Site specific N-glycan profiling of NeuAc(alpha 2-6)-Gal/GalNAc-binding bark Sambucus nigra agglutinin using LC-MSn revealed differential glycosylation

The bark of Sambucus nigra contains a complex mixture of glycoproteins that are characterized as chimeric lectins known as type II ribosome inactivating proteins and holo lectins. These type II ribosome inactivating proteins possess RNA N-glycosidase activity in subunit A and lectin activity associated with subunit B exhibiting distinct sugar specificities to NeuAc(alpha 2-6)-Gal/GalNAc and Gal/GalNAc. In the present study we have determined the N-glycosylation pattern of type II ribosome inactivating protein specific to NeuAc(alpha 2-6)-Gal/GalNAc (Sambucus nigra agglutinin I) by subjecting it to digestion with multiple proteases. The resulting mixture of peptides and N-glycopeptides were analyzed on liquid chromatography coupled to electro spray ionization-iontrap mass spectrometry in MSn mode. MS2 of precursor ions was carried out using CID which provided information on glycan sequence. In subsequent MS3 of Y-1/Y-1 alpha ions (peptide + HexNAc)(+n) of corresponding N-glycopeptides, resulted in the fragmentation of peptide backbone confirming the site of attachment. We observed microheterogeneity in each glycan occupied site with subunit A possessing four N-glycans out of six sites with complex and paucimannose types while subunit B comprises occupancy of two sites with a paucimannose and a high mannose type. The differential N-glycosylation of subunits in SNA is discussed in the context of other type II RIPs glycans

Comprehensive analysis of alpha 2-3-linked sialic acid specific Maackia amurensis leukagglutinin reveals differentially occupied N-glycans and C-terminal processing

Seeds of Maackia amurensis constitutes two sialic acid specific agglutinins known as leukagglutinin and hemagglutinin. Maackia amurensis leukagglutinin (MAL) recognizes alpha 12-3-linked sialic acid present mainly in N-glycans and composed of two disulfide linked monomers. It exhibits potential N-glycosylation sites (four PNGs) which have been assumed to undergo differential occupancy. In this study we have characterized the site specific macro- and microheterogeneity of monomers in detail by analysing N-glycopeptides and peptides through liquid chromatography coupled to ion trap mass spectrometer in MS3 mode (LC-MSn). We observed the presence of mainly paucimannose N-glycans at Asn(61), Asn(113) and Asn(191) whereas a high mannose type with varying Man(5-9) occurs at Asn(179). Interestingly Asn(179) and Asn(191) exhibited differential occupancy which was evident by the presence of non-glycosylated peptides. This has contributed to the difference in molecular mass of monomers upon SDS-PAGE. Further the presence of disulfide linked peptides confirmed the covalent linkage of monomers which also undergoes uniform C-terminal processing. (C) 2016 Elsevier B.V. All rights reserved

Site specific N-glycan profiling of NeuAc(alpha 2-6)-Gal/GalNAc-binding bark Sambucus nigra agglutinin using LC-MSn revealed differential glycosylation

The bark of Sambucus nigra contains a complex mixture of glycoproteins that are characterized as chimeric lectins known as type II ribosome inactivating proteins and holo lectins. These type II ribosome inactivating proteins possess RNA N-glycosidase activity in subunit A and lectin activity associated with subunit B exhibiting distinct sugar specificities to NeuAc(alpha 2-6)-Gal/GalNAc and Gal/GalNAc. In the present study we have determined the N-glycosylation pattern of type II ribosome inactivating protein specific to NeuAc(alpha 2-6)-Gal/GalNAc (Sambucus nigra agglutinin I) by subjecting it to digestion with multiple proteases. The resulting mixture of peptides and N-glycopeptides were analyzed on liquid chromatography coupled to electro spray ionization-iontrap mass spectrometry in MSn mode. MS2 of precursor ions was carried out using CID which provided information on glycan sequence. In subsequent MS3 of Y-1/Y-1 alpha ions (peptide + HexNAc)(+n) of corresponding N-glycopeptides, resulted in the fragmentation of peptide backbone confirming the site of attachment. We observed microheterogeneity in each glycan occupied site with subunit A possessing four N-glycans out of six sites with complex and paucimannose types while subunit B comprises occupancy of two sites with a paucimannose and a high mannose type. The differential N-glycosylation of subunits in SNA is discussed in the context of other type II RIPs glycans

Determination of antibacterial activity and metabolite profile of Ruta graveolens against Streptococcus mutans and Streptococcus sobrinus

BACKGROUND: Ruta graveolens is one of the most used phytomedicines. To date, there is no report of determining the bioactivity of R. graveolens against cariogenic causing bacteria (Streptococcus mutans and Streptococcus sobrinus). OBJECTIVE: The objective of the present study was to determine the antibacterial activity and metabolite profile of R. graveolens against S. mutans and S. sobrinus. MATERIAL AND METHODS: R. graveolens plant material was collected and processed in the month of February. The plant material was extracted by Soxhlet apparatus using methanol solvent. Two strains of S. mutans and two strains of S. sobrinus were isolated from dental caries-active participants and cultured on mitis salivarius-bacitracin agar. The antibacterial susceptibility testing of methanolic extract of R. graveolens was performed by disc diffusion method. The metabolite profile of the plant extract was determined using electrospray ionization-tandem mass spectrometry. RESULTS: The methanolic extract of R. graveolens showed a promising antibacterial activity against S. mutans and S. sobrinus. Two compounds named γ-fagarine and kokusaginine were identified from the methanolic extract of R. graveolens. CONCLUSIONS: The study concluded that R. graveolens contains significant antibacterial activity. However, further investigations are suggested to understand the anticaries properties of these pure compounds

Immuno-affinity purification of PglPGIP1, a polygalacturonase-inhibitor protein from pearl millet: studies on its inhibition of fungal polygalacturonases and role in resistance against the downy mildew pathogen

Polygalacturonase-inhibitor proteins (PGIPs) are important plant defense proteins which modulate the activity of microbial polygalacturonases (PGs) leading to elicitor accumulation. Very few studies have been carried out towards understanding the role of PGIPs in monocot host defense. Hence, present study was taken up to characterize a native PGIP from pearl millet and understand its role in resistance against downy mildew. A native glycosylated PGIP (PglPGIP1) of similar to 43 kDa and pI 5.9 was immunopurified from pearl millet. Comparative inhibition studies involving PglPGIP1 and its non-glycosylated form (rPglPGIP1; recombinant pearl millet PGIP produced in Escherichia coli) against two PGs, PG-II isoform from Aspergillus niger (AnPGII) and PG-III isoform from Fusarium moniliforme, showed both PGIPs to inhibit only AnPGII. The protein glycosylation was found to impact only the pH and temperature stability of PGIP, with the native form showing relatively higher stability to pH and temperature changes. Temporal accumulation of both PglPGIP1 protein (western blot and ELISA) and transcripts (real time PCR) in resistant and susceptible pearl millet cultivars showed significant Sclerospora graminicola-induced accumulation only in the incompatible interaction. Further, confocal PGIP immunolocalization results showed a very intense immuno-decoration with highest fluorescent intensities observed at the outer epidermal layer and vascular bundles in resistant cultivar only. This is the first native PGIP isolated from millets and the results indicate a role for PglPGIP1 in host defense. This could further be exploited in devising pearl millet cultivars with better pathogen resistance

Molecular Mapping of Genes and QTLs in Pigeonpea

Pigeonpea is one of the most important grain legume crops grown in arid and semiarid regions of the world. There is an increasing demand for the development of new cultivars with high yield potential and better adaptability to adverse environmental conditions. Recent advances in genomics tools and techniques have helped to develop large repertoire of molecular markers and genotypic platforms. The availability of molecular markers facilitated the development of high-density genetic maps that have been used in discovery of important/major QTLs for targeted traits in pigeonpea. In addition, the availability of high-throughput genotypic platforms helped to generate whole genome genotypic data in high-throughput manner necessary for whole genome scanning/genome-wide association mapping of economically important traits. The advances in comparative genomics, transcriptomics, and whole genome sequencing have uncovered thousands of useful genes including some genes unique to pigeonpea crop. The availability of wealth of genomics resources/information will facilitate molecular breeding aimed at improving production and productivity of pigeonpea in extreme environments of arid and semiarid regions of the world