Lectin microarray analysis of LV extracts and plasma.

<p>Reactivity of fucose-binding lectins AOL and AAL, and mucin-type <i>O</i>-glycan-binding lectin ACA was analyzed in LV extracts (A) and plasma depleted of high-abundance proteins (B) of DS rats (n = 3). Entire lectin microarray datasets are shown in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150210#pone.0150210.s004" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150210#pone.0150210.s005" target="_blank">S4</a> Tables. Data are presented as normalized intensity. *, <i>p <</i> 0.05 (Tukey-HSD).</p

Altered <i>O</i>-glycosylation on CSRP3 in the LV of DS hypertensive rats.

<p>(A) ACA lectin blot analysis and SYPRO Ruby staining of fractions from sialidase-treated LV extracts. Arrow indicates the ACA-positive band, which is observed strongly in fraction 3 of HS (<i>H</i>) but weakly in that of the LS (<i>L</i>) group. (B) Two-dimensional PAGE images of sialidase-treated LV fraction 3. Proteins transferred to membranes were subjected to SYPRO Ruby staining, and then to ACA lectin blotting. Insets show magnified images of two spots used for protein identification. (C) Western blot (<i>WB</i>) and ACA lectin blot (<i>LB</i>) analyses of recombinant human CSRP3. Recombinant proteins expressed in <i>E</i>. <i>coli</i> (unglycosylated negative control) and in HEK293 cells (potentially glycosylated reference) were analyzed after treatment with sialidase and <i>O</i>-glycosidase. (D) Relative expression levels of <i>Csrp3</i> in the LV tissues. qPCR data were normalized to <i>Tbp</i> expression levels. The numbers of examined rats were n = 12 and n = 15 for HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (E) Protein levels of CSRP3 in LV extracts. Densitometry analysis data of western blotting are shown (n = 6). (D,E) The data are presented as the fold change compared with LS rats at 12 weeks. (F) Western blot (<i>WB</i>) and ACA lectin blot (<i>LB</i>) analyses of CSRP3 from LV extracts of DS rats. CSRP3 in LV extracts was immunoprecipitated, denatured, separated by SDS-PAGE, and analyzed. Recombinant human CSRP3 was used as an experimental control of immunoprecipitation with anti-CSRP3 antibody (+) or normal IgG (-). Lower panel shows densitometry analysis data; the intensity of each band in LB was normalized to that in WB (n = 6). (G) Effects of glycosidases on CSRP3 dimerization. LV extracts from three HS (<i>H</i>) or LS (<i>L</i>) rats at 16 weeks were treated with three glycosidases as indicated and then analyzed by western blotting for CSRP3. Arrows indicate the bands corresponding to monomers and dimers. Lower panels show densitometry analysis from five experiments; dimer/monomer ratios are presented as the fold change compared with LS rats without glycosidase treatment. (D-G) *, <i>p <</i> 0.05 (Tukey-HSD). In (G), statistical comparison of HS and LS groups in the same condition and that of HS groups in five conditions are indicated.</p

Upregulation of glycogene expression in the LV of DS hypertensive rats.

<p>(A) Relative expression levels of glycogenes selected after qPCR array in the LV of DS rats fed HS and LS diets were quantified by qPCR. <i>Rps18</i> was used as an internal control. The numbers of examined rats were n = 12 and n = 15 for the HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. Expression levels were normalized to that of TATA box-binding protein (<i>Tbp</i>). (B) Protein levels of glycogenes in LV extracts were analyzed by western blotting. β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as internal controls. Representative results from three rats per group are indicated. (C) Densitometry analysis of immunoblots shown in (B). Intensity of each band was normalized to that of GAPDH. Data are presented as the fold change compared with LS rats at 12 weeks (n = 6). (A,C) *, <i>p <</i> 0.05 (Tukey-HSD).</p

Summary of the lectin microarray results of LV extracts and plasma in DS rats.

<p>Summary of the lectin microarray results of LV extracts and plasma in DS rats.</p

Decrease of core fucosylation on <i>N</i>-glycans in DS hypertensive rats.

<p>(A) Lectin blot analysis of LV extracts using AOL. Representative images demonstrate AOL-reactive glycoproteins and SYPRO Ruby-stained proteins of three individual rats in each group. Right panel shows densitometry analysis data; intensity of each band was normalized to total protein. Data are presented as the fold change (n = 6) compared with LS rats at 12 weeks. (B) Relative expression levels of the genes responsible for core fucosylation (<i>Fut8</i>) and defucosylation (<i>Fuca1</i> and <i>Fuca2</i>) on <i>N</i>-glycans were examined by qPCR; levels in the LV and liver were normalized to that of <i>Tbp</i> and <i>Actb</i>, respectively. Data are presented as the fold change compared with the LS group at 12 weeks. (C) AFU activity in the LV and liver extracts, and plasma. Data were normalized to protein content. (D) Plasma levels of FUCA1 and FUCA2. (E) Correlation of AFU activity in LV extracts shown in (C) with relative ANP expression shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150210#pone.0150210.g003" target="_blank">Fig 3B</a>. (F,G) Correlation of plasma AFU activity shown in (C) with plasma NT-proANP concentration (<i>F</i>) and LV anterior wall thickness during diastole (LVAWd) (G). (B-G) The numbers of examined rats were n = 12 and n = 15 for HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (A-D) *, <i>p <</i> 0.05 (Tukey-HSD).</p

Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure

<div><p>Targeted proteomics focusing on post-translational modifications, including glycosylation, is a useful strategy for discovering novel biomarkers. To apply this strategy effectively to cardiac hypertrophy and resultant heart failure, we aimed to characterize glycosylation profiles in the left ventricle and plasma of rats with cardiac hypertrophy. Dahl salt-sensitive hypertensive rats, a model of hypertension-induced cardiac hypertrophy, were fed a high-salt (8% NaCl) diet starting at 6 weeks. As a result, they exhibited cardiac hypertrophy at 12 weeks and partially impaired cardiac function at 16 weeks compared with control rats fed a low-salt (0.3% NaCl) diet. Gene expression analysis revealed significant changes in the expression of genes encoding glycosyltransferases and glycosidases. Glycoproteome profiling using lectin microarrays indicated upregulation of mucin-type <i>O</i>-glycosylation, especially disialyl-T, and downregulation of core fucosylation on <i>N</i>-glycans, detected by specific interactions with <i>Amaranthus caudatus</i> and <i>Aspergillus oryzae</i> lectins, respectively. Upregulation of plasma α-l-fucosidase activity was identified as a biomarker candidate for cardiac hypertrophy, which is expected to support the existing marker, atrial natriuretic peptide and its related peptides. Proteomic analysis identified cysteine and glycine-rich protein 3, a master regulator of cardiac muscle function, as an <i>O</i>-glycosylated protein with altered glycosylation in the rats with cardiac hypertrophy, suggesting that alternations in <i>O</i>-glycosylation affect its oligomerization and function. In conclusion, our data provide evidence of significant changes in glycosylation pattern, specifically mucin-type <i>O</i>-glycosylation and core defucosylation, in the pathogenesis of cardiac hypertrophy and heart failure, suggesting that they are potential biomarkers for these diseases.</p></div

Free Thiol of Transthyretin in Human Plasma Most Accessible to Modification/Oxidation

Free-thiol(s) in proteins, especially, when located on the surface of the molecule, are susceptible to oxidation/modification, which may cause loss of function or an alteration in the ternary structure. This suggests that the status of thiol group(s) of cysteine residue(s) in a protein, i.e., free-thiol versus an oxidized/modified form, in vivo, could reflect the physiological state of the molecule with respect to susceptibility to oxidative stress. To address this issue, we established an efficient method for isolating proteins that contain free thiol groups from a complex mixture, which permits the amount of free-thiol form(s) to modified/oxidized forms to be estimated. Albumin, which accounts for 55% of the total plasma proteins and has such a free thiol and has been reported to scavenge various reactive oxygen species (ROS) in vivo. The developed method was used to isolate the free form of albumin from fresh plasma. However, contrary to our expectations, transthyretin (TTR), which also has a single free thiol, was found to be the major protein that was the most susceptible to modification/oxidation. In addition, the free-thiol form could be separated from oxidized or modified molecules, permitting the relative abundance of the free-thiol form to be estimated. The findings show that the levels of the free-thiol form of TTR in plasma was significantly lowered after a hydrogen peroxide treatment, even at low concentrations (0.1 mM), suggesting that TTR could be a useful biomarker for monitoring a ROS imbalance in relation to various oxidative stress conditions

Peptidomics for Studying Limited Proteolysis

Limited proteolysis is a pivotal mechanism regulating protein functions. Identifying physiologically or pathophysiologically relevant cleavage sites helps to develop molecular tools that can be used for diagnostics or therapeutics. During proteolysis of secretory and membrane proteins, part of the cleaved protein is liberated and destined to undergo degradation but should retain original cleavage sites created by proteolytic enzymes. We profiled endogenous peptides accumulated for 4 h in media conditioned by primary cultured rat cardiac fibroblasts. A total of 3916 redundant peptide sequences from 94 secretory proteins and membrane proteins served to identify limited cleavage sites, both annotated and unannotated, for signal peptide or propeptide removal, peptide hormone processing, ectodomain shedding, and regulated intramembrane proteolysis. Incorrectly predicted signal cleavage sites are found in typical proteins such as extracellular matrix proteins and the peptide hormone precursor adrenomedullin ADM. The revealed signal peptide cleavage site for ADM was experimentally verified by identifying the major molecular form of flanking proadrenomedullin N-terminal peptide. We suggest that profiling of endogenous peptides, like transcriptome sequence reads, makes sense in regular cells such as fibroblasts and that peptidomics provides insight into proteolysis-regulated protein functions

Discovery of Potent Hexapeptide Agonists to Human Neuromedin U Receptor 1 and Identification of Their Serum Metabolites

Neuromedin U (NMU) and S (NMS) display various physiological activities, including an anorexigenic effect, and share a common C-terminal heptapeptide-amide sequence that is necessary to activate two NMU receptors (NMUR1 and NMUR2). On the basis of this knowledge, we recently developed hexapeptide agonists <b>2</b> and <b>3</b>, which are highly selective to human NMUR1 and NMUR2, respectively. However, the agonists are still less potent than the endogenous ligand, hNMU. Therefore, we performed an additional structure–activity relationship study, which led to the identification of the more potent hexapeptide <b>5d</b> that exhibits similar NMUR1-agonistic activity as compared to hNMU. Additionally, we studied the stability of synthesized agonists, including <b>5d</b>, in rat serum, and identified two major biodegradation sites: Phe²-Arg³ and Arg⁵-Asn⁶. The latter was more predominantly cleaved than the former. Moreover, substitution with 4-fluorophenylalanine, as in <b>5d</b>, enhanced the metabolic stability at Phe²-Arg³. These results provide important information to guide the development of practical hNMU agonists

Discovery of a Human Neuromedin U Receptor 1‑Selective Hexapeptide Agonist with Enhanced Serum Stability

Neuromedin U (NMU) activates two NMU receptors (NMUR1 and NMUR2) and is a useful antiobesity drug lead. We report discovery of a hexapeptide agonist, 2-thienylacetyl-Trp¹-Phe­(4-F)²-Arg³-Pro⁴-Arg⁵-Asn⁶-NH₂ (<b>4</b>). However, the NMUR1 selectivity and serum stability of this agonist were unsatisfactory. Through a structure–activity relationship study focused on residue 2 of agonist <b>4</b>, serum stability, and pharmacokinetic properties, we report here the discovery of a novel NMUR1 selective hexapeptide agonist <b>7b</b> that suppresses body weight gain in mice