Structure-based Comparative Analysis and Prediction of N-linked Glycosylation Sites in Evolutionarily Distant Eukaryotes

The asparagine-X-serine/threonine (NXS/T) motif, where X is any amino acid except proline, is the consensus motif for N-linked glycosylation. Significant numbers of high-resolution crystal structures of glycosylated proteins allow us to carry out structural analysis of the N-linked glycosylation sites (NGS). Our analysis shows that there is enough structural information from diverse glycoproteins to allow the development of rules which can be used to predict NGS. A Python-based tool was developed to investigate asparagines implicated in N-glycosylation in five species: Homo sapiens, Mus musculus, Drosophila melanogaster, Arabidopsis thaliana and Saccharomyces cerevisiae. Our analysis shows that 78% of all asparagines of NXS/T motif involved in N-glycosylation are localized in the loop/turn conformation in the human proteome. Similar distribution was revealed for all the other species examined. Comparative analysis of the occurrence of NXS/T motifs not known to be glycosylated and their reverse sequence (S/TXN) shows a similar distribution across the secondary structural elements, indicating that the NXS/T motif in itself is not biologically relevant. Based on our analysis, we have defined rules to determine NGS. Using machine learning methods based on these rules we can predict with 93% accuracy if a particular site will be glycosylated. If structural information is not available the tool uses structural prediction results resulting in 74% accuracy. The tool was used to identify glycosylation sites in 108 human proteins with structures and 2247 proteins without structures that have acquired NXS/T site/s due to non-synonymous variation. The tool, Structure Feature Analysis Tool (SFAT), is freely available to the public at http://hive.biochemistry.gwu.edu/tools/sfat.     

Analysis and modeling of heat-labile enterotoxins of Escherichia coli suggests a novel space with insights into receptor preference

Features of heat-labile enterotoxins of Escherichia coli which make them fit to use as novel receptors for antidiarrheals are not completely explored. Data-set of 14 different serovars of enterotoxigenic Escherichia coli producing heat-labile toxins were taken from NCBI Genbank database and used in the study. Sequence analysis showed mutations in different subunits and also at their interface residues. As these toxins lack crystallography structures, homology modeling using Modeller 9.11 led to the structural approximation for the E. coli producing heat-labile toxins. Interaction of modeled toxin subunits with proanthocyanidin, an antidiarrheal showed several strong hydrogen bonding interactions at the cost of minimized energy. The hits were subsequently characterized by molecular dynamics simulation studies to monitor their binding stabilities. This study looks into novel space where the ligand can choose the receptor preference not as a whole but as an individual subunit. Mutation at interface residues and interaction among subunits along with the binding of ligand to individual subunits would help to design a non-toxic labile toxin and also to improve the therapeutics.     

High Heregulin Expression Is Associated with Activated HER3 and May Define an Actionable Biomarker in Patients with Squamous Cell Carcinomas of the Head and Neck

Purpose

Tumors with oncogenic dependencies on the HER family of receptor tyrosine kinases (RTKs) often respond well to targeted inhibition. Our previous work suggested that many cell lines derived from squamous cell carcinomas of the head and neck (SCCHNs) depend on autocrine signaling driven by HER2/3 dimerization and high-level co-expression of HRG. Additionally, results from a Phase I trial of MEHD7495A, a dual-action antibody that blocks ligand binding to EGFR and HER3, suggest that high-level HRG expression was associated with clinical response in SCCHN patients. Here we explore the hypothesis that high-level HRG expression defines a subpopulation of SCCHNs with activated HER3.

Experimental Design

qRT-PCR expression profiling was performed on >750 tumors of diverse origin, including >150 therapy-naïve, primary, and recurrent SCCHNs. Activated HER3, defined by immunoprecipitation of phospho-HER3, was compared to HRG expression in SCCHN samples. Paracrine versus autocrine expression was evaluated using RNA-in situ hybridization.

Results

SCCHN tumors express the highest levels of HRG compared to a diverse collection of other tumor types. We show that high HRG expression is associated with activated HER3, whereas low HRG expression is associated with low HER3 activation in SCCHN tumors. Furthermore, HRG expression is higher in recurrent SCCHN compared to patient-matched therapy naïve specimens.

Conclusions

HRG expression levels define a biologically distinct subset of SCCHN patients. We propose that high-level expression of HRG is associated with constitutive activation of HER3 in SCCHN and thus defines an actionable biomarker for interventions targeting HER3.     

Receptor-ligand analyses define minimal killer cell Ig-like receptor (KIR) in humans

Interactions between inhibitory killer cell immunoglobulin-like receptors (iKIR) and human leukocyte antigen (HLA) class I molecules regulate natural killer (NK) cell responses to eliminate infected and transformed cells while maintaining tolerance to healthy cells. Unlinked polymorphic gene families encode KIR receptors and HLA class I ligands and their independent segregation results in a variable number and type of iKIR + HLA pairs inherited in individuals. The diversity in the co-inheritance of iKIR + HLA pairs and activating KIR (aKIR) genes in 759 unrelated individuals from four ethnic populations was analyzed. Every individual studied inherited a minimum of one iKIR + HLA pair; suggesting that major histocompatibility complex class I-dependent inhibitory KIR signaling is essential for human NK cell function. In contrast, 13.4% of the study group lacked all aKIR genes. Twenty percent of the study group carried only one of the four iKIR + HLA pairs. Interestingly, 3% of the study group carrying only KIR2DL3 + HLA-C1 as an iKIR + HLA pair lacked aKIR genes. These data suggest that a single iKIR can constitute the minimal KIR repertoire for human NK cells. Genotypes carrying an equal number of iKIR + HLA pairs and aKIR genes represented 20% of the study group. The remaining individuals had either a dominant inhibitory KIR genotype (iKIR + HLA > aKIR) or a dominant activating KIR genotype (iKIR + HLA < aKIR). Genotypes encoding these imbalanced inhibitory and activating interactions may contribute to susceptibility or resistance to human diseases.     

Exploitation of Dunaliella for β-carotene production

Halotolerant microalga Dunaliella, which is exploited for the production of dried biomass or cell extract, is used as a medicinal food. With the advancement in this field in recent years, the production of bio-organic compounds such as β-carotene is established in many countries. Large-scale production of β-carotene is controlled by numerous stress factors like high light intensity, high salinity, temperature and availability of nutrients. The state-of-the-art strategies in industries in closed systems under new set of inductive factors will additionally promote the ease of commercial production of β-carotene. This review mainly focuses on the different methodologies employed recently for the optimum production of β-carotene from Dunaliella species.     

Pyramiding transgenic resistance in elite indica rice cultivars against the sheath blight and bacterial blight

Elite indica rice cultivars were cotransformed with genes expressing a rice chitinase (chi11) and a thaumatin-like protein (tlp) conferring resistance to fungal pathogens and a serine-threonine kinase (Xa21) conferring bacterial blight resistance, through particle bombardment, with a view to pyramiding sheath blight and bacterial blight resistance. Molecular analyses of putative transgenic lines by polymerase chain reaction, Southern Blot hybridization, and Western Blotting revealed stable integration and expression of the transgenes in a few independent transgenic lines. Progeny analyses showed the stable inheritance of transgenes to their progeny. Coexpression of chitinase and thaumatin-like protein in the progenies of a transgenic Pusa Basmati1 line revealed an enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, as compared to that in the lines expressing the individual genes. A transgenic Pusa Basmati1 line pyramided with chi11, tlp, and Xa21 showed an enhanced resistance to both sheath blight and bacterial blight. S. Maruthasalam and K. Kalpana have contributed to this article equally.     

Patterns of simple sequence repeats in cultivated blueberries (Vaccinium section Cyanococcus spp.) and their use in revealing genetic diversity and population structure

Blueberry (Vaccinium section Cyanococcus) is an important small fruit crop native to North America. Relationships among the primary species known as ‘blueberry’ has been a source of speculation due to the out-breeding nature of the crop, the use of intra-specific hybridization and open-pollinated selection in breeding programs, and the lack of genomic resources to adequately address the issue. The objectives of this study were to characterize simple sequence repeats (SSRs) from an emerging genomic draft sequence, develop useful molecular markers and provide an in-depth analysis of genetic diversity and population structure in blueberry using a broad range of cultivated blueberry accessions representing multiple species, ploidy levels and sources of origin. Genomic scaffolding was assembled from the whole-genome sequencing of a diploid V. corymbosum accession ‘W8520.’ From the assembled 358 Mb sequence, a total number of 43,594 SSRs were identified (122 per Mb). Among genomic regions, SSRs were longest and occurred most frequently in predicted 5′ untranslated regions (5′ UTR), while SSRs were shortest and least common in the predicted coding sequences. AG/CT and AAG/CTT were the most frequent motifs while CG/CG and CCG/CGG motifs were the rarest di- and trinucleotide motifs, respectively. For analysis of genetic diversity and population structure, 42 genomic SSR and EST–SSR markers with an average of 14.2 alleles and 56.0 allele phenotypes per locus were used to genotype a diverse blueberry population of 150 accessions. Cluster analysis grouped the 150 accessions in a manner consistent with known information regarding species, ploidy levels and pedigree. The analysis of population structure among blueberry accessions revealed inter- and intra-specific levels of stratification. Rabbiteye blueberry (V. virgatum) represents a genetically distinct subgroup within Cyanococcus. Three additional subpopulations were detected among highbush varieties that are largely attributable to distinctions between northern and southern highbush and founder effects of a single cultivar (‘Weymouth’). The identification of substructure that correlates with known pedigree information, and the availability of new genomic molecular markers will facilitate future evolutionary and genetic studies in blueberry.