded the effect of forest management. Secondary forest management practices alter the vegetation structure and fruit availability, important factors associated with differences in frugivorous bat assemblage structure, and fruit consumption and can therefore modify forest succession. Our objective was to elucidate factors (forest structural variables and fruit availability) determining bat diversity, abundance, composition and species-specific abundance of bats in (i) secondary forests managed by Lacandon farmers dominated by Ochroma pyramidale, in (ii) secondary forests without management, and in (iii) mature rain forests in Chiapas, Southern Mexico. Frugivorous bat species diversity (Shannon H’) was similar between forest types. However, bat abundance was highest in rain forest and O. pyramidale forests. Bat species composition was different among forest types with more Carollia sowelli and Sturnira lilium captures in O. pyramidale forests. Overall, bat fruit consumption was dominated by early-successional shrubs, highest late-successional fruit consumption was found in rain forests and more bats consumed early-successional shrub fruits in O. pyramidale forests. Ochroma pyramidale forests presented a higher canopy openness, tree height, lower tree density and diversity of fruit than secondary forests. Tree density and canopy openness were negatively correlated with bat species diversity and bat abundance, but bat abundance increased with fruit abundance and tree height. Hence, secondary forest management alters forests’ structural characteristics and resource availability, and shapes the frugivorous bat community structure, and thereby the fruit consumption by bats.     

Towards more efficient longline fisheries: fish feeding behaviour,bait characteristics and development of alternative baits

Better knowledge of food search behaviour in fish is essential for studies that aim to improve longline fishing, particularly through bait development. This review provides an overview of our understanding of how fish detect and locate sources of food odour, focusing on the stimuli and sensory modalities involved, and on factors that affect feeding activity. Studies that identify feeding attractants and efforts to develop alternative longline baits are presented. The review reveals that such studies are few in number, and that to date there are no alternatives to traditional baits in commercial longlining despite the growing demand for these resources, which are also used for human consumption. The chemical compounds that elicit food search behaviour differ from species to species, and species selectivity could be improved by incorporating specific feeding attractants in manufactured baits. The unique properties of chemical stimuli and odour dispersal form the basis for improving longline efficiency through the development of a long-lasting bait. Vision is important in prey capture, and manufactured baits can be made more visible than natural baits by increasing the contrast (e.g. via fluorescent or polarising coatings) and creating motion through buoyancy. Physical properties such as size, shape, texture and strength can also be manipulated in a manufactured bait to improve catch efficiency. Knowledge obtained from studies of various aspects of food search behaviour is of paramount importance for future research aimed at alternative bait development and improving longline fishing.     

Frontiers in glycomics; bioinformatics and biomarkers in disease. September 11-13, 2006 Natcher Conference Center, NIH Campus, Bethesda, MD, USA

This is a short summary of a meeting entitled "The Frontiers in Glycomics; Bioinformatics and Biomarkers in Disease" which was jointly organized by the NIH Consortium for Functional Glycomics (CFG), Human Disease Glycomics/Proteome Initiative (HGPI), National Cancer Institute, National Institute of General Medical Sciences, Japan Society for the Promotion of Science and National Center for Research Resources held at the NIH Campus, Bethesda, MD, Natcher Conference Center in September 11-13, 2006.     

A synthetic sialic acid analogue is recognized by influenza C virus as a receptor determinant but is resistant to the receptor-destroying enzyme.

Synthetic sialic acid analogues varying in the substitutents at position C-9 were analyzed for their ability to replace the natural receptor determinant for influenza C virus, N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). By incubation of erythrocytes with sialyltransferase and the CMP-activated analogues, the cell surface was modified to contain sialic acid with one of the following C-9 substituents: an azido, an amino, an acetamido, or a hexanoylamido group. Among these, only 9-acetamido-N-acetylneuraminic acid (9-acetamido-Neu5Ac) was able to function as a receptor determinant for influenza C virus as indicated by the ability of the virus to agglutinate the modified red blood cells. In contrast to the natural receptors, 9-acetamido-Neu5Ac-containing receptors were found to be resistant against the action of sialate 9-O-acetylesterase, the viral receptor-destroying enzyme. No difference in the hemolytic activity of influenza C virus was detected when analyzed with erythrocytes containing either Neu5,9Ac2 or 9-acetamido-Neu5Ac on their surface. This finding indicates that cleavage of the receptor is not required for the viral fusion activity. The sialic acid analogues should be useful for analyzing not only the importance of the receptor-destroying enzyme of influenza C virus, but also other biological processes involving sialic acid.