A Novel Method for the Simultaneous Enrichment,Identification, and Quantification of Phosphopeptides and Sialylated Glycopeptides Applied to a Temporal Profile of Mouse Brain Development

We describe a method that combines an optimized titanium dioxide protocol and hydrophilic interaction liquid chromatography to simultaneously enrich, identify and quantify phosphopeptides and formerly N-linked sialylated glycopeptides to monitor changes associated with cell signaling during mouse brain development. We initially applied the method to enriched membrane fractions from HeLa cells, which allowed the identification of 4468 unique phosphopeptides and 1809 formerly N-linked sialylated glycopeptides. We subsequently combined the method with isobaric tagging for relative quantification to compare changes in phosphopeptide and formerly N-linked sialylated glycopeptide abundance in the developing mouse brain. A total of 7682 unique phosphopeptide sequences and 3246 unique formerly sialylated glycopeptides were identified. Moreover 669 phosphopeptides and 300 formerly N-sialylated glycopeptides differentially regulated during mouse brain development were detected. This strategy allowed us to reveal extensive changes in post-translational modifications from postnatal mice from day 0 until maturity at day 80. The results of this study confirm the role of sialylation in organ development and provide the first extensive global view of dynamic changes between N-linked sialylation and phosphorylation.The development of novel methods to simultaneously monitor multiple protein post-translational modifications (PTMs)¹ is an attractive tool for researchers. There is increasing evidence that both phosphorylation and glycosylation play important roles in cellular signaling networks during development and transformation of cells. Development of the mammalian brain is initiated during the embryonic stage and continues until adulthood. The brain originates through the proliferation of the telencephalon, the anterior part of the neural tube. Following differentiation, cells begin to migrate and associate into different brain structures. The brain structures are reorganized with the extension of axons and dendrites to communicate via synaptic terminal interactions (1, 2). These molecular interactions are governed by cell surface receptors that are often post-translationally modified with both N-linked glycans and phosphate groups, and studies have suggested that extracellular glycans play vital roles in the regulation of signal transduction pathways (3). For example, the myelin-associated glycoprotein (MAG) binds to cell surface glyco-conjugates GD1a, GT1b and Nogo receptors to form signaling complexes that inhibit axon outgrowth, whereas inhibition of Rho kinase reverses this process in a number of nerve cell types (4). There is growing evidence that both the differentiation and migration of neurons and the guidance of axons are regulated by sialic acid-containing glycoconjugates (5–7). Dietary supplementation of sialic acid leads to increases in sialic acid-containing glycoproteins in the frontal cortex and is associated with faster learning and memory in piglets (8). The nervous system contains an abundant array of sialylated molecules and it is therefore not surprising that changes in the sialiome (the content of sialylated glycoproteins (9)) of a neuron can regulate activity. Removal of sialic acids from membrane proteins by NEU3 in primary neurons leads to actin depolymerization and axonal growth through TrkA-mediated signaling (10). Moreover, the modulation of phosphorylation events because of changes in cell membrane sialylation has been described in cancer (11, 12). Tumors induced in sialyltransferase-deficient animals show altered expression of genes associated with focal adhesion signaling and display decreased phosphorylation of focal adhesion kinase, a target of β1-integrins (13). Sialylated glycoconjugates include N-linked glycans (attached to asparagine residues), O-linked glycans (attached to hydroxylated residues) and glycolipids. N-linked and O-linked glycans are predominantly processed through the endoplasmic reticulum and Golgi, and their protein targets are generally membrane associated, cell-surface or found in extracellular environments. Additional glycoconjugates include single sugar modifications such as O-linked N-acetylglucosamine, glycosaminoglycans, large lipopolysaccharides, and peptidoglycans.The ability to identify and quantify PTM in proteins using mass spectrometry (MS) relies on specific enrichment techniques to purify modified peptides-of-interest from among a complex mixture. As modified peptides are normally present in sub-stoichiometric levels compared with nonmodified peptides, they are generally not detected by MS without such specific enrichment. Many methods are available to enrich for single PTM, including phosphorylation and glycosylation. Titanium dioxide (TiO₂) chromatography was originally described for enrichment of phophopeptides from peptide mixtures using similar peptide loading conditions as used for immobilized metal affinity chromatography (14–16). However, using this procedure resulted in significant co-enrichment of nonphosphorylated peptides. Later we demonstrated that TiO₂ was able to selectively purify phosphorylated peptides and sialic acid-containing N-glycopeptides (9, 17) if peptide samples are loaded onto the TiO₂ resin in a buffer containing high organic solvent, very low pH and a multifunctional acid, such as 2,5- dihydroxybenzoic acid or glycolic acid.A recent study demonstrated the first simultaneous enrichment of N-glycopeptides and phosphopeptides from a complex peptide mixture (18). Peptides from mouse brain were separated using electrostatic repulsion hydrophilic interaction chromatography to identify 738 unique glycosylation sites representing 446 glycoproteins, and 915 unique phosphorylation sites from 382 phosphoproteins. This method however, required 3 mg of starting material and did not demonstrate the ability to selectively enrich sialylated glycopeptides from glycopeptides displaying neutral glycans. Furthermore, only a comparatively low number of phosphopeptides could be identified considering the generous protein load investigated. The method was also unable to separate deglycosylated peptides from phosphopeptides and no quantitative capabilities were shown.Here we report a novel multidimensional strategy that employs TiO₂ chromatography to enrich for sialylated glycopeptides and phosphopeptides followed by PNGase F treatment of the eluent and μHPLC hydrophilic interaction liquid chromatography (HILIC) to fractionate and separate formerly N-linked sialylated glycopeptides and phosphopeptides from complex membrane protein preparations of a variety of biological samples. The development of a quantitative N-linked sialiomics and phosphoproteomic strategy that is able to simultaneously monitor cell-(extracellular)-cell interactions and receptor signaling will be a valuable tool to study tissue development and cell stimulation.     

Lipid body biogenesis and the role of microtubules in lipid synthesis in Ornithogalum umbellatum lipotubuloids

Lipid bodies present in lipotubuloids of Ornithogalum umbellatum ovary epidermis take the form of a lens between leaflets of ER (endoplasmic reticulum) membrane filled with a highly osmiophilic substance. The two enzymes, DGAT1 [DAG (diacylglycerol) acyltransferase 1] and DGAT2 (DAG acyltransferase 2), involved in this process are synthesized on rough ER and localized in the ER near a monolayer surrounding entities like lipid bodies. After reaching the appropriate size, newly formed lipid bodies transform into mature spherical lipid bodies filled with less osmiophilic content. They appear to be surrounded by a half-unit membrane, with numerous microtubules running adjacently in different directions. The ER, no longer continuous with lipid bodies, makes contact with them through microtubules. At this stage, lipid synthesis takes place at the periphery of lipid bodies. This presumption, and a hypothesis that microtubules are involved in lipid synthesis delivering necessary components to lipid bodies, is based on strong arguments: (i) silver grains first appear over microtubules after a short [3H]palmitic acid incubation and before they are observed over lipid bodies; (ii) blockade of [3H]palmitic acid incorporation into lipotubuloids by propyzamide, an inhibitor of microtubule function; and (iii) the presence of gold grains above the microtubules after DGAT1 and DGAT2 reactions, as also near microtubules after an immunogold method that identifies phospholipase D1.     

Shape and evolution of the fundamental niche in marine Vibrio

Hutchinson''s fundamental niche, defined by the physical and biological environments in which an organism can thrive in the absence of inter-species interactions, is an important theoretical concept in ecology. However, little is known about the overlap between the fundamental niche and the set of conditions species inhabit in nature, and about natural variation in fundamental niche shape and its change as species adapt to their environment. Here, we develop a custom-made dual gradient apparatus to map a cross-section of the fundamental niche for several marine bacterial species within the genus Vibrio based on their temperature and salinity tolerance, and compare tolerance limits to the environment where these species commonly occur. We interpret these niche shapes in light of a conceptual model comprising five basic niche shapes. We find that the fundamental niche encompasses a much wider set of conditions than those strains typically inhabit, especially for salinity. Moreover, though the conditions that strains typically inhabit agree well with the strains'' temperature tolerance, they are negatively correlated with the strains'' salinity tolerance. Such relationships can arise when the physiological response to different stressors is coupled, and we present evidence for such a coupling between temperature and salinity tolerance. Finally, comparison with well-documented ecological range in V. vulnificus suggests that biotic interactions limit the occurrence of this species at low-temperature—high-salinity conditions. Our findings highlight the complex interplay between the ecological, physiological and evolutionary determinants of niche morphology, and caution against making inferences based on a single ecological factor.     

Variable rates of random genetic drift in protected populations of English yew: implications for gene pool conservation

Protecting populations in their natural habitat allows for the maintenance of naturally evolved adaptations and ecological relationships. However, the conservation of genetic resources often requires complementary practices like gene banks, translocations or reintroductions. In order to minimize inbreeding depression and maximize the adaptive potential of future populations, populations chosen for ex situ conservation should be selected according to criteria that will result in a reduction of global coancestry in the population. Generally, large populations should reveal lower coancestry and higher genetic variation than small populations. If detailed knowledge about coancestry is lacking, census population number (N _c ) can be used as a proxy for required characteristics. However, a simple measure of N _c may be misleading in particular cases as genetic processes rely on effective population size (N _e ) rather than N _c and these two measures may differ substantially due to demographic processes. We used an example of English yew to address whether N _c can be a good predictor of genetic parameters when used in conservation programs. Using microsatellite markers, we estimated allelic richness, inbreeding and coancestry coefficients of six relatively large yew populations in Poland. Each population was characterized by N _e using the linkage disequilibrium method. Our results showed that populations of English yew were subject to substantial divergence and genetic drift, with both being inversely proportional to the effective subpopulation size (N _e ). Additionally, allelic richness appeared proportional to N _e but not to N _c . However, the N _e /N ratio differed greatly among populations, which was possibly due to different population histories. From the results we concluded that choosing source populations based only on their census size can be fairly misleading. Implications for conservation are briefly discussed.     

Quantum mechanical studies of lincosamides

Lincosamides are a class of antibiotics used both in clinical and veterinary practice for a wide range of pathogens. This group of drugs inhibits the activity of the bacterial ribosome by binding to the 23S RNA of the large ribosomal subunit and blocking protein synthesis. Currently, three X-ray structures of the ribosome in complex with clindamycin are available in the Protein Data Bank, which reveal that there are two distinct conformations of the pyrrolidinyl propyl group of the bound clindamycin. In this work, we used quantum mechanical methods to investigate the probable conformations of clindamycin in order to explain the two binding modes in the ribosomal 23S RNA. We studied three lincosamide antibiotics: clindamycin, lincomycin, and pirlimycin at the B3LYP level with the 6-31G** basis set. The focus of our work was to connect the conformational landscape and electron densities of the two clindamycin conformers found experimentally with their physicochemical properties. For both functional conformers, we applied natural bond orbital (NBO) analysis and the atoms in molecules (AIM) theory, and calculated the NMR parameters. Based on the results obtained, we were able to show that the structure with the intramolecular hydrogen bond C=O…H-O is the most stable conformer of clindamycin. The charge transfer between the pyrrolidine-derivative ring and the six-atom sugar (methylthiolincosamide), which are linked via an amide bond, was found to be the dominant factor influencing the high stability of this conformer.     

Consequences of one-electron oxidation and one-electron reduction for 4-aminopyrimidine—DFT studies

The consequences of one-electron oxidation and one-electron reduction were studied for 4-aminopyrimidine (4APM), which displays prototropic tautomerism. Since experimental techniques are incapable of detecting less than 0.1% of minor tautomers, quantum-chemical calculations [DFT(B3LYP)/6-311+G(d,p)] were carried out for all possible tautomers of neutral 4AMP and its redox forms, 4APM (+ ?) and 4APM (- ?). Four tautomers were considered: one amine and three imine tautomers (two NH and one CH form). Geometric isomerism of the exo?=?NH group was also taken into account. One-electron oxidation (4APM - e → 4APM (+??)) has no significant effect on the tautomeric preferences; it influences solely the composition of the tautomeric mixture. The amine tautomer is favored for both 4APM (+??) and 4APM. An interesting change in the tautomeric preference occurs for 4APM (- ?). One-electron reduction (4APM?+?e → 4APM (- ?)) favors the C5 atom for the labile proton. The preference of the imine CH tautomer in the tautomeric mixture of 4APM (- ?) may partially explain the origin of CH tautomers in nucleobases.     

Expression of the androgen receptor in the testis of mice with a Sertoli cell specific knock-out of the connexin 43 gene (SCCx43KO−/−)

Gap junctions are intercellular channels that connect the cytoplasm of adjacent cells, allowing the passage of small molecules (<1 kDa) and thereby the regulation of many different processes. In the male gonad, the most abundant protein that builds gap junctions is connexin 43 (Cx43, GJA1). Specific knock-out of Sertoli cells (SCCx43KO^?/?) results in an impaired spermatogenesis up to the Sertoli cell only syndrome. The aim of this study was to compare the testicular expression pattern of the androgen receptor (AR) in wild type (WT) and SCCx43KO^?/? mice. In both WT and SCCx43KO^?/? testes, the AR staining was restricted to the nuclei of Sertoli, Leydig, and peritubular cells. However, the staining intensity varied between control and mutant mice. In the latter, the AR expression depended on the level of the seminiferous tubule impairment. In tubules with qualitatively normal spermatogenesis, the AR protein expression was similar to that observed in the testes of WT mice. Conversely, seminiferous tubules with an arrest of spermatogenesis at the level of spermatogonial or spermatocyte phase expressed the AR at a lower intensity. In Sertoli cell only tubules (no germ cells in the tubules), the AR immunoreaction was mainly weak or undetectable. Moreover, AR staining was lower in Sertoli and Leydig cells (p < 0.001 and p < 0.05, respectively) of SCCx43KO^?/? mice compared to WT mice, as revealed by a semiquantitative analysis. In conclusion, the deletion of Cx43 leads to a partial disruption of the AR signaling pathway, indicating a possible reason for the observed impaired spermatogenesis.     

Intraspecific variation in alkaloid profile of four lupine species with implications for the pea aphid probing behaviour

The alkaloid profile of white lupine Lupinus albus L. cv. ‘Boros’ and ‘Butan’, narrow-leaf lupine L. angustifolius L. cv. ‘Bojar’, ‘Graf’, ‘Karo’, ‘Mirela’, and ‘Sonet’, yellow lupine L. luteus L. cv. ‘Dukat’, ‘Parys’, ‘Perkoz’, and ‘Talar’, and wild species big-leaf lupine L. polyphyllus Lindl. was studied. The pea aphid Acyrthosiphon pisum Harris probing behaviour was monitored using the electrical penetration graph (EPG) technique.Four types of feeding behaviour were recognized. First, on highly acceptable L. luteus cv. ‘Dukat’, the total and mean duration of probing, time to reach phloem phase, and the duration of the first phloem phase were comparable to those in aphids on control plant Pisum sativum. Second, on partially acceptable L. luteus cv. ‘Talar’, pathway activities were slightly impeded, and the probes were more numerous and slightly shorter than on control plants. The phloem phase occurred sporadically, and feeding was terminated early after a brief period of ingestion. Third, on unpalatable but acceptable L. angustifolius cv. ‘Bojar’ and ‘Sonet’, and L. luteus cv. ‘Parys’ and ‘Perkoz’, the probes were numerous and usually epidermal. The phloem phase occurred rarely, and when it did, it was short and consisted mainly of watery salivation. Finally, on unacceptable L. albus cv. ‘Boros’ and ‘Butan’, L. angustifolius cv. ‘Graf’, ‘Karo’, ‘Mirela’, and L. polyphyllus, total probing time and individual probes were short and phloem phase did not occur.Eighteen alkaloids were identified: one piperidine alkaloid (ammodendrine), one indole (gramine), ten quinolizidine alkaloids (one tricyclic and nine tetracyclic compounds), and six esters. All lupine varieties that contained lupanine, its derivatives and especially their esters appeared to be unacceptable to the pea aphid, independent of the total concentration of any specific lupanine alkaloid. In contrast, sparteine and its derivatives did not seem to affect aphid probing significantly.     

Ds-like restless deletion derivatives occur in Tolypocladium inflatum and two foreign hosts, Neurospora crassa and Penicillium chrysogenum

Single copies of the transposon Restless from Tolypocladium inflatum were introduced into Neurospora crassa and Penicillium chrysogenum. Excision of Restless from its donor site was investigated in N. crassa and in P. chrysogenum using direct selective conditions. In N. crassa, forward selection was also analyzed. Deleted Restless elements were frequently obtained in addition to the expected complete removal of Restless from its donor site. Similar deleted elements were also identified in T. inflatum employing a PCR amplification strategy. These deleted Restless copies strongly resemble maize Ds elements of various types, and direct repeated sequences of 3 to 16 bp were found to flank the truncated regions. In addition Ds1-like Restless elements were identified that carried foreign sequences between the inverted repeats. We discuss how Ds-like Restless elements might be generated by inaccurate excision from an active transposon copy.