An Improved Method for Cell Type-Selective Glycomic Analysis of Tissue Sections Assisted by Fluorescence Laser Microdissection

Lectin microarray (LMA) is a highly sensitive technology used to obtain the global glycomic profiles of endogenous glycoproteins in biological samples including formalin-fixed paraffin-embedded tissue sections. Here, we describe an effective method for cell type-selective glycomic profiling of tissue fragments collected by laser microdissection (LMD) under fluorescent histochemical visualization. We optimized each step of histochemical staining and confirmed the reliability and validity of glycomic profiling. Using the optimized procedure, glycomic profiles were obtained with 0.5 mm2 of stained thymic sections (5-μm-thick) from 8-week-old C57BL/6J male mice. The glycomic profiles of Ulex europaeus agglutinin-I (UEA-I)-stained medullary regions showed higher UEA-I signals than those of the morphologically determined medulla regions, indicating the utility of this method for UEA-I(+) cell-selective analysis. To further evaluate this method, tissue fragments was serially collected from stained and unstained areas of medullary epithelial cell probes (UEA-I and anti-cytokeratin 5 antibody) and a cortex-staining probe (peanut agglutinin). The medullary regions assigned by the three probes showed significantly different glycomic profiles, highlighting the difference in subpopulation recognition among the three probes, which was consistent with previous reports. In conclusion, our fluorescence LMD-LMA method enabled cell type-selective tissue glycomic analysis of pathological specimens and animal models, especially for glyco-biomarker discovery

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

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

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

Tachykinin-Related Peptides Share a G Protein-Coupled Receptor with Ion Transport Peptide-Like in the Silkworm Bombyx mori.

Recently, we identified an orphan Bombyx mori neuropeptide G protein-coupled receptor (BNGR)-A24 as an ion transport peptide-like (ITPL) receptor. BNGR-A24 belongs to the same clade as BNGR-A32 and -A33, which were recently identified as natalisin receptors. Since these three BNGRs share high similarities with known receptors for tachykinin-related peptides (TRPs), we examined whether these BNGRs can function as physiological receptors for five endogenous B. mori TRPs (TK-1-5). In a heterologous expression system, BNGR-A24 acted as a receptor for all five TRPs. In contrast, BNGR-A32 responded only to TK-5, and BNGR-A33 did not respond to any of the TRPs. These findings are consistent with recent studies on the ligand preferences for B. mori natalisins. Furthermore, we evaluated whether the binding of ITPL and TRPs to BNGR-A24 is competitive by using a Ca2+ imaging assay. Concomitant addition of a TRP receptor antagonist, spantide I, reduced the responses of BNGR-A24 not only to TK-4 but also to ITPL. The results of a binding assay using fluorescent-labeled BNGR-A24 and ligands demonstrated that the binding of ITPL to BNGR-A24 was inhibited by TK-4 as well as by spantide I, and vice versa. In addition, the ITPL-induced increase in cGMP levels of BNGR-A24-expressing BmN cells was suppressed by the addition of excess TK-4 or spantide I. The intracellular levels of cAMP and cGMP, as second messenger candidates of the TRP signaling, were not altered by the five TRPs, suggesting that these peptides act via different signaling pathways from cAMP and cGMP signaling at least in BmN cells. Taken together, the present findings suggest that ITPL and TRPs are endogenous orthosteric ligands of BNGR-A24 that may activate discrete signaling pathways. This receptor, which shares orthosteric ligands, may constitute an important model for studying ligand-biased signaling

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

<i>In vitro</i> competitive binding assay of rITPL and TK-4 to BNGR-A24.

<p>(A–D) Microscopic imaging. The binding of RR-labeled ligands (A and C, rITPL; B and D, TK-4) and CHO cells expressing EGFP-fused BNGR-A24 was examined in the presence of potential competitors (A, TK-4; B, rITPL; C and D, spantide I [SP]). EGFP and RR fluorescences were observed by confocal microscopy. Co-localization is presented as yellow in the merged images (Merge). Representative images of cells from at least two independent experiments are shown. (E–H) Relative fluorescent intensity obtained from experiment images (A)–(D), respectively. RR fluorescent intensity was normalized by EGFP intensity per image and is indicated as fold change over that without competitor (mean + S.D.; <i>n</i> = 6). Asterisks indicate significant differences compared to ‘no competitor’ data (<i>P</i> < 0.05, Dunnett’s test).</p

EC₅₀ of BNGR responses to <i>B</i>. <i>mori</i> TRPs.

<p>EC₅₀ of BNGR responses to <i>B</i>. <i>mori</i> TRPs.</p