Detection of malignancy-associated phosphoproteome changes in human colorectal cancer induced by cell surface binding of growth-inhibitory galectin-4.

Emerging evidence on efficient tumor growth regulation by endogenous lectins directs interest to determine on a proof-of-principle level the range of information on alterations provided by full-scale analysis using phosphoproteomics. In our pilot study, we tested galectin-4 (gal-4) that is a growth inhibitor for colon cancer cells (CRC), here working with the LS 180 line. In order to cover monitoring of short- and long-term effects stable isotope labeling by amino acids in cell culture-based quantitative phosphoproteomic analyses were conducted on LS 180 cell preparations collected 1 and 72 h after adding gal-4 to the culture medium. After short-term treatment, 981 phosphosites, all of them S/T based, were detected by phosphoproteomics. Changes higher than 1.5-fold were seen for eight sites in seven proteins. Most affected were the BET1 homolog (BET1), whose level of phosphorylation at S50 was about threefold reduced, and centromere protein F (CENPF), extent of phosphorylation at S3119 doubling in gal-4-treated cells. Phosphoproteome analysis after 72 h of treatment revealed marked changes at 33 S/T-based phosphosites from 29 proteins. Prominent increase of phosphorylation was observed for cofilin-1 at position S3. Extent of phosphorylation of the glutamine transporter SLC1A5 at position S503 was decreased by a factor of 3. Altered phosphorylation of BET1, CENPF, and cofilin-1 as well as a significant effect of gal-4 treatment on glutamine uptake by cells were substantiated by independent methods in the Vaco 432, Colo 205, CX 1, and HCT 116 cell lines. With the example of gal-4 which functions as a tumor suppressor in CRC cells, we were able to prove that cell surface binding of the lectin not only markedly influences the cell proteome, but also has a bearing on malignancy-associated intracellular protein phosphorylation. These results underscore the potential of this approach to give further work on elucidating the details of signaling underlying galectin-triggered growth inhibition a clear direction. © 2018 IUBMB Life, 71(3):364-375, 2019.

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.

Evaluation of the bactericidal activity of galectins.

Over a century ago, Karl Landsteiner discovered that blood group antigens could predict the immunological outcome of red blood cell transfusion. While the discovery of ABO(H) blood group antigens revolutionized transfusion medicine, many questions remain regarding the development and regulation of naturally occurring anti-blood group antibody formation. Early studies suggested that blood group antibodies develop following stimulation by bacteria that express blood group antigens. While this may explain the development of anti-blood group antibodies in blood group negative individuals, how blood group positive individuals, who cannot generate anti-blood group antibodies, protect themselves against blood group positive microbes remained unknown. Recent studies suggest that several members of the galectin family specifically target blood group positive microbes, thereby providing innate immune protection against blood group antigen positive microbes regardless of the blood group status of an individual. Importantly, subsequent studies suggest that this unique form of immunity may not be limited to blood group expressing microbes, but may reflect a more generalized form of innate immunity against molecular mimicry. As this form of antimicrobial activity represents a unique and unprecedented form of immunity, we will examine important considerations and methodological approaches that can be used when seeking to ascertain the potential antimicrobial activity of various members of the galectin family.

Galectin-4, a novel neuronal regulator of myelination.

Myelination of axons by oligodendrocytes (OLGs) is essential for proper saltatory nerve conduction, i.e., rapid transmission of nerve impulses. Among others, extracellular matrix (ECM) molecules, neuronal signaling, and axonal adhesion regulate the biogenesis and maintenance of myelin membranes, driven by polarized transport of myelin-specific proteins and lipids. Galectin-4, a tandem-repeat-type lectin with affinity to sulfatide and nonsialylated termini of N-glycans, has the ability to regulate adhesion of cells to ECM components and is also involved in polarized membrane trafficking. We, therefore, anticipated that galectin-4 might play a role in myelination. Here, we show that in developing postnatal rat brains galectin-4 expression is downregulated just before the onset of myelination. Intriguingly, when immature OLGs were treated with galectin-4, OLG maturation was retarded, while a subset of the immature OLGs reverted to a morphologically less complex progenitor stage, displaying concomitantly an increase in proliferation. Similarly, myelination was inhibited when galectin-4 or anti-galectin-4 antibodies were added to co-cultures of dorsal root ganglion neurons and OLGs. Neurons and OLGs were identified as a possible source of galectin-4, both in vitro and in vivo. In culture, neurons but not OLGs released galectin-4. Interestingly, in co-cultures, a reduced release of endogenous galectin-4 correlated with the onset of myelination. Moreover, galectin-4-reactive sites are transiently expressed on processes of premyelinating primary OLGs, but not on neurons. Taken together, these results identify neuronal galectin-4 as a candidate for a soluble regulator of OLG differentiation and, hence, myelination. © 2012 Wiley Periodicals, Inc.

Human galectin-1, -2, and -4 induce surface exposure of phosphatidylserine in activated human neutrophils but not in activated T cells.

Cellular turnover is associated with exposure of surface phosphatidylserine (PS) in apoptotic cells, leading to their phagocytic recognition and removal. But recent studies indicate that surface PS exposure is not always associated with apoptosis. Here we show that several members of the human galectin family of glycan binding proteins (galectins-1, -2, and -4) induce PS exposure in a carbohydrate-dependent fashion in activated, but not resting, human neutrophils and in several leukocyte cell lines. PS exposure is not associated with apoptosis in activated neutrophils. The exposure of PS in cell lines treated with these galectins is sustained and does not affect cell viability. Unexpectedly, these galectins bind well to activated T lymphocytes, but do not induce either PS exposure or apoptosis, indicating that galectin's effects are cell specific. These results suggest novel immunoregulatory contribution of galectins in regulating leukocyte turnover independently of apoptosis.