Proteome Analysis of Pod and Seed Development in the Model Legume Lotus japonicus

Legume pods serve important functions during seed development and are themselves sources of food and feed. Compared to seeds, the metabolism and development of pods are not well-defined. The present characterization of pods from the model legume Lotus japonicus, together with the detailed analyses of the pod and seed proteomes in five developmental stages, paves the way for comparative pathway analysis and provides new metabolic information. Proteins were analyzed by two-dimensional gel electrophoresis and tandem-mass spectrometry. These analyses lead to the identification of 604 pod proteins and 965 seed proteins, including 263 proteins distinguishing the pod. The complete data set is publicly available at http://www.cbs.dtu.dk/cgi-bin/lotus/db.cgi, where spots in a reference map are linked to experimental data, such as matched peptides, quantification values, and gene accessions. Identified pod proteins represented enzymes from 85 different metabolic pathways, including storage globulins and a late embryogenesis abundant protein. In contrast to seed maturation, pod maturation was associated with decreasing total protein content, especially proteins involved in protein biosynthesis and photosynthesis. Proteins detected only in pods included three enzymes participating in the urea cycle and four in nitrogen and amino group metabolism, highlighting the importance of nitrogen metabolism during pod development. Additionally, five legume seed proteins previously unassigned in the glutamate metabolism pathway were identified

A Lotus japonicus cytoplasmic kinase connects Nod factor perception by the NFR5 LysM receptor to nodulation

The establishment of nitrogen-fixing root nodules in legume-rhizobia symbiosis requires an intricate communication between the host plant and its symbiont. We are, however, limited in our understanding of the symbiosis signaling process. In particular, how membrane-localized receptors of legumes activate signal transduction following perception of rhizobial signaling molecules has mostly remained elusive. To address this, we performed a coimmunoprecipitation-based proteomics screen to identify proteins associated with Nod factor receptor 5 (NFR5) in Lotus japonicus. Out of 51 NFR5-associated proteins, we focused on a receptor-like cytoplasmic kinase (RLCK), which we named NFR5-interacting cytoplasmic kinase 4 (NiCK4). NiCK4 associates with heterologously expressed NFR5 in Nicotiana benthamiana, and directly binds and phosphorylates the cytoplasmic domains of NFR5 and NFR1 in vitro. At the cellular level, Nick4 is coexpressed with Nfr5 in root hairs and nodule cells, and the NiCK4 protein relocates to the nucleus in an NFR5/NFR1-dependent manner upon Nod factor treatment. Phenotyping of retrotransposon insertion mutants revealed that NiCK4 promotes nodule organogenesis. Together, these results suggest that the identified RLCK, NiCK4, acts as a component of the Nod factor signaling pathway downstream of NFR5

177^{177}Lu-OPS201 targeting somatostatin receptors: in vivo biodistribution and dosimetry in a pig model

Background $^{177}$Lu is used in peptide receptor radionuclide therapies for the treatment of neuroendocrine tumors. Based on the recent literature, SST2 antagonists are superior to agonists in tumor uptake. The compound OPS201 is the novel somatostatin antagonist showing the highest SST2 affinity. The aim of this study was to measure the in vivo biodistribution and dosimetry of $^{177}$Lu-OPS201 in five anesthetized Danish Landrace pigs as an appropriate substitute for humans to quantitatively assess the absorbed doses for future clinical applications. Results $^{177}$Lu-OPS201 was obtained with a specific activity ranging from 10 to 17 MBq/μg. Prior to administration, the radiochemical purity was measured as s > 99.7 % in all cases. After injection, fast clearance of the compound from the blood stream was observed. Less than 5 % of the injected activity was presented in blood 10 min after injection. A series of SPECT/CT and whole-body scans conducted until 10 days after intravenous injection showed uptake mostly in the liver, spine, and kidneys. There was no visible uptake in the spleen. Blood samples were taken to determine the time-activity curve in the blood. Time-activity curves and time-integrated activity coefficients were calculated for the organs showing visible uptake. Based on these data, the absorbed organ dose coefficients for a 70-kg patient were calculated with OLINDA/EXM. For humans after an injection of 5 GBq $^{177}$Lu-OPS201, the highest predicted absorbed doses are obtained for the kidneys (13.7 Gy), the osteogenic cells (3.9 Gy), the urinary bladder wall (1.8 Gy), and the liver (1.0 Gy). No metabolites of 177Lu-OPS201 were found by radio HPLC analysis. None of the absorbed doses calculated will exceed organ toxicity levels. Conclusions The $^{177}$Lu-OPS201 was well tolerated and caused no abnormal physiological or behavioral signs. In vivo distributions and absorbed doses of pigs are comparable to those observed in other publications. According to the biodistribution data in pigs, presented in this work, the expected radiation exposure in humans will be within the acceptable range

Fractionation of Echinacea purpurea L. (Moench) proteins and assessment of Glycosylated proteins distribution in fractions

Plant lectins, non-immune origin glycoproteins, got their attention for possible beneficial effects to health just 3 decades ago and are quiet new group of compounds with pharmacological interest. Echinacea purpurea L. (Moench) is a well-known medical plant. However, pharmacologically interesting proteins in this plant aren’t well characterized. In this study we extracted and fractionated proteins of Echinacea purpurea L. (Moench) roots and checked estimation of glycosylated proteins in all fractions. Materials and Methods 30 g of fresh roots were homogenized in mortar with liquid nitrogen and extracted in phosphate buffer saline pH 7.4 in a ratio 1:5 for 2 hours at 4 °C. Proteins were precipitated from crude extract by adding 4 volumes of cold (-20 °C) 10 % Trichloracetic acid/acetone solution, containing 0.2 % β - mercaptoethanol. Protein pellets were washed with acetone solution, containing 0.2 % β – mercaptoethanol for 3 times and resuspended in phosphate buffer 7.4. Resuspended protein sample was loaded on HiTrap Q FF 1 mL column with gradient elution from 0 M to 1 M NaCl. 5 mL fractions were collected, dialyzed in phosphate buffer 7.4 and immunoblotted with anti-xylose antibody. Results After anion exchange fractionation we’ve collected 18 protein fractions, 11 of them contained glycosylated proteins. Glycoproteins started eluting from the column when sodium chloride concentration in eluent reached 0.4 M. Elution of glycosylated proteins decreased when sodium chloride concentration reached 0.7 M. Conclusions Protein fractions extracted from Echinacea purpurea L. (Moench) roots contained glycosylated proteins. Glycosylated protein patternBotanikos sodasLietuvos sveikatos mokslų universitetas. Medicinos akademijaLietuvos sveikatos mokslų universitetas. Veterinarijos akademijaLietuvos žemdirbystės instituto Agrocheminių tyrimų centrasVytauto Didžiojo universiteta

Combined N-glycome and N-glycoproteome analysis of the lotus japonicus seed globulin fraction shows conservation of protein structure and glycosylation in legumes

Legume food allergy, such as allergy toward peanuts and soybeans, is a health issue predicted to worsen as dietary advice recommends higher intake of legume-based foods. Lotus japonicus (Lotus) is an established legume plant model system for studies of symbiotic and pathogenic microbial interactions and, due to its well characterized genotype/phenotype and easily manipulated genome, may also be suitable for studies of legume food allergy. Here we present a comprehensive study of the Lotus N-glycoproteome. The global and site-specific N-glycan structures of Lotus seed globulins were analyzed using mass spectrometry-based glycomics and glycoproteomics techniques. In total, 19 N-glycan structures comprising high mannose (∼20%), pauci-mannosidic (∼40%), and complex forms (∼40%) were determined. The pauci-mannosidic and complex N-glycans contained high amounts of the typical plant determinants β-1,2-xylose and α-1,3-fucose. Two abundant Lotus seed N-glycoproteins were site-specifically profiled; a predicted lectin containing two fully occupied N-glycosylation sites carried predominantly pauci-mannosidic structures in different distributions. In contrast, Lotus convicilin storage protein 2 (LCP2) carried exclusively high mannose N-glycans similar to its homologue, Ara h 1, which is the major allergen in peanut. In silico investigation confirmed that peanut Ara h 1 and Lotus LCP2 are highly similar at the primary and higher protein structure levels. Hence, we suggest that Lotus has the potential to serve as a model system for studying the role of seed proteins and their glycosylation in food allergy.10 page(s