Xiangchun Wang

Core fucosylation regulates epidermal growth factor receptor-mediated intracellular signaling

α1,6-Fucosyltransferase (Fut8) catalyzes the transfer of a fucose residue to N-linked oligosaccharides on glycoproteins via an α1,6-linkage to form core fucosylation in mammals. We recently found that disruption of the Fut8 gene induces severe growth retardation and early postnatal death. To investigate the molecular mechanism involved, we have established embryonic fibroblasts of Fut8+/+ and Fut8–/–, derived from wild-type and Fut8-null mice, respectively. Interestingly, the epidermal growth factor (EGF)-induced phosphorylation levels of the EGF receptor (EGFR) were substantially blocked in Fut8–/– cells, compared with Fut8+/+ cells, while there are no significant changes in the total activities of tyrosine phosphatase for phosphorylated EGFR between two cells. The inhibition of EGFR phosphorylation was completely restored by re-introduction of the Fut8 gene to Fut8–/– cells. Consistent with this, EGFR-mediated JNK or ERK activation was significantly suppressed in Fut8–/– cells. Finally, we found that the core fucosylation of N-glycans is required for the binding of the EGF to its receptor, whereas no effect was observed for the expression levels of EGFR on the cell surface. Collectively, these results strongly suggest that core fucosylation is essential for EGF receptor-mediated biological functions.

Carbohydrate Binding Specificity of a Fucose-specific Lectin from Aspergillus oryzae A NOVEL PROBE FOR CORE FUCOSE

The α1,6-fucosyl residue (core fucose) of glycoproteins is widely distributed in mammalian tissues and is altered under pathological conditions. A probe that specifically detects core fucose is important for understanding the role of this oligosaccharide structure. Aleuria aurantia lectin (AAL) and Lens culimaris agglutinin-A (LCA) have been often used as carbohydrate probes for core fucose in glycoproteins. Here we show, by using surface plasmon resonance (SPR) analysis, that Aspergillus oryzae l-fucose-specific lectin (AOL) has strongest preference for the α1,6-fucosylated chain among α1,2-, α1,3-, α1,4-, and α1,6-fucosylated pyridylaminated (PA)-sugar chains. These results suggest that AOL is a novel probe for detecting core fucose in glycoproteins on the surface of animal cells. A comparison of the carbohydrate-binding specificity of AOL, AAL, and LCA by SPR showed that the irreversible binding of AOL to the α1,2-fucosylated PA-sugar chain (H antigen) relative to the α1,6-fucosylated chain was weaker than that of AAL, and that the interactions of AOL and AAL with α1,6-fucosylated glycopeptide (FGP), which is considered more similar to in vivo glycoproteins than PA-sugar chains, were similar to their interactions with the α1,6-fucosylated PA-sugar chain. Furthermore, positive staining of AOL, but not AAL, was completely abolished in the cultured embryo fibroblast (MEF) cells obtained from α1,6-fucosyltransferase (Fut8) knock-out mice, as assessed by cytological staining. Taken together, these results suggest that AOL is more suitable for detecting core fucose than AAL or LCA.

Deletion of Core Fucosylation on α3β1 Integrin Down-regulates Its Functions

The core fucosylation (α1,6-fucosylation) of glycoprotein is widely distributed in mammalian tissues. Recently α1,6-fucosylation has been further reported to be very crucial by the study of α1,6-fucosyltransferase (Fut8)-knock-out mice, which shows the phenotype of emphysema-like changes in the lung and severe growth retardation. In this study, we extensively investigated the effect of core fucosylation on α3β1 integrin and found for the first time that Fut8 makes an important contribution to the functions of this integrin. The role of core fucosylation in α3β1 integrin-mediated events has been studied by using Fut8+/+ and Fut8–/– embryonic fibroblasts, respectively. We found that the core fucosylation of α3β1 integrin, the major receptor for laminin 5, was abundant in Fut8+/+ cells but was totally abolished in Fut8–/– cells, which was associated with the deficient migration mediated by α3β1 integrin in Fut8–/– cells. Moreover integrin-mediated cell signaling was reduced in Fut8–/– cells. The reintroduction of Fut8 potentially restored laminin 5-induced migration and intracellular signaling. Collectively, these results suggested that core fucosylation is essential for the functions of α3β1 integrin.

Cell-Cell Interaction-dependent Regulation of N-Acetylglucosaminyltransferase III and the Bisected N-Glycans in GE11 Epithelial Cells INVOLVEMENT OF E-CADHERIN-MEDIATED CELL ADHESION

Changes in oligosaccharide structures are associated with numerous physiological and pathological events. In this study, the effects of cell-cell interactions on N-linked oligosaccharides (N-glycans) were investigated in GE11 epithelial cells. N-glycans were purified from whole cell lysates by hydrazinolysis and then detected by high performance liquid chromatography and mass spectrometry. Interestingly, the population of the bisecting GlcNAc-containing N-glycans, the formation of which is catalyzed by N-acetylglucosaminyltransferase III (GnT-III), was substantially increased in cells cultured under dense conditions compared with those cultured under sparse conditions. The expression levels and activities of GnT-III but not other glycosyltransferases, such as GnT-V and α1,6-fucosyltransferase, were also consistently increased in these cells. However, this was not observed in mouse embryonic fibroblasts or MDA-MB231 cells, in which E-cadherin is deficient. In contrast, perturbation of E-cadherin-mediated adhesion by treatment with EDTA or a neutralizing anti-E-cadherin antibody abolished the up-regulation of expression of GnT-III. Furthermore, we observed the significant increase in GnT-III activity under dense growth conditions after restoration of the expression of E-cadherin in MDA-MB231 cells. Our data together indicate that a E-cadherin-dependent pathway plays a critical role in regulation of GnT-III expression. Given the importance of GnT-III and the dynamic regulation of cell-cell interaction during tissue development and homeostasis, the changes in GnT-III expression presumably contribute to intracellular signaling transduction during such processes.

Phenotype changes of Fut8 knockout mouse: core fucosylation is crucial for the function of growth factor receptor(s).

Alpha1,6-fucosyltransferase (Fut8) catalyzes the transfer of a fucose residue to N-linked oligosaccharides on glycoproteins by means of an alpha1,6-linkage to form core fucosylation in mammals. In mice, disruption of Fut8 induces severe growth retardation, early death during postnatal development, and emphysema-like changes in the lung. A marked dysregulation of TGF-beta1 receptor activation and signaling in Fut8-null mice lung results in overexpression of matrix metalloproteinases (MMPs), such as MMP12 and MMP13, and a down-regulation of extracellular matrix (ECM) proteins such as elastin, which contributes to the destructive emphysema-like phenotype observed in Fut8-null mice. Furthermore, therapeutic administration of exogenous TGF-beta1 rescued the null mice from the emphysema-like phenotype. On the other hand, absence of Fut8 on EGF or PDGF receptor results in down-regulation of the receptor-mediated signaling, which is a plausible factor that may be responsible for the growth retardation. Reintroduction of the Fut8 gene to Fut8-null cells potentially rescued these receptor-mediated signaling impaired in null cells. Collectively, these results suggest that core fucosylation is crucial for growth factor receptors such as TGF-beta1 and EGF receptor-mediated biological functions.

Requirement of Fut8 for the expression of vascular endothelial growth factor receptor-2: a new mechanism for the emphysema-like changes observed in Fut8-deficient mice.

alpha1,6-Fucosylation plays key roles in many biological functions, as evidenced by the study of alpha1,6-fucosyltransferase (Fut8) knockout (Fut8(-/-)) mice. Phenotypically, Fut8(-/-) mice exhibit emphysema-like changes in the lung, and severe growth retardation. Fut8(-/-) cells also show marked dysregulation of the TGF-beta1 receptor, EGF receptor, integrin activation and intracellular signalling, all of which can be rescued by reintroduction of Fut8. The results of the present study demonstrated that vascular endothelial growth factor receptor-2 (VEGFR-2) expression was significantly suppressed in Fut8(-/-) mice, suggesting that Fut8 was required for VEGFR-2 expression. The expression of VEGFR-2 mRNA and protein was consistently down-regulated by knockdown of the Fut8 gene with small interference RNA in A549 cells, as well as in TGP49 cells, suggesting that suppression occurs at the level of transcription. In contrast, the expression level of ceramide, an inducer of cell apoptosis, was increased in the lungs of Fut8(-/-) mice. The terminal transferase dUTP nick end-labelling (TUNEL) assay was used to identify apoptotic cells. The number of TUNEL-positive septal epithelia and endothelia cells was significantly increased in the alveolar septa of lungs from Fut8(-/-) mice when in comparison with lungs from wild-type mice. It is well known that, in emphysema, ceramide expression can be greatly enhanced by blockade of the VEGFR-2. Thus, suppression of VEGFR-2 expression may provide a novel explanation for the emphysema-like changes in Fut8(-/-) mice.