Detection of Proteome Changes in Human Colon Cancer Induced by Cell Surface Binding of Growth-Inhibitory Human Galectin‑4 Using Quantitative SILAC-Based Proteomics

Endogenous lectins have the capacity to translate glycan-encoded information on the cell surface into effects on cell growth. As test cases to examine changes in protein presence associated with tumor growth inhibition, we applied SILAC-based proteomics on human colon carcinoma cells treated with galectin-4 (Gal-4). The five tested linesLS 180, Vaco 432, Colo 205, CX 1, and HCT 116responded with differentiation and reduced proliferation to Gal-4 binding. In proteomic analysis (mass spectral data deposited with PRIDE, PXD003489), 2654 proteins were quantified, of which 190 were down-regulated and 115 were up-regulated (>2-fold). 1D annotation analysis of the results indicated down-regulation of DNA replication-associated processes, while protein presence for secretory and transport functions appeared increased. The strongest induction was found for CALB2 (calretinin; ∼24-fold), TGM2 (protein-glutamine γ-glutamyltransferase 2; ∼11-fold), S100A3 (∼10-fold), and GSN (gelsolin; 9.5-fold), and the most pronounced decreases were seen for CDKN2A (tumor suppressor ARF; ∼6-fold), EPCAM (epithelial cell adhesion molecule; ∼6-fold), UBE2C (ubiquitin-conjugating enzyme E2 C; ∼5-fold), KIF2C (kinesin-like protein KIF2C; 5-fold), and LMNB1 (lamin-B1; ∼5-fold). The presence of the common proliferation marker Ki-67 was diminished about 4-fold. By tracing significant alterations of protein expression likely relevant for the observed phenotypic effects, the capacity of a galectin to affect the proteome of human colon cancer cells at multiple sites is revealed

Molecular Recognition of Complex-Type Biantennary <i>N</i>‑Glycans by Protein Receptors: a Three-Dimensional View on Epitope Selection by NMR

The current surge in defining glycobiomarkers by applying lectins rekindles interest in definition of the sugar-binding sites of lectins at high resolution. Natural complex-type <i>N</i>-glycans can present more than one potential binding motif, posing the question of the actual mode of interaction when interpreting, for example, lectin array data. By strategically combining <i>N</i>-glycan preparation with saturation-transfer difference NMR and modeling, we illustrate that epitope recognition depends on the structural context of both the sugar and the lectin (here, wheat germ agglutinin and a single hevein domain) and cannot always be predicted from simplified model systems studied in the solid state. We also monitor branch-end substitutions by this strategy and describe a three-dimensional structure that accounts for the accommodation of the α2,6-sialyl­ated terminus of a biantennary <i>N</i>-glycan by viscumin. In addition, we provide a structural explanation for the role of terminal α2,6-sialyl­ation in precluding the interaction of natural <i>N</i>-glycans with lectin from Maackia amurensis. The approach described is thus capable of pinpointing lectin-binding motifs in natural <i>N</i>-glycans and providing detailed structural explanations for lectin selectivity

Thermodynamic Switch in Binding of Adhesion/Growth Regulatory Human Galectin‑3 to Tumor-Associated TF Antigen (CD176) and MUC1 Glycopeptides

A shift to short-chain glycans is an observed change in mucin-type O-glycosylation in premalignant and malignant epithelia. Given the evidence that human galectin-3 can interact with mucins and also weakly with free tumor-associated Thomsen-Friedenreich (TF) antigen (CD176), the study of its interaction with MUC1 (glyco)­peptides is of biomedical relevance. Glycosylated MUC1 fragments that carry the TF antigen attached through either Thr or Ser side chains were synthesized using standard Fmoc-based automated solid-phase peptide chemistry. The dissociation constants (<i>K</i>_d) for interaction of galectin-3 and the glycosylated MUC1 fragments measured by isothermal titration calorimetry decreased up to 10 times in comparison to that of the free TF disaccharide. No binding was observed for the nonglycosylated control version of the MUC1 peptide. The most notable feature of the binding of MUC1 glycopeptides to galectin-3 was a shift from a favorable enthalpy to an entropy-driven binding process. The comparatively diminished enthalpy contribution to the free energy (Δ<i>G</i>) was compensated by a considerable gain in the entropic term. ¹H–¹⁵N heteronuclear single-quantum coherence spectroscopy nuclear magnetic resonance data reveal contact at the canonical site mainly by the glycan moiety of the MUC1 glycopeptide. Ligand-dependent differences in binding affinities were also confirmed by a novel assay for screening of low-affinity glycan–lectin interactions based on AlphaScreen technology. Another key finding is that the glycosylated MUC1 peptides exhibited activity in a concentration-dependent manner in cell-based assays revealing selectivity among human galectins. Thus, the presentation of this tumor-associated carbohydrate ligand by the natural peptide scaffold enhances its affinity, highlighting the significance of model studies of human lectins with synthetic glycopeptides

Immunofluorescence staining of <i>Hydra</i> polyps with anti-PPOD4 antibody.

<p>Polyps were fixed with PFA (A-I) or Lawdowsky’s fixative (J–O), stained with anti-PPOD4 or anti-GFP antibody and examined in the confocal microscope. Schematic diagrams indicate the positions of the optical sections shown. Polyps in A-I were not permeabilized and show staining of the extracellular surface. Polyps in J-O were permeabilized to permit staining of intracellular vesicles. See text for details.</p

PAS cytochemistry of <i>Hydra</i> cuticle.

<p>Periodic acid-thiocarbohydrazide-silver proteinate staining was performed on Epon sections from <i>H. vulgaris</i>. A: Cuticle layers c1–5 react positively as do apical secretory granules (s) and glycogen particles (arrow-heads) in a neighbouring nematocyst; the asterisk marks a vacuole. Scale bar: 500 nm. B: Negative control for the PAS-reaction (omission of periodic acid oxidation); faint unspecific staining results from binding of thiocarbohydrazide to the osmium tetroxide used for freeze-substitution. Scale bar: 500 nm.</p

Internal repeats and three-dimensional structure of <i>Hydra</i> PPOD.

<p>A) Alignment of six internal repeats detected within the PPOD4 sequence using the RADAR algorithm. B) Structural model of a single β-trefoil domain in PPOD4 as inferred by Phyre. Three internal sequence repeats (coloured ribbon models) correspond to three repeated supersecondary structures that form a single β-trefoil fold.</p

PPOD agglutinates erythrocytes.

<p>Haemagglutination assays with rabbit erythrocytes. A: Addition of increasing amounts PPOD4 protein agglutinates erythrocytes and prevents their sedimentation (dark dot at 0 µl PPOD4 indicates sedimentation of erythrocytes, which are not agglutinated). Addition of mannose or glucose does not prevent PPOD4 induced agglutination. B: Erythrocytes sediment in the absence of PPOD4 protein (lower row). Addition of PPOD4 prevents this due to agglutination. Addition of heparin or chondroitin (GalNAc-4-SO4) to PPOD prevents PPOD4 induced agglutination in a concentration dependent manner.</p

Soluble protein content of hypertonic salt wash.

<p>A: SDS-PAGE gel of 0.2 M NaCl wash stained with coomassie. Major bands were excised and identified by mass spectrometry. Positions of the PPOD 2, 3 and 4 bands are indicated. Bands 1–4 represent members of SWT protein family. See text for details. B: Immunoblot for this gel stained with anti-PPOD4 antibody.</p

SWT (sweet tooth) proteins identified in the salt wash by MS/MS.

<p>SWT (sweet tooth) proteins identified in the salt wash by MS/MS.</p

Identification of PPOD proteins in the SDS-PAGE gel by mass spectrometry.

*<p>Numbers in parentheses refer to numbers of peptides identified by MS-spectra and MS/MS spectra respectively.</p>**<p>Numbers give the position of the peptides in the protein sequence.</p>***<p>There are two slightly different PPOD4 gene models (XP_002161930 and XP_002159894) based on NCBI annotation of the <i>Hydra</i> genome. Unique peptides corresponding to both gene models were found in the 27 kDa band. By comparison, only one PPOD4 gene model (Hma2.211683) corresponding to XP_002161930 is found in the <i>Hydra</i> genome browser (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052278#pone.0052278-Chapman1" target="_blank">[11]</a> for details of two genome assemblies).</p