Galectin-12 colocalizes with splicing factor-rich speckles and shuttles between the nucleus and cytoplasm in colon cancer cells

Several members of the glycan-binding family of galectins have been linked to colon cancer initiation and progression while the role of galectin-12 in this malignancy is largely unexplored. In previous studies we observed expression of galectin-12 in normal colon epithelium in contrast to its lack of expression in colorectal cancer tissues. In order to gain insight into its molecular function we established a genetically engineered colon cancer model cell line, which facilitates inducible and reconstituted expression of LGALS12 transgene at physiological levels in an isogenic background. Regulation of transgene expression by doxycycline was confirmed at the transcript- and protein level in a time- and dose-dependent manner for two independent clones. Reconstituted galectin-12 expression did not affect cell morphology and proliferation. Analysis of its subcellular distribution showed that galectin-12 resides in and shuttles between the nucleus and cytoplasm. More detailed analysis by double-immunofluorescence and confocal microscopy revealed colocalization of galectin-12 with splicing-factor rich nuclear speckles, hinting towards a potential role of galectin-12 in pre-mRNA splicing and processing. Therefore, the established model cell line represents a powerful tool to investigate the functional impact of galectin-12 reconstitution on colon cancer tumorigenesis

TGFBR2-dependent alterations of exosomal cargo and functions in DNA mismatch repair-deficient HCT116 colorectal cancer cells

Background: Colorectal cancers (CRCs) that lack DNA mismatch repair function exhibit the microsatellite unstable (MSI) phenotype and are characterized by the accumulation of frameshift mutations at short repetitive DNA sequences (microsatellites). These tumors recurrently show inactivating frameshift mutations in the tumor suppressor Transforming Growth Factor Beta Receptor Type 2 (TGFBR2) thereby abrogating downstream signaling. How altered TGFBR2 signaling affects exosome-mediated communication between MSI tumor cells and their environment has not been resolved. Here, we report on molecular alterations of exosomes shed by MSI cells and the biological response evoked in recipient cells. Methods: Exosomes were isolated and characterized by electron microscopy, nanoparticle tracking, and western blot analysis. TGFBR2-dependent effects on the cargo and functions of exosomes were studied in a MSI CRC model cell line enabling reconstituted and inducible TGFBR2 expression and signaling. Microsatellite frameshift mutations in exosomal and cellular DNA were examined by PCR-based DNA fragment analysis and exosomal protein profiles were identified by mass spectrometry. Uptake of fluorescent-labeled exosomes by hepatoma recipient cells was monitored by confocal microscopy. TGFBR2-dependent exosomal effects on secreted cytokine levels of recipient cells were analyzed by Luminex technology and ELISA. Results: Frameshift mutation patterns in microsatellite stretches of TGFBR2 and other MSI target genes were found to be reflected in the cargo of MSI CRC-derived exosomes. At the proteome level, reconstituted TGFBR2 expression and signaling uncovered two protein subsets exclusively occurring in exosomes derived from TGFBR2-deficient (14 proteins) or TGFBR2-proficient (five proteins) MSI donor cells. Uptake of these exosomes by recipient cells caused increased secretion (2–6 fold) of specific cytokines (Interleukin-4, Stem Cell Factor, Platelet-derived Growth Factor-B), depending on the TGFBR2 expression status of the tumor cell. Conclusion: Our results indicate that the coding MSI phenotype of DNA mismatch repair-deficient CRC cells is maintained in their exosomal DNA. Moreover, we uncovered that a recurrent MSI tumor driver mutation like TGFBR2 can reprogram the protein content of MSI cell-derived exosomes and in turn modulate the cytokine secretion profile of recipient cells. Apart from its diagnostic potential, these TGFBR2-dependent exosomal molecular and proteomic signatures might help to understand the signaling routes used by MSI tumors. Graphical Abstract Fricke et al. uncovered coding microsatellite instability-associated mutations of colorectal tumor driver genes like TGFBR2 in MSI tumor cellderived exosomes. Depending on the TGFBR2 expression status of their donor cells, shed exosomes show distinct proteomic signatures and promote altered cytokine secretion profiles in recipient cells

Prototype chicken galectins revisited: characterization of a third protein with distinctive hydrodynamic behaviour and expression pattern in organs of adult animals

Prototype galectins are versatile modulators of cell adhesion and growth via their reactivity to certain carbohydrate and protein ligands. These functions and the galectins' marked developmental regulation explain their attractiveness as models to dissect divergent evolution after gene duplication. Only two members have so far been assumed to constitute this group in chicken, namely the embryonic muscle/liver form {C-16 or CLL-I [16 kDa; chicken lactose lectin, later named CG-16 (chicken galectin-16)]} and the embryonic skin/intestine form (CLL-II or C-14; later named CG-14). In the present study, we report on the cloning and expression of a third prototype CG. It has deceptively similar electrophoretic mobility compared with recombinant C-14, the protein first isolated from embryonic skin, and turned out to be identical with the intestinal protein. Hydrodynamic properties unusual for a homodimeric galectin and characteristic traits in the proximal promoter region set it apart from the two already known CGs. Their structural vicinity to galectin-1 prompts their classification as CG-1A (CG-16)/CG-1B (CG-14), whereas sequence similarity to mammalian galectin-2 gives reason to refer to the intestinal protein as CG-2. The expression profiling by immunohistochemistry with specific antibodies discerned non-overlapping expression patterns for the three CGs in several organs of adult animals. Overall, the results reveal a network of three prototype galectins in chicken. © The Authors

Studying the Structural Significance of Galectin Design by Playing a Modular Puzzle: Homodimer Generation from Human Tandem-Repeat-Type (Heterodimeric) Galectin-8 by Domain Shuffling

Tissue lectins are emerging (patho) physiological effectors with broad significance. The capacity of adhesion/growth-regulatory galectins to form functional complexes with distinct cellular glycoconjugates is based on molecular selection of matching partners. Engineering of variants by changing the topological display of carbohydrate recognition domains (CRDs) provides tools to understand the inherent specificity of the functional pairing. We here illustrate its practical implementation in the case of human tandem-repeat-type galectin-8 (Gal-8). It is termed Gal-8 (NC) due to presence of two different CRDs at the N-and C-terminal positions. Gal-8N exhibits exceptionally high affinity for 3'-sialylated/sulfated beta-galactosides. This protein is turned into a new homodimer, i. e., Gal-8 (NN), by engineering. The product maintained activity for lactose-inhibitable binding of glycans and glycoproteins. Preferential association with 3'-sialylated/sulfated (and 6-sulfated) beta-galactosides was seen by glycan-array analysis when compared to the wild-type protein, which also strongly bound to ABH-type epitopes. Agglutination of erythrocytes documented functional bivalency. This result substantiates the potential for comparative functional studies between the variant and natural Gal-8 (NC)/Gal-8N

Small intestinal mucosa expression of putative chaperone fls485

<p>Abstract</p> <p>Background</p> <p>Maturation of enterocytes along the small intestinal crypt-villus axis is associated with significant changes in gene expression profiles. <it>fls485 </it>coding a putative chaperone protein has been recently suggested as a gene involved in this process. The aim of the present study was to analyze <it>fls48</it>5 expression in human small intestinal mucosa.</p> <p>Methods</p> <p><it>fls485 </it>expression in purified normal or intestinal mucosa affected with celiac disease was investigated with a molecular approach including qRT-PCR, Western blotting, and expression strategies. Molecular data were corroborated with several <it>in situ </it>techniques and usage of newly synthesized mouse monoclonal antibodies.</p> <p>Results</p> <p>fls485 mRNA expression was preferentially found in enterocytes and chromaffine cells of human intestinal mucosa as well as in several cell lines including Rko, Lovo, and CaCo2 cells. Western blot analysis with our new anti-fls485 antibodies revealed at least two fls485 proteins. In a functional CaCo2 model, an increase in fls485 expression was paralleled by cellular maturation stage. Immunohistochemistry demonstrated fls485 as a cytosolic protein with a slightly increasing expression gradient along the crypt-villus axis which was impaired in celiac disease Marsh IIIa-c.</p> <p>Conclusions</p> <p>Expression and synthesis of fls485 are found in surface lining epithelia of normal human intestinal mucosa and deriving epithelial cell lines. An interdependence of enterocyte differentiation along the crypt-villus axis and fls485 chaperone activity might be possible.</p

Reaction of a programmable glycan presentation of glycodendrimersomes and cells with engineered human lectins to show the sugar functionality of the cell surface

10 p.-6 fig.-22 p. inf. supl. Kopitz, Jürgen et al.Chemical and biological tools are harnessed to investigate the impact of spatial factors for functional pairing of human lectins with counterreceptors. The homodimeric adhesion/growth-regulatory galectin-1 and a set of covalently linked homo-oligomers from di- to tetramers serve as proof-of-principle test cases. Glycodendrimersomes provide a versatile and sensitive diagnostic platform to reveal thresholds for ligand density and protein concentration in aggregation assays (trans-activity), irrespective of linker length between lectin domains. Monitoring the affinity of cell binding and ensuing tumor growth inhibition reveal the linker length to be a bidirectional switch for cis-activity. The discovery that two aspects of lectin functionality (trans- versus cis-activity) respond non-uniformly to a structural change underscores the power of combining synthetic and biological tools to advance understanding of the sugar functionality of the cell surface.Financial support from the National Science Foundation (grants DMR-1066116 and DMR-1120901), the P. Roy Vagelos Chair at the University of Pennsylvania (all to V.P.), and the National Science Foundation (grant DMR-1120901 to M.L.K.) are gratefully acknowledgedPeer reviewe

Proteomic analysis of vitreous humor in retinal vein occlusion

Purpose To analyze the protein profile of human vitreous of untreated patients with retinal vein occlusion (RVO). Methods Sixty-eight vitreous humor (VH) samples (44 from patients with treatment naïve RVO, 24 controls with idiopathic floaters) were analyzed in this clinical-experimental study using capillary electrophoresis coupled to mass spectrometer and tandem mass spectrometry. To define potential candidate protein markers of RVO, proteomic analysis was performed on RVO patients (n = 30) and compared with controls (n = 16). To determine validity of potential biomarker candidates in RVO, receiver operating characteristic (ROC) was performed by using proteome data of independent RVO (n = 14) and control samples (n = 8). Results Ninety-four different proteins (736 tryptic peptides) could be identified. Sixteen proteins were found to be significant when comparing RVO and control samples (P = 1.43E-05 to 4.48E-02). Five proteins (Clusterin, Complement C3, Ig lambda-like polypeptide 5 (IGLL5), Opticin and Vitronectin), remained significant after using correction for multiple testing. These five proteins were also detected significant when comparing subgroups of RVO (central RVO, hemi-central RVO, branch RVO) to controls. Using independent samples ROC-Area under the curve was determined proving the validity of the results: Clusterin 0.884, Complement C3 0.955, IGLL5 1.000, Opticin 0.741, Vitronectin 0.786. In addition, validation through ELISA measurements was performed. Conclusion The results of the study reveal that the proteomic composition of VH differed significantly between the patients with RVO and the controls. The proteins identified may serve as potential biomarkers for pathogenesis induced by RVO

Combining Recombinase-Mediated Cassette Exchange Strategy with Quantitative Proteomic and Phosphoproteomic Analyses to Inspect Intracellular Functions of the Tumor Suppressor Galectin-4 in Colorectal Cancer Cells

Galectin-4 (Gal4) has been suggested to function as a tumor suppressor in colorectal cancer (CRC). In order to systematically explore its function in CRC, we established a CRC cell line where Gal4 expression can be regulated via the doxycycline (dox)-inducible expression of a single copy wildtype LGALS4 transgene generated by recombinase-mediated cassette exchange (RMCE). Using this model and applying in-depth proteomic and phosphoproteomic analyses, we systematically screened for intracellular changes induced by Gal4 expression. Overall, 3083 cellular proteins and 2071 phosphosites were identified and quantified, of which 1603 could be matched and normalized to their protein expression levels. A bioinformatic analysis revealed that most of the regulated proteins and phosphosites can be localized in the nucleus and are categorized as nucleic acid-binding proteins. The top candidates whose expression was modulated by Gal4 are PURB, MAPKAPK3, BTF3 and BCAR1, while the prime candidates with altered phosphorylation included ZBTB7A, FOXK1, PURB and CK2beta. In order to validate the (phospho)proteomic data, we confirmed these candidates by a radiometric metabolic-labelling and immunoprecipitation strategy. All candidates exert functions in the transcriptional or translational control, indicating that Gal4 might be involved in these processes by affecting the expression or activity of these proteins

Reconstitution of TGFBR2-Mediated Signaling Causes Upregulation of GDF-15 in HCT116 Colorectal Cancer Cells.

Although inactivating frameshift mutations in the Transforming growth factor beta receptor type 2 (TGFBR2) gene are considered as drivers of microsatellite unstable (MSI) colorectal tumorigenesis, consequential alterations of the downstream target proteome are not resolved completely. Applying a click-it chemistry protein labeling approach combined with mass spectrometry in a MSI colorectal cancer model cell line, we identified 21 de novo synthesized proteins differentially expressed upon reconstituted TGFBR2 expression. One candidate gene, the TGF-ß family member Growth differentiation factor-15 (GDF-15), exhibited TGFBR2-dependent transcriptional upregulation causing increased intracellular and extracellular protein levels. As a new TGFBR2 target gene it may provide a link between the TGF-ß branch and the BMP/GDF branch of SMAD-mediated signaling