Glycoproteomic Alterations in Drug-Resistant Nonsmall Cell Lung Cancer Cells Revealed by Lectin Magnetic Nanoprobe-Based Mass Spectrometry.

Understanding the functional role of glycosylation-mediated pathogenesis requires deep characterization of glycoproteome, which remains extremely challenging due to the inherently complex nature of glycoproteins. We demonstrate the utility of lectin-magnetic nanoprobe (MNP@lectin) coupled to Orbitrap HCD-CID-MS/MS for complementary glycotope-specific enrichment and site-specific glycosylation analysis of the glycoproteome. By three nanoprobes, MNP@ConA, MNP@AAL, and MNP@SNA, our results revealed the first large-scale glycoproteome of nonsmall cell lung cancer (NSCLC) with 2290 and 2767 nonredundant glycopeptides confidently identified (Byonic score ≥100) in EGFR-TKI-sensitive PC9 and -resistant PC9-IR cells, respectively, especially with more fucosylated and sialylated glycopeptides in PC9-IR cells. The complementary enrichment was demonstrated with only five glycopeptides commonly enriched in three MNP@lectins. Glycotope specificity of 79 and 62% for enrichment was achieved using MNP@AAL and MNP@SNA, respectively. Label-free quantitation revealed predominant fucosylation in PC9-IR cells, suggesting its potential role associated with NSCLC resistance. Moreover, without immunoprecipitation, this multilectin nanoprobe allows the sensitive identification of 51 glycopeptides from 10 of 12 reported sites from onco-protein EGFR. Our results not only demonstrated a sensitive approach to study the vastly under-represented N-glycoprotome but also may pave the way for a glycoproteomic atlas to further explore the site-specific function of glycoproteins associated with drug resistance in NSCLC.

Prognostic Value of Copeptin in Chronic Kidney Disease: From General Population to End-Stage Renal Disease.

Arginine vasopressin (AVP), also known as antidiuretic hormone (ADH), is released in response to osmotic and non-osmotic stimuli and plays a key role in many physiologic and pathologic processes. The main function of AVP is the control of fluid homeostasis by inducing water conservation by the kidney, but it also stimulates arteriolar vasoconstriction and the release of adrenocorticotropic hormone (ACTH). These actions are mediated by different AVP receptors located on various target cells. Produced in hypothalamus from a larger precursor, pre-proAVP, AVP is produced in equimolar amounts to copeptin, a glycopeptide with yet unknown biologic function. Copeptin remains stable in plasma and its circulating concentrations correlate directly with those of AVP. Because AVP is unstable in isolated plasma or serum and its half-life is short, copeptin has become an easily measured surrogate marker reflecting vasopressin concentration. Recently, associations between high circulating copeptin and decline in glomerular filtration rate as well as greater risk of new-onset chronic kidney disease (CKD) have been reported. In addition, copeptin has been shown to be associated with increased risk of complications such as myocardial infarction, heart failure, diabetes mellitus and metabolic syndrome. In this brief review, studies on the prognostic value of copeptin measurement in the general population and in CKD are presented and discussed.

Less increase of copeptin and MR-proADM due to intervention with selenium and coenzyme Q10 combined: Results from a 4-year prospective randomized double-blind placebo-controlled trial among elderly Swedish citizens.

Intervention with selenium and coenzyme Q10 have recently been found to reduce mortality and increase cardiac function. The mechanisms behind these effects are unclear. As selenium and coenzyme Q10 is involved in the anti-oxidative defence, the present study aimed to evaluate effects of selenium and coenzyme Q10 on copeptin and adrenomedullin as oxidative stress biomarkers. Therefore 437 elderly individuals were included and given intervention for 4 years. Clinical examination and blood samples were undertaken at start and after 18 and 48 months. Evaluations of copeptin and MR-proADM changes were performed using repeated measures of variance. Cardiovascular mortality was evaluated using a 10-year-period of follow-up, and presented in Kaplan-Meier plots. A significant increase in copeptin level could be seen in the placebo group during the intervention period (from 9.4 pmol/L to 15.3 pmol/L), compared to the active treatment group. The difference between the groups was confirmed in the repeated measurement of variance analyses (P = 0.031) with less copeptin increase in the active treatment group. Furthermore, active treatment appeared to protect against cardiovascular death both in those with high and with low copeptin levels at inclusion. Less increase of MR-proADM could also be seen during the intervention in the active treatment group compared to controls (P = 0.026). Both in those having an MR-proADM level above or below median level, significantly less cardiovascular mortality could be seen in the active treatment group (P = 0.0001, and P = 0.04 respectively). In conclusion supplementation with selenium and coenzyme Q10 during four years resulted in less concentration of both copeptin and MR-proADM. A cardioprotective effect of the supplementation was registered, irrespective of the initial levels of these biomarkers, and this protection was recognized also after 10 years of observation.

Hydroxyproline-rich glycopeptide signals in potato elicit signalling associated with defense against insects and pathogens.

HypSys peptides are 18-20 amino acids glycopeptide defense signal first discovered in tobacco and tomato that activate expression of defensive genes against insect-herbivores. Discovery of their orthologs in other Solanaceaous and nonsolanaceous plants demonstrated their possible ubiquitous nature and species specific functional diversity. In our continued search to establish the paradigm of defense signalling by HypSys peptides, we isolated a cDNA from potato leaves encoding putative analogs of tomato HypSys peptides flanked by conserved proteolytic cleavage sites. The gene encoding the cDNA was a member of a gene family in the tetraploid genome of potato and its expression was transcriptionally activated by wounding and methyl jasmonate. The deduced precursor protein contained a leader peptidase splice site and three putative HypSys peptides with conserved N- and C-termini along with central proline-rich motifs. In defense signalling, the three HypSys peptides elicit H2O2 generation in vivo and activate several antioxidant defensive enzymes in young potato leaves. Similar to potato systemin, the HypSys peptides activate the expression of octadecanoid pathway genes and protease inhibitors for insect defense. In addition, the HypSys peptides also activate the essential genes of the innate pathogen defense response in young potato leaves, acting as common elicitors of signalling associated with anti-herbivore and anti-pathogen defense in potato.

Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato.

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Man alpha 1-6(Man alpha 1-3)(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.

A chimeric lectin formed from Bauhinia purpurea lectin and Lens culinaris lectin recognizes a unique carbohydrate structure.

Lectins are carbohydrate-binding proteins widely used in biochemical, immunochemical, and histochemical studies. Bauhinia purpurea lectin (BPA) is a leguminous lectin with an affinity for galactose and lactose. Nine amino acids, DTWPNTEWS, corresponding to the amino acid sequence from aspartic acid-135 to serine-143 in the primary structure of BPA were replaced with the corresponding amino acid residues from the mannose-binding Lens culinaris lectin (LCA), and the chimeric lectin obtained was expressed in Escherichia coli cells. The carbohydrate-binding specificity of the recombinant chimeric lectin was investigated in detail by comparing the elution profiles of various glycopeptides and oligosaccharides with defined carbohydate structures from immobilized lectin columns. Glycopeptides carrying three constitutive carbohydrate sequences of Galbeta1-3GalNAc-Ser/Thr and a complex-type biantennary glycopeptide, which show a high affinity for BPA or LCA, were shown to have no affinity for the chimeric lectin. In contrast, hybrid-type and high mannose-type glycopeptides with a Manalpha1-6(Manalpha1-3)Manalpha1-6Man sequence were found to have a moderate affinity for the chimeric lectin. This result demonstrates that a novel type of lectin with a unique carbohydrate-binding specificity can be constructed from BPA by substituting several amino acid residues in its metal-binding region with other amino acid residues. Additional lectin(s) with distinctly different carbohydrate-binding specificities will provide a powerful tool for many studies.

Biological activities of bovine glycomacropeptide.

Biological activity of bovine kappa-caseino glycomacropeptide (GMP) has received much attention in recent years. Research has focused on the ability of GMP to bind cholera and Escherichia coli enterotoxins, inhibit bacterial and viral adhesion, suppress gastric secretions, promote bifidobacterial growth and modulate immune system responses. Of these, protection against toxins, bacteria, and viruses and modulation of the immune system are the most promising applications.

Conformations and internal mobility of a glycopeptide derived from bromelain using molecular dynamics simulations and NOESY analysis.

The conformation and internal flexibility of a glycopeptide Manalpha1-6 (Xylbeta1-2)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3) GlcNAcbeta1-N(Asn-Glu-Ser-Ser), prepared from pineapple stem bromelain, have been analyzed using a combination of molecular dynamics (MD) simulations in water with NOESY 1H NMR spectroscopy. Theoretical NOESY cross-peak intensities were calculated by the CROSREL program on the basis of models, obtained from MD simulations, using a full relaxation matrix approach. Special attention was paid to the description of internal flexibility of the hexasaccharide moiety by the use of generalized order parameters, in combination with the application of an individual rotation correlation tme for each monosaccharide residue. The tetrapeptide moiety appeared to be very mobile during the MD simulations, which was confirmed by the absence of NOE cross peaks. For the oligosaccharide part a model was developed to estimate characteristic times for large reorientational motions around the glycosidic linkages, associated with conformational transitions. For the Manalpha1-6Man and the Fucalpha1-3GlcNAc linkages such a flexibility was found with a characteristic time of 2 ns. In contrast, the Xylbeta1-2Manbeta1-4GlcNAcbeta1-4GlcNAc part of the glycan appears to be relatively rigid.

Age-related development of a heterozygous phenotype in solitary neurons of the homozygous Brattleboro rat.

A single-base deletion in the single-copy vasopressin gene is the cause of diabetes insipidus in the homozygous Brattleboro rat (di/di). It results in the synthesis of an altered vasopressin precursor of which the axonal transport is blocked. Paradoxically, a small number of solitary hypothalamic neurons displays all the immunoreactivities of the wild-type vasopressin precursor (i.e., vasopressin, neurophysin, and a glycopeptide). In the present paper we provide evidence that these neurons have undergone a switch to a genuine heterozygous (di/+) phenotype; i.e., they contain the immunoreactivities of both the wild-type and the mutated vasopressin precursors. In the neural lobe, glycopeptide fibers are also present, showing that axonal transport of the wild-type precursor is restored. Moreover, the number of neurons displaying this di/+ phenotype increases markedly and in a linear way (from 0.1% up to 3% of the vasopressin cells) with age. These findings indicate that after mitotic division has ceased, genomic alterations occur in somatic neurons in vivo. The molecular event generating the di/+ phenotype in the di/di animal could involve a somatic intrachromosomal gene conversion between the homologous exons of the vasopressin and the related oxytocin genes.