Kaikki aineistot
Lisää
Abstract Carbohydrate chains are the most abundant and diverse of nature’s biopolymers and represent one of the four fundamental macromolecular building blocks of life together with proteins, nucleic acids, and lipids. Indicative of their essential roles in cells and in multicellular organisms, genes encoding proteins associated with glycosylation account for approximately 2% of the human genome. It has been estimated that 50–80% of all human proteins carry carbohydrate chains—glycans—as part of their structure. Despite cells utilize only nine different monosaccharides for making their glycans, their order and conformational variation in glycan chains together with chain branching differences and frequent post-synthetic modifications can give rise to an enormous repertoire of different glycan structures of which few thousand is estimated to carry important structural or functional information for a cell. Thus, glycans are immensely versatile encoders of multicellular life. Yet, glycans do not represent a random collection of unpredictable structures but rather, a collection of predetermined but still dynamic entities that are present at defined quantities in each glycosylation site of a given protein in a cell, tissue, or organism. In this chapter, we will give an overview of what is currently known about N-glycan synthesis in higher eukaryotes, focusing not only on the processes themselves but also on factors that will affect or can affect the final outcome—the dynamicity and heterogeneity of the N-glycome. We hope that this review will help understand the molecular details underneath this diversity, and in addition, be helpful for those who plan to produce optimally glycosylated antibody-based therapeutics.
Abstract Several distinct metastasis-associated glycosylation changes have been shown to promote cancer cell invasion and metastasis, the main cause of death of cancer patients. However, it is unclear whether their presence reflects cell- or tissue-specific variations for metastasis, or species needed to drive different phases of the metastatic cascade. To address this issue from a different perspective, we investigated here whether different cancer cell lines share any glycotopes that are common and important for their invasive phenotype. By using lectin microarray glycan profiling and an established myoma tissue-based 3D invasion assay, we identified a single glycotope recognized by Helix Pomatia agglutinin (HPA), whose expression level in different cancer cells correlated significantly with their invasive potential. Lectin pull-down assay and LC-MS/MS analysis in highly- (A431 and SW-48) and poorly invasive (HepG2 and RCC4) cancer cells revealed ~85 glycoproteins of which several metastasis-promoting members of the integrin family of cell adhesion receptors, the epidermal growth factor receptor (EGFR) and the matrix metalloproteinase-14 (MMP-14) were among the abundant ones. Moreover, we showed that the level of the GalNAc glycotope in MMP-14, EGFR, αV-, β1- and β4 integrin in highly and poorly invasive cancer cells correlated positively with their invasive potential. Collectively, our findings suggest that altered glycosylation of several metastasis-associated glycoproteins with terminal GalNAc drives the highly invasive cancer cell phenotype.
Abstract Aims: Carcinoembryonic antigen (CEACAM5, CEA) is a known tumor marker for colorectal cancer that localizes in a polarized manner to the apical surface in normal colon epithelial cells whereas in cancer cells it is present at both the apical and basolateral surfaces of the cells. Since the Golgi apparatus sorts and transports most proteins to these cell surface domains, we set out here to investigate whether any of the factors commonly associated with tumorigenesis, including hypoxia, generation of reactive oxygen species (ROS), altered redox homeostasis, or an altered Golgi pH, are responsible for mistargeting of CEA to the basolateral surface in cancer cells. Results: Using polarized nontumorigenic Madin-Darby canine kidney (MDCK) cells and CaCo-2 colorectal cancer cells as targets, we show that apical delivery of CEA is not affected by hypoxia, ROS, nor changes in the Golgi redox state. Instead, we find that an elevated Golgi pH induces basolateral targeting of CEA and increases its TX-100 solubility, indicating impaired association of CEA with lipid rafts. Moreover, disruption of lipid rafts by methyl-β-cyclodextrin induced accumulation of the CEA protein at the basolateral surface in MDCK cells. Experiments with the glycosylphosphatidylinositol (GPI)-anchorless CEA mutant and CEA-specific GPI-anchored enhanced green fluorescent protein (EGFP-GPI) fusion protein revealed that the GPI-anchor was critical for the pH-dependent apical delivery of the CEA in MDCK cells. Innovation and Conclusions: The findings indicate that an abnormal Golgi pH homeostasis in cancer cells is an important factor that causes mistargeting of CEA to the basolateral surface of cancer cells via inhibiting its GPI-anchor-mediated association with lipid rafts.
Abstract Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-environment that is known to be dictated by the rates of V-ATPase-mediated H+ pumping and its leakage back to the cytoplasm via an elusive “H+ leak” pathway. Here, we show that this proton leak across Golgi membranes is mediated by the AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange, as it is strictly dependent on bicarbonate import (in exchange for chloride export) and the expression level of the Golgi-localized AE2a anion exchanger. In the acidic Golgi lumen, imported bicarbonate anions and protons then facilitate a common buffering reaction that yields carbon dioxide and water before their egress back to the cytoplasm via diffusion or water channels. The flattened morphology of the Golgi cisternae helps this process, as their high surface-volume ratio is optimal for water and gas exchange. Interestingly, this net acid efflux pathway is often upregulated in cancers and established cancer cell lines, and responsible for their markedly elevated Golgi resting pH and attenuated glycosylation potential. Accordingly, AE2 knockdown in SW-48 colorectal cancer cells was able to restore these two phenomena, and at the same time, reverse their invasive and anchorage-independent growth phenotype. These findings suggest a possibility to return malignant cells to a benign state by restoring Golgi resting pH.
Abstract NDST1 (glucosaminyl N-deacetylase/N-sulfotransferase) is a key enzyme in heparan sulfate (HS) biosynthesis, where it is responsible for HS N-deacetylation and N-sulfation. In addition to the full length human enzyme of 882 amino acids, here designated NDST1A, a shorter form containing 825 amino acids (NDST1B) is synthesized after alternative splicing of the NDST1 mRNA. NDST1B is mostly expressed at a low level, but increased amounts are seen in several types of cancer where it is associated with shorter survival. In this study, we aimed at characterizing the enzymatic properties of NDST1B and its effect on HS biosynthesis. Purified recombinant NDST1B lacked both N-deacetylase and N-sulfotransferase activities. Interestingly, HEK293 cells overexpressing NDST1B synthesized HS with reduced sulfation and altered domain structure. Fluorescence resonance energy transfer-microscopy demonstrated that both NDST1A and NDST1B had the capacity to interact with the HS copolymerase subunits EXT1 and EXT2 and also to form NDST1A/NDST1B dimers. Since lysates from cells overexpressing NDST1B contained less NDST enzyme activity than control cells, we suggest that NDST1B works in a dominant negative manner, tentatively by replacing the active endogenous NDST1 in the enzyme complexes taking part in biosynthesis.