Core fucosylation is one of the most important glycosylation events in the progression of liver cancer. For this study, we used an easily handled L-fucose analog, 2-fluoro-L-fucose (2FF), which interferes with the normal synthesis of GDP-fucose, and verified its potential roles in regulating core fucosylation and cell behavior in the HepG2 liver cancer cell line. Results obtained from lectin blot and flow cytometry analysis clearly showed that 2FF treatment dramatically inhibited core fucosylation, which was also confirmed via mass spectrometry analysis. Cell proliferation and integrin-mediated cell migration were significantly suppressed in cells treated with 2FF. We further analyzed cell colony formation in soft agar and tumor xenograft efficacy, and found that both were greatly suppressed in the 2FF-treated cells, compared with the control cells. Moreover, the treatment with 2FF decreased the core fucosylation levels of membrane glycoproteins such as EGF receptor and integrin β1, which in turn suppressed downstream signals that included phospho-EGFR, -AKT, -ERK, and -FAK. These results clearly described the roles of 2FF and the importance of core fucosylation in liver cancer progression, suggesting 2FF shows promise for use in the treatment of hepatoma.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug that leads to severe hepatotoxicity at excessive doses. Fucoidan, a sulfated polysaccharide derived from brown seaweeds, possesses a wide range of pharmacological properties. However, the impacts of fucoidan on APAP-induced liver injury have not been sufficiently addressed. In the present study, male Institute of Cancer Research (ICR) mice aged 6 weeks were subjected to a single APAP (500 mg/kg) intraperitoneal injection after 7 days of fucoidan (100 or 200 mg/kg/day) or bicyclol intragastric administration. The mice continued to be administered fucoidan or bicyclol once per day, and were sacrificed at an indicated time. The indexes evaluated included liver pathological changes, levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum, levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT) in the liver, and related proteins levels (CYP2E1, pJNK and Bax). Furthermore, human hepatocyte HL-7702 cell line was used to elucidate the potential molecular mechanism of fucoidan. The mitochondrial membrane potential (MMP) and nuclear factor-erythroid 2-related factor (Nrf2) translocation in HL-7702 cells were determined. The results showed that fucoidan pretreatment reduced the levels of ALT, AST, ROS, and MDA, while it enhanced the levels of GSH, SOD, and CAT activities. Additionally, oxidative stress-induced phosphorylated c-Jun N-terminal protein kinase (JNK) and decreased MMP were attenuated by fucoidan. Although the nuclear Nrf2 was induced after APAP incubation, fucoidan further enhanced Nrf2 in cell nuclei and total expression of Nrf2. These results indicated that fucoidan ameliorated APAP hepatotoxicity, and the mechanism might be related to Nrf2-mediated oxidative stress.

The α2,3‐sialylation of N‐glycans is considered important but complicated because the functions of the three β‐galactoside α2,3‐sialyltransferases, ST3GAL3, ST3GAL4, and ST3GAL6, could be compensating for one another. To distinguish their specific functions, we established each individual knockout (KO) cell line. Loss of either the ST3GAL3 or ST3GAL6 genes decreased cell proliferation and colony formation, as opposed to the effect in the ST3GAL4 KO cells. The phosphorylation levels of ERK and AKT were significantly suppressed in the ST3GAL6 KO and ST3GAL3 KO cells, respectively. The cell aggregations were clearly observed in the KO cells, particularly the ST3GAL3 KO and ST3GAL6 KO cells, and the expression levels of E‐cadherin and claudin‐1 were enhanced in both those cell lines, but were suppressed in the ST3GAL4 KO cells. Those alterations were reversed with an overexpression of each corresponding gene in rescued cells. Of particular interest, the α2,3‐sialylation levels of β1 integrin were clearly suppressed in the ST3GAL4 KO cells, but these were increased in the ST3GAL3 KO and ST3GAL6 KO cells, whereas the α2,3‐sialylation levels of EGFR were significantly decreased in the ST3GAL6 KO cells. The decrease in α2,3‐sialylation increased the α2,6‐sialylation on β1, but not EGFR. Furthermore, a cross‐restoration of each of the three genes in ST3GAL6 KO cells showed that overexpression of ST3GAL6 sufficiently rescued the total α2,3‐sialylation levels, cell morphology, and α2,3‐sialylation of EGFR, whereas the α2,3‐sialylation levels of β1 were greatly enhanced by an overexpression of ST3GAL4. These results clearly demonstrate that the three α2,3‐sialyltransferases modify characteristic target proteins and regulate cell biological functions in different ways.

N ‐Glycosylation of integrin α5β1 plays important roles in cell biologic functions; however, the mechanisms that underlie those roles remain poorly understood. Here, we present evidence that themembrane‐proximal N ‐glycosylation on integrin β1 could positively regulate cell migration by promoting β1 activation. The S4–6 β1 mutant contains only 3 N ‐glycosylation sites, which are essential for α5 and β1 heterodimer formation, and despite only a small difference in expression levels of α5β1 between wild‐type and S4–6 mutant, cell spreading and migration of the S4–6 mutant was significantly decreased compared with that of control. Consistent with these phenotypes, β1 ‐mediated cellular signaling and its activation were clearly suppressed in the S4–6 mutant. Of note, these developments could be rescued by restoration of N ‐glycosylation sites in the membrane‐proximal domain. Further study on the regulatory mechanisms suggested that membrane‐proximal N ‐glycosylation is critical for intermolecular interactions between integrin β1 and other cell membrane proteins, such as syndecan‐4 and epidermal growth factor receptor. Moreover, α2, 6‐sialylation is required for β1 activation. These data suggest a novel regulatory mechanism where in N ‐glycosy lationnear the cell membrane on β1 may serve as a platform that facilitates its complex formation on the cell membrane, thereby affecting integrin‐mediated functions.—Hou, S., Hang, Q., Isaji, T., Lu, J., Fukuda, T., Gu, J. Importance ofmembrane‐proximal N ‐glycosylation on integrin β1 in its activation and complex formation. FASEB J. 30, 4120–4131 (2016). www.fasebj.org

The N-glycosylation of integrin α5β1 is thought to control many fundamental aspects of cell behavior, including cell adhesion and migration. However, the mechanism of how N-glycans function remains largely obscure. Here, we used a loss-of-function approach. Wild-type (WT) integrin α5 and N-glycosylation mutant S3-5 (sites 3 to 5) integrin α5, which contains fewer N-glycans, were stably reconstituted in α5 knockout cancer cells. We found that the migration ability of S3-5 cells was decreased in comparison with that of the WT. Interestingly, the levels of phosphorylated focal adhesion kinase and actin stress fiber formation were greatly enhanced in the S3-5 mutant. In a mechanistic manner, the internalization of active but not total integrin α5β1 was inhibited in S3-5 cells, which is a process that is related to the enhanced expression of active integrin α5β1 on the cell surface. Importantly, restoration of N-glycosylation on the β-propeller domain of α5 reinstated the cell migration ability, active α5β1 expression, and internalization. Moreover, these N-glycans are critical for α5–syndecan-4 complex formation. These findings indicate that N-glycosylation on the β-propeller domain functions as a molecular switch to control the dynamics of α5β1 on the cell surface that in turn is required for optimum adhesion for cell migration.

N-Glycosylation of integrin α5β1 is involved in multiple cell behaviors. We previously reported that the N-glycosylations of the calf domain on integrin α5 (S3–5,10–14) are essential for its inhibitory effect on EGFR signaling in regulating cell proliferation. However, the importance of the individual N-glycosylation and the underlying mechanisms of inhibition remain unclear. Here, we characterize the S3–5,10–14 mutants in detail and found that the N-glycosylation of site-11 (Asn712) is key for cell growth. The restoration of site-11, unlike the other individual sites, significantly suppressed cell growth and EGFR signaling in a manner that was similar to that of wild-type (WT). Mechanistically, this N-glycosylation inhibited the response abilities upon EGF stimulation and EGFR dimerization. Interestingly, we found this N-glycosylation controlled the EGFR complex formation with integrin α5β1 or α6β4; i.e., the loss of site-11 switched EGFR-α5β1 to EGFR-α6β4, which is well known to promote cellular signaling for cell growth. Moreover, the site-11 N-glycan exhibited a more branching structure compared with other sites, which may be required for EGFR-α5β1 formation. Taken together, these data clearly demonstrate that the site-11 N-glycosylation on α5 is most important for its inhibitory effect on EGFR signaling, which may provide a novel regulatory mechanism for crosstalks between integrins and EGFR.