Distribution of glycan motifs at the surface of midgut cells in the cotton leafworm (Spodoptera littoralis) demonstrated by lectin binding

Glycans are involved in many biological phenomena, including signal transduction, cell adhesion, immune response or differentiation. Although a few papers have reported on the role of glycans in the development and proper functioning of the insect midgut, no data are available regarding the localization of the glycan structures on the surface of the cells in the gut of insects. In this paper, we analyzed the spatial distribution of glycans present on the surface of the midgut cells in larvae of the cotton leafworm Spodoptera littoralis, an important agricultural pest insect worldwide. For this purpose, we established primary midgut cell cultures, probed these individual cells that are freely suspended in liquid medium with a selection of seven fluorescently labeled lectins covering a range of different carbohydrate binding specificities [mannose oligomers (GNA and HHA), GalNAc/Gal (RSA and SSA), GlcNAc (WGA and Nictaba) and Neu5Ac(alpha-2,6)Gal/GalNAc (SNA-I)], and visualized the interaction of these lectins with the different zones of the midgut cells using confocal microscopy. Our analysis focused on the typical differentiated columnar cells with a microvillar brush border at their apical side, which are dominantly present in the Lepidopteran midgut and function in food digestion and absorption, and as well as on the undifferentiated stem cells that are important for midgut development and repair. Confocal microscopy analyses showed that the GalNAc/Gal-binding lectins SSA and RSA and the terminal GlcNAc-recognizing WGA bound preferentially to the apical microvillar zone of the differentiated columnar cells as compared to the basolateral pole. The reverse result was observed for the mannose-binding lectins GNA and HHA, as well as Nictaba that binds preferentially to GlcNAc oligomers. Furthermore, differences in lectin binding to the basal and lateral zones of the cell membranes of the columnar cells were apparent. In the midgut stem cells. GNA and Nictaba bound more strongly to the membrane of these undifferentiated cells compared to the microvillar pole of the columnar cells, while SSA, HHA, WGA, and SNA-I showed stronger binding to the microvilli. Our results indicated that polarization of the midgut cells is also reflected by a specific distribution of glycans, especially between the basal and microvillar pole. The data are discussed in relation to the functioning and development of the insect midgut

RNAi-mediated silencing of pgants shows core 1 O-glycans are required for pupation in Tribolium castaneum

Protein glycosylation is one of the most common and most important post-translational modifications. Despite the growing knowledge on N-glycosylation, the research on O-glycosylation is lagging behind. This study investigates the importance of O-glycosylation in the post-embryonic development of insects using the red flour beetle, Tribolium castaneum, as a model. We identified 28 O-glycosylation-related genes (OGRGs) in the genome of the red flour beetle. 14 OGRGs were selected for functional analysis based on their involvement in the initial attachment of the carbohydrate in the different O-glycosylation pathways or the further elongation of the most abundant O-glycans and, in addition, showing severe RNAi-induced phenotypes in Drosophila melanogaster. The expression profile of these OGRGs was mapped throughout the developmental stages of the insect and in the different tissues of the pupa and adult. Subsequently, these genes were silenced using RNA interference (RNAi) to analyze their role in development. A broad spectrum of phenotypes was observed: from subtle effects and disrupted wing formation when silencing the genes involved in O-mannosylation, to blockage of pupation and high mortality after silencing of the genes involved in O-GalNAc and core 1 O-glycan (O-GalNAc-Gal) synthesis. RNAi experiments were also performed to assess the effects of blocking multiple pathways of O-glycosylation. However, the observed phenotypes induced by multiple RNAi were similar to those of the single gene RNAi experiments. The silencing of OGRGs often resulted in high mortality and wing phenotypes, indicating the importance of O-glycosylation for the survival of the insect and the formation of wings during the post-embryonic development of T. castaneum

Comparative study of lectin domains in model species : new insights into evolutionary dynamics

Lectins are present throughout the plant kingdom and are reported to be involved in diverse biological processes. In this study, we provide a comparative analysis of the lectin families from model species in a phylogenetic framework. The analysis focuses on the different plant lectin domains identified in five representative core angiosperm genomes (Arabidopsisthaliana, Glycine max, Cucumis sativus, Oryza sativa ssp. japonica and Oryza sativa ssp. indica). The genomes were screened for genes encoding lectin domains using a combination of Basic Local Alignment Search Tool (BLAST), hidden Markov models, and InterProScan analysis. Additionally, phylogenetic relationships were investigated by constructing maximum likelihood phylogenetic trees. The results demonstrate that the majority of the lectin families are present in each of the species under study. Domain organization analysis showed that most identified proteins are multi-domain proteins, owing to the modular rearrangement of protein domains during evolution. Most of these multi-domain proteins are widespread, while others display a lineage-specific distribution. Furthermore, the phylogenetic analyses reveal that some lectin families evolved to be similar to the phylogeny of the plant species, while others share a closer evolutionary history based on the corresponding protein domain architecture. Our results yield insights into the evolutionary relationships and functional divergence of plant lectins

Open access to sequence: Browsing the Pichia pastoris genome

The first genome sequences of the important yeast protein production host Pichia pastoris have been released into the public domain this spring. In order to provide the scientific community easy and versatile access to the sequence, two web-sites have been installed as a resource for genomic sequence, gene and protein information for P. pastoris: A GBrowse based genome browser was set up at and a genome portal with gene annotation and browsing functionality at . Both websites are offering information on gene annotation and function, regulation and structure

Alpha-gal and cross-reactive carbohydrate determinants in the N-glycans of salivary glands in the lone star tick, Amblyomma americanum

Ticks are important ectoparasites and vectors of numerous human and animal pathogens. Ticks secrete saliva that contains various bioactive materials to evade the host defense system, and often facilitates the pathogen transmission. In addition, the Lone star tick saliva is thought to be the sensitizer in red meat allergy that is characterized by an allergic reaction to glycan moieties carrying terminal galactose-alpha-1,3-galactose (aGal). To assess N-glycome of Amblyomma americanum, we examined the N-glycan structures in male and female salivary glands at three different feeding stages and in carcasses of partially fed lone star ticks. We also surveyed the genes involved in the N-glycosylation in the tick species. The aGal epitopes and cross-reactive carbohydrate determinants (CCD) increases over time after the onset of blood feeding in both male and female A. americanum. These CCDs include xylosylation of the core mannose, 1,3-mono and 1,3- and 1,6-difucosylations of the basal GlcNac and mono- or diantennary aGal. Combinations of both xylosylation and aGal and fucosylation and aGal were also found on the N-glycan structures. While the enzymes required for the early steps of the N-glycosylation pathway are quite conserved, the enzymes involved in the later stages of N-glycan maturation in the Golgi apparatus are highly diverged from those of insects. Most of all, we propose that the aGal serves as a molecular mimicry of bioactive proteins during tick feedings on mammalian hosts, while it contributes as a sensitizer of allergy in atypical host human

RNA-seq analysis of gene expression changes during pupariation in Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)

Background: The oriental fruit fly, Bactrocera dorsalis (Hendel) has been considered to be one of the most important agricultural pest around the world. As a holometabolous insect, larvae must go through a metamorphosis process with dramatic morphological and structural changes to complete their development. To better understand the molecular mechanisms of these changes, RNA-seq of B. dorsalis from wandering stage (WS), late wandering stage (LWS) and white puparium stage (WPS) were performed. Results: In total, 11,721 transcripts were obtained, out of which 1914 genes (578 up-regulated and 1336 down-regulated) and 2047 genes (655 up-regulated and 1392 down-regulated) were found to be differentially expressed between WS and LWS, as well as between WS and WPS, respectively. Of these DEGs, 1862 and 1996 genes were successfully annotated in various databases. The analysis of RNA-seq data together with qRT-PCR validation indicated that during this transition, the genes in the oxidative phosphorylation pathway, and genes encoding P450s, serine protease inhibitor, and cuticular proteins were down-regulated, while the serine protease genes were up-regulated. Moreover, we found some 20-hydroxyecdysone (20E) biosynthesis and signaling pathway genes had a higher expression in the WS, while the genes responsible for juvenile hormone (JH) synthesis, degradation, signaling and transporter pathways were down-regulated, suggesting these genes might be involved in the process of larval pupariation in B. dorsalis. For the chitinolytic enzymes, the genes encoding chitinases (chitinase 2, chitinase 5, chitinase 8, and chitinase 10) and chitin deacetylase might play the crucial role in the degradation of insect chitin with their expressions significantly increased during the transition. Here, we also found that chitin synthase 1A might be involved in the chitin synthesis of cuticles during the metamorphosis in B. dorsalis. Conclusions: Significant changes at transcriptional level were identified during the larval pupariation of B. dorsalis. Importantly, we also obtained a vast quantity of RNA-seq data and identified metamorphosis associated genes, which would all help us to better understand the molecular mechanism of metamorphosis process in B. dorsalis

Protein-Carbohydrate Interactions, and Beyond …

Carbohydrates are ubiquitous and play an intriguing role inside the cell as well as on the cell surface.[...

Pichia surface display: a tool for screening single domain antibodies

Yeast surface display is being employed as an efficient tool for the isolation and engineering of traditional antibody fragments, both scFv and Fab, as well as single domain antibodies. Here we describe the protocols for a yeast surface display system developed in the methylothrophic yeast Pichia pastoris , the most commonly used yeast species for protein production. In this system the immune or maturated library of single domain antibodies is fused to the C-terminal domain of the Saccharomyces cerevisiae alpha-agglutinin gene ( SAG1 ) and expressed on the surface of P. pastoris cells. Labeling with ligands enables rapid and quantitative analysis in conjunction with isolation of single domain antibodies with the desired characteristics