Site-specific N-glycosylation of integrin α2 mediates collagen-dependent cell survival.
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ABSTRACT:
SUBMITTER: Huang YL
PROVIDER: S-EPMC8501769 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Although type IV collagen is heavily glycosylated, the influence of this post-translational modification on integrin binding has not been investigated. In the present study, galactosylated and nongalactosylated triple-helical peptides have been constructed containing the α1(IV)382-393 and α1(IV)531-543 sequences, which are binding sites for the α2β1 and α3β1 integrins, respectively. All peptides had triple-helical stabilities of 37 °C or greater. The galactosylation of Hyl(393) in α1(IV)382-393 and Hyl(540) and Hyl(543) in α1(IV)531-543 had a dose-dependent influence on melanoma cell adhesion that was much more pronounced in the case of α3β1 integrin binding. Molecular modeling indicated that galactosylation occurred on the periphery of α2β1 integrin interaction with α1(IV)382-393 but right in the middle of α3β1 integrin interaction with α1(IV)531-543. The possibility of extracellular deglycosylation of type IV collagen was investigated, but no β-galactosidase-like activity capable of collagen modification was found. Thus, glycosylation of collagen can modulate integrin binding, and levels of glycosylation could be altered by reduction in expression of glycosylation enzymes but most likely not by extracellular deglycosylation activity.
| S-EPMC4118119 | biostudies-literature
Project description:The extracellular matrix (ECM) plays critical roles in tumor progression and metastasis. However, the contribution of ECM proteins to early metastatic onset in the peritoneal cavity remains unexplored. Here, we suggest a new route of metastasis through the interaction of integrin alpha 2 (ITGA2) with collagens enriched in the tumor coinciding with poor outcome in patients with ovarian cancer. Using multiple gene-edited cell lines and patient-derived samples, we demonstrate that ITGA2 triggers cancer cell adhesion to collagen, promotes cell migration, anoikis resistance, mesothelial clearance, and peritoneal metastasis in vitro and in vivo. Mechanistically, phosphoproteomics identify an ITGA2-dependent phosphorylation of focal adhesion kinase and mitogen-activated protein kinase pathway leading to enhanced oncogenic properties. Consequently, specific inhibition of ITGA2-mediated cancer cell-collagen interaction or targeting focal adhesion signaling may present an opportunity for therapeutic intervention of metastatic spread in ovarian cancer.
| S-EPMC7541088 | biostudies-literature
Project description:We have characterized site-specific N-glycosylation of the HeLa cell line glycoproteins, using a complex workflow based on high and low energy tandem mass spectrometry of glycopeptides. The objective was to obtain highly reliable data on common glycoforms, so rigorous data evaluation was performed. The analysis revealed the presence of a high amount of bovine serum contaminants originating from the cell culture media - nearly 50% of all glycans were of bovine origin. Unaccounted, the presence of bovine serum components causes major bias in the human cellular glycosylation pattern; as is shown when literature results using released glycan analysis are compared. We have reliably identified 43 (human) glycoproteins, 69 N-glycosylation sites, and 178 glycoforms. HeLa glycoproteins were found to be highly (68.7%) fucosylated. A medium degree of sialylation was observed, on average 46.8% of possible sialylation sites were occupied. High-mannose sugars were expressed in large amounts, as expected in the case of a cancer cell line. Glycosylation in HeLa cells is highly variable. It is markedly different not only on various proteins but also at the different glycosylation sites of the same protein. Our method enabled the detailed characterization of site-specific N-glycosylation of several glycoproteins expressed in HeLa cell line.
| S-EPMC6794373 | biostudies-literature
Project description:Previous reports demonstrate that the α2-integrin (α2) mediates pancreatic ductal adenocarcinoma (PDAC) cell interactions with collagens. We found that while well-differentiated cells use α2 exclusively to adhere and migrate on collagenI, poorly differentiated PDAC cells demonstrate reduced reliance on, or complete loss of, α2. Since well-differentiated PDAC lines exhibit reduced in vitro invasion and α2-blockade suppressed invasion of well-differentiated lines exclusively, we hypothesized that α2 may suppress the malignant phenotype in PDAC. Accordingly, ectopic expression of α2 retarded in vitro invasion and maintenance on collagenI exacerbated this effect. Affymetrix profiling revealed that kallikrein-related peptidase-5 (KLK5) was specifically upregulated by α2, and reduced α2 and KLK5 expression was observed in poorly differentiated PDAC cells in situ. Accordingly, well-differentiated PDAC lines express KLK5, and KLK5 blockade increased the invasion of KLK5-positive lines. The α2-cytoplasmic domain was dispensable for these effects, demonstrating that the α2-ectodomain and KLK5 coordinately regulate a less invasive phenotype in PDAC.
| S-EPMC3245846 | biostudies-literature
Project description:BackgroundThe mechanism of small-molecule stabilised protein-protein interactions is of growing interest in the pharmacological discovery process. A plethora of different substances including the aromatic sulphonamide E7820 have been identified to act by such a mechanism. The process of E7820 induced CAPERα degradation and the resultant transcriptional down regulation of integrin α2 expression has previously been described for a variety of different cell lines and been made responsible for E7820's antiangiogenic activity. Currently the application of E7820 in the treatment of various malignancies including pancreas carcinoma and breast cancer is being investigated in pre-clinical and clinical trials. It has been shown, that integrin α2 deficiency has beneficial effects on bone homeostasis in mice. To transfer E7820 treatment to bone-related pathologies, as non-healing fractures, osteoporosis and bone cancer might therefore be beneficial. However, at present no data is available on the effect of E7820 on osseous cells or skeletal malignancies.MethodsPre-osteoblastic (MC3T3 and Saos-2) cells and endothelial (eEnd2 cells and HUVECs) cells, each of human and murine origin respectively, were investigated. Vitality assay with different concentrations of E7820 were performed. All consecutive experiments were done at a final concentration of 50 ng/ml E7820. The expression and production of integrin α2 and CAPERα were investigated by quantitative real-time PCR and western blotting. Expression of CAPERα splice forms was differentiated by semi-quantitiative reverse transcriptase PCR.ResultsHere we present the first data showing that E7820 can increase integrin α2 expression in the pre-osteoblast MC3T3 cell line whilst also reproducing canonical E7820 activity in HUVECs. We show that the aberrant activity of E7820 in MC3T3 cells is likely due to differential activity of CAPERα at the integrin α2 promoter, rather than due to differential CAPERα degradation or differential expression of CAPERα spliceforms.ConclusionThe results presented here indicate that E7820 may not be suitable to treat certain malignancies of musculoskeletal origin, due to the increase in integrin α2 expression it may induce. Further investigation of the differential functioning of CAPERα and the integrin α2 promoter in cells of various origin would however be necessary to more clearly differentiate between cell lines that will positively respond to E7820 from those that will not.
| S-EPMC8132423 | biostudies-literature
Project description:Integrins are cell adhesion molecules that are composed of an alpha (α) subunit and a beta (β) subunit with affinity for different extracellular membrane components. The integrin family includes 24 known members that actively regulate cellular growth, differentiation, and apoptosis. Each integrin heterodimer has a particular function in defined contexts as well as some partially overlapping features with other members in the family. As many reviews have covered the general integrin family in molecular and cellular studies in life science, this review will focus on the specific regulation, function, and signaling of integrin α2 subunit (CD49b, VLA-2; encoded by the gene ITGA2) in partnership with β1 (CD29) subunit in normal and cancer cells. Its roles in cell adhesion, cell motility, angiogenesis, stemness, and immune/blood cell regulations are discussed. The pivotal role of integrin α2 in many diseases such as cancer suggests its potential to be used as a novel therapeutic target.
| S-EPMC6411621 | biostudies-literature
Project description:Vascular endothelial growth factor receptor-2 (VEGFR-2) is an important receptor tyrosine kinase (RTK) that plays critical roles in both physiologic and pathologic angiogenesis. The extracellular domain of VEGFR-2 is composed of seven immunoglobulin-like domains, each with multiple potential N-glycosylation sites (sequons). N-glycosylation plays a central role in RTK ligand binding, trafficking, and stability. However, despite its importance, the functional role of N-glycosylation of VEGFR-2 remains poorly understood. The objectives of the present study were to characterize N-glycosylation sites in VEGFR-2 via enzymatic release of the glycans and concomitant incorporation of 18O into formerly N-glycosylated sites followed by tandem mass spectrometry (MS/MS) analysis to determine N-glycosylation site occupancy and the site-specific N-glycan heterogeneity of VEGFR-2 glycopeptides. The data demonstrated that all seven VEGFR-2 immunoglobulin-like domains have at least one occupied N-glycosylation site. MS/MS analyses of glycopeptides and deamidated, deglycosylated (PNGase F-treated) peptides from ectopically expressed VEGFR-2 in porcine aortic endothelial (PAE) cells identified N-glycans at the majority of the 17 potential N-glycosylation sites on VEGFR-2 in a site-specific manner. The data presented here provide direct evidence for site-specific, heterogeneous N-glycosylation and N-glycosylation site occupancy on VEGFR-2. The study has important implications for the therapeutic targeting of VEGFR-2, ligand binding, trafficking, and signaling.
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Project description:Fibronectin (FN), a major extracellular matrix component, enables integrin-mediated cell adhesion via binding of α5β1, αIIbβ3 and αv-class integrins to an RGD-motif. An additional linkage for α5 and αIIb is the synergy site located in close proximity to the RGD motif. We report that mice with a dysfunctional FN-synergy motif (Fn1syn/syn) suffer from surprisingly mild platelet adhesion and bleeding defects due to delayed thrombus formation after vessel injury. Additional loss of β3 integrins dramatically aggravates the bleedings and severely compromises smooth muscle cell coverage of the vasculature leading to embryonic lethality. Cell-based studies revealed that the synergy site is dispensable for the initial contact of α5β1 with the RGD, but essential to re-enforce the binding of α5β1/αIIbβ3 to FN. Our findings demonstrate a critical role for the FN synergy site when external forces exceed a certain threshold or when αvβ3 integrin levels decrease below a critical level.
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Project description:Yes-associated protein (YAP) is a transcriptional co-activator and a major effector of the Hippo pathway that promotes cell proliferation and stemness, while inhibiting apoptosis. YAP plays a central role in organ size control, and its deregulation strongly promotes cancer initiation and progression. However, the mechanisms by which YAP promotes cell invasion and metastasis are not fully understood. Here, we report that YAP induces leukocyte-specific integrin β2 (ITGB2) expression in cancer cells, thereby promoting cell invasion through the endothelium in a manner mimicking leukocytes. Through independent biochemical purification and a functional screen, we further identified PR/SET domain 4 (PRDM4) as a transcription factor interacting with the WW domains of YAP to mediate ITGB2 expression and cell invasion. Consistently, ITGB2 and PRDM4 mRNA levels are significantly increased in metastatic prostate cancer. In addition, PRDM4 contributes to YAP-induced tumorigenesis possibly via mediating the expression of other YAP target genes. Our results demonstrate that YAP promotes cell invasion by inducing leukocyte-specific integrin expression, and identify PRDM4 as a novel transcription factor for YAP targets.
| S-EPMC5989780 | biostudies-literature
Project description:Here, we present for the first time, a site-specific N-glycosylation analysis of proteins from a Brazilian Zika virus (ZIKV) strain. The virus was propagated with high yield in an embryo-derived stem cell line (EB66, Valneva SE), and concentrated by g-force step-gradient centrifugation. Subsequently, the sample was proteolytically digested with different enzymes, measured via a LC-MS/MS-based workflow, and analyzed in a semi-automated way using the in-house developed glyXtoolMS software. The viral non-structural protein 1 (NS1) was glycosylated exclusively with high-mannose structures on both potential N-glycosylation sites. In case of the viral envelope (E) protein, no specific N-glycans could be identified with this method. Nevertheless, N-glycosylation could be proved by enzymatic de-N-glycosylation with PNGase F, resulting in a strong MS-signal of the former glycopeptide with deamidated asparagine at the potential N-glycosylation site N444. This confirmed that this site of the ZIKV E protein is highly N-glycosylated but with very high micro-heterogeneity. Our study clearly demonstrates the progress made towards site-specific N-glycosylation analysis of viral proteins, i.e. for Brazilian ZIKV. It allows to better characterize viral isolates, and to monitor glycosylation of major antigens. The method established can be applied for detailed studies regarding the impact of protein glycosylation on antigenicity and human pathogenicity of many viruses including influenza virus, HIV and corona virus.
| S-EPMC7933209 | biostudies-literature