Altered glycosylation of several metastasis-associated glycoproteins with terminal GalNAc defines the highly invasive cancer cell phenotype

Publisher Copyright: © 2022 Khosrowabadi et al.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.Peer reviewe

The Pro-Oncogenic Adaptor CIN85 Acts as an Inhibitory Binding Partner of Hypoxia-Inducible Factor Prolyl Hydroxylase 2

The EGFR adaptor protein, CIN85, has been shown to promote breast cancer malignancy and hypoxia-inducible factor (HIF) stability. However, the mechanisms underlying cancer promotion remain ill defined. Here we show that CIN85 is a novel binding partner of the main HIF-prolyl hydroxylase, PHD2, but not of PHD1 or PHD3. Mechanistically, the N-terminal SRC homology 3 domains of CIN85 interacted with the proline-arginine-rich region within the N-terminus of PHD2, thereby inhibiting PHD2 activity and HIF degradation. This activity is essential in vivo, as specific loss of the CIN85-PHD2 interaction in CRISPR/Cas9-edited cells affected growth and migration properties, as well as tumor growth in mice. Overall, we discovered a previously unrecognized tumor growth checkpoint that is regulated by CIN85-PHD2 and uncovered an essential survival function in tumor cells by linking growth factor adaptors with hypoxia signaling. Significance: This study provides unprecedented evidence for an oxygen-independent mechanism of PHD2 regulation that has important implications in cancer cell survival.Peer reviewe

Golgi pH, ion and redox homeostasis:how much do they really matter?

Abstract Exocytic and endocytic compartments each have their own unique luminal ion and pH environment that is important for their normal functioning. A failure to maintain this environment — the loss of homeostasis — is not uncommon. In the worst case, all the main Golgi functions, including glycosylation, membrane trafficking and protein sorting, can be perturbed. Several factors contribute to Golgi homeostasis. These include not only ions such as H⁺, Ca²⁺, Mg²⁺, Mn²⁺, but also Golgi redox state and nitric oxide (NO) levels, both of which are dependent on the oxygen levels in the cells. Changes to any one of these factors have consequences on Golgi functions, the nature of which can be dissimilar or similar depending upon the defects themselves. For example, altered Golgi pH homeostasis gives rise to Cutis laxa disease, in which glycosylation and membrane trafficking are both affected, while altered Ca²⁺ homeostasis due to the mutated SCPA1 gene in Hailey–Hailey disease, perturbs various protein sorting, proteolytic cleavage and membrane trafficking events in the Golgi. This review gives an overview of the molecular machineries involved in the maintenance of Golgi ion, pH and redox homeostasis, followed by a discussion of the organelle dysfunction and disease that frequently result from their breakdown. Congenital disorders of glycosylation (CDGs) are discussed only when they contribute directly to Golgi pH, ion or redox homeostasis. Current evidence emphasizes that, rather than being mere supporting factors, Golgi pH, ion and redox homeostasis are in fact key players that orchestrate and maintain all Golgi functions

Hypoxia and reactive oxygen species as modulators of endoplasmic reticulum and Golgi homeostasis

Abstract Significance: Eukaryotic cells execute various functions in subcellular compartments or organelles for which cellular redox homeostasis is of importance. Apart from mitochondria, hypoxia and stress-mediated formation of reactive oxygen species (ROS) were shown to modulate endoplasmic reticulum (ER) and Golgi apparatus (GA) functions. Recent Advances: Research during the last decade has improved our understanding of disulfide bond formation, protein glycosylation and secretion, as well as pH and redox homeostasis in the ER and GA. Thus, oxygen (O₂) itself, NADPH oxidase (NOX) formed ROS, and pH changes appear to be of importance and indicate the intricate balance of intercompartmental communication. Critical Issues: Although the interplay between hypoxia, ER stress, and Golgi function is evident, the existence of more than 20 protein disulfide isomerase family members and the relative mild phenotypes of, for example, endoplasmic reticulum oxidoreductin 1 (ERO1)- and NOX4-knockout mice clearly suggest the existence of redundant and alternative pathways, which remain largely elusive. Future Directions: The identification of these pathways and the key players involved in intercompartmental communication needs suitable animal models, genome-wide association, as well as proteomic studies in humans. The results of those studies will be beneficial for the understanding of the etiology of diseases such as type 2 diabetes, Alzheimer's disease, and cancer, which are associated with ROS, protein aggregation, and glycosylation defects

N-glycan biosynthesis:basic principles and factors affecting its outcome

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

Crystal structures of eukaryote glycosyltransferases reveal biologically relevant enzyme homooligomers

Abstract Glycosyltransferases (GTases) transfer sugar moieties to proteins, lipids or existing glycan or polysaccharide molecules. GTases form an important group of enzymes in the Golgi, where the synthesis and modification of glycoproteins and glycolipids take place. Golgi GTases are almost invariably type II integral membrane proteins, with the C-terminal globular catalytic domain residing in the Golgi lumen. The enzymes themselves are divided into 103 families based on their sequence homology. There is an abundance of published crystal structures of GTase catalytic domains deposited in the Protein Data Bank (PDB). All of these represent either of the two main characteristic structural folds, GT-A or GT-B, or present a variation thereof. Since GTases can function as homomeric or heteromeric complexes in vivo, we have summarized the structural features of the dimerization interfaces in crystal structures of GTases, as well as considered the biochemical data available for these enzymes. For this review, we have considered all 898 GTase crystal structures in the Protein Data Bank and highlight the dimer formation characteristics of various GTases based on 24 selected structures

Altered glycosylation of several metastasis-associated glycoproteins with terminal GalNAc defines the highly invasive cancer cell phenotype

Publisher Copyright: © 2022 Khosrowabadi et al.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.Peer reviewe