[2 Phenotype Changes of Fut8 Knockout Mouse Core Fucosylation Is Crucial for the Function of Growth Factor Receptors

By Xiangchun Wang, Jianguo Gu, Eiji Miyoshi, Koichi Honke, and Naoyuki Taniguchi

Abstract a1,6-Fucosyltransferase (Fut8) catalyzes the transfer of a fucose residue to N-linked oligosaccharides on glycoproteins by means of an a1,6-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-^1 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-^1 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-^1 and EGF receptor-mediated biological functions.

Overview

The remodeling of cell surface growth factor receptors by modification of their oligosaccharide structures is associated with certain functions and biological events (Akiyama et al., 1989; Gregoriou, 1993; Hakomori Si, 2002;

METHODS IN ENZYMOLOGY, VOL. 417 Copyright 2006, Elsevier Inc. All rights reserved.

0076-6879/06 $35.00 DOI: 10.1016/S0076-6879(06)17002-0

Taniguchi et al., 2001; Zheng et al., 1994). Certain N-glycan structures of a number of glycoproteins seem to contribute to the folding, stability, and sorting of glycoproteins (Dwek, 1995; Wyss et al., 1995). They have a core structure, and their branching patterns are determined by glycosyltrans-ferases (Dennis et al., 1999; Schachter, 1986; Taniguchi et al., 1999).

GDP-l-Fuc:N-acetyl-^-d-glucosaminide a1,6-fucosyltransferase (Fut8, E.C.2.4.1.152) catalyzes the transfer of a fucose residue from GDP-fucose to position 6 of the innermost GlcNAc residue of hybrid and complex types of N-linked oligosaccharides on glycoproteins to form core fucosylation in mammals, as shown in Fig.1A. Fut8 is the only core FucT in mammals, but there are core a1,3-Fuc residues in plants, insects, and probably other species. The Fut8 gene is expressed in most rat organs with a relatively high level expression in brain and small intestine (Miyoshi et al., 1997). a1,6-Fucosylated glycoproteins are widely distributed in mammalian tissues and are altered under some pathological conditions. For example, the level of core fucosylation is elevated in both liver and serum during the process of hepatocarcinogenesis (Hutchinson et al., 1991). The presence of core fucosylation of a-fetoprotein, a well-known tumor marker for hepatocellular carcinoma (HCC), is known to distinguish patients with HCC from those with chronic hepatitis and liver cirrhosis (Sato et al., 1993; Taketa et al., 1993). It has recently been reported that the deletion of the core fucose from the IgG1 molecule enhances antibody-dependent cellular cytotoxicity activity by up to 50-100 fold (Shields et al., 2002; Shinkawa et al., 2003) and, therefore, is thought to have considerable potential for use in antibody therapy against cancer. These findings strongly suggested that core fucosylation of N-glycans modifies the function of the glycoproteins.

To define the physiological roles of Fut8, Fut8-null mice were generated by gene targeting technology. A targeted disruption of Fut8 was generated through homologous recombination in embryonic stem cells. The targeting vector was constructed by replacing exon 2 of Fut8, which contains the translation initiation site, with an IRES-LacZ-Neo-pA cassette (Fig. 1B). Disruption of Fut8 induces severe growth retardation, early death during postnatal development, and emphysema-like changes in the lung (Wang et al., 2005). Fut8~'~ mice were born apparently healthy with almost the expected Mendelian inheritance: Of 277 pups, there were 59 (21.3%) Fut8~/~, 147 (53.1%) Fut8+/~, and 71 (25.6%) Fut8+/+ mice. The appearance of Fut8~'~ mice could not be distinguished from Fut8+'~ and Fut8+/+ mice within 3 days of age, but approximately 70% of them died during this period (Fig. 2A). Most of the survivors manifested severe growth retardation (Fig. 2B). In fact, we found that dysregulation of TGF-^1 receptor activation leads to abnormal lung development and emphysema-like phenotype in Fu8-null mice (Wang et al., 2005) and

Fig. 1. Reaction pathway of core fucose synthesis and targeted disruption of Fut8 locus. (A) Man, mannose; Fuc, fucose; GDP-Fuc, guanosinediphosphofucopyranoside; Asn, asparagine. (B) The Fut8 gene (wild-type allele; top), the targeting vector (middle), and the disrupted Fut8 locus (mutant allele; bottom). The box in the Fut8 gene represents exon 2, which includes the translation-initiation site (ATG). A 184-bp deletion of exon 2 containing the translation-initiation site was replaced with an IRES-LacZ-Neo-pA cassette. The expected size of the Pst I digestion products of the gene, hybridized with the indicated probe, is shown for the wild-type allele (7.5 kb) and for the mutant allele (11.0 kb). Restriction enzyme sites: P, Pst I; Xb, Xba I; S, Sac I; H, Hind III; Xh, Xho I.

Fig. 1. Reaction pathway of core fucose synthesis and targeted disruption of Fut8 locus. (A) Man, mannose; Fuc, fucose; GDP-Fuc, guanosinediphosphofucopyranoside; Asn, asparagine. (B) The Fut8 gene (wild-type allele; top), the targeting vector (middle), and the disrupted Fut8 locus (mutant allele; bottom). The box in the Fut8 gene represents exon 2, which includes the translation-initiation site (ATG). A 184-bp deletion of exon 2 containing the translation-initiation site was replaced with an IRES-LacZ-Neo-pA cassette. The expected size of the Pst I digestion products of the gene, hybridized with the indicated probe, is shown for the wild-type allele (7.5 kb) and for the mutant allele (11.0 kb). Restriction enzyme sites: P, Pst I; Xb, Xba I; S, Sac I; H, Hind III; Xh, Xho I.

Mice age

Fig. 2. Semilethality and growth retardation in Fut8~'~ mice. (A) Survival ratio of Fut8~'~ (-/-, striped bar), Fut8+/~ (+/-, gray bar) mice after birth. (B) A 16-day-old Fut8~'~ pup (-/-) with a Fut8+/+ litter mate (+/+).

Mice age

Fig. 2. Semilethality and growth retardation in Fut8~'~ mice. (A) Survival ratio of Fut8~'~ (-/-, striped bar), Fut8+/~ (+/-, gray bar) mice after birth. (B) A 16-day-old Fut8~'~ pup (-/-) with a Fut8+/+ litter mate (+/+).

down-regulation of EGF receptor, as well as PDGF receptor activation, are plausible factors that may be responsible for the growth retardation (Wang et al., 2006). Here, roles of core fucosylation on TGF-,1 and EGF receptors are described.

Lacking Core Fucosylation on TGF- p Receptor Type II Leads to Emphysema-Like Changes in Fut8-null Mice Lung

The lungs of Fut8~'~ mice apparently displayed generalized air space enlargement and dilated alveolar ducts compared with those of Fut8+/+ mice (Fig. 3). By calculation of mean linear intercept (MLI), diameters of the pulmonary alveoli of Fut8~'~ mice were increased significantly from postnatal day 7 (Wang et al., 2005).

Pulmonary emphysema is believed to result from decreased structural integrity of connective tissues because of a defect in their formation or to an abnormal proteolysis. Elastin and fibrillar collagen are major components of the extracellular matrix (ECM), which sustains the normal lung architecture. On the other hand, matrix metalloproteinases (MMPs) are a

Tgf Receptor Fucose

Fig. 3. Lacking core fucosylation on TGF-, receptor type II leads to emphysema-like changes in Fut8-null mice lung. In mice lacking core fucosylation of TGF-,3 type II receptor, the reduced binding ability of TGF-,1 to its receptor results in down-regulation of Samd2 activation. This disturbed the homeostasis of ECM and MMPs. The increase in proteolytic potential leads to destruction of lung tissue and emphysema in Fut8-null mice.

Fig. 3. Lacking core fucosylation on TGF-, receptor type II leads to emphysema-like changes in Fut8-null mice lung. In mice lacking core fucosylation of TGF-,3 type II receptor, the reduced binding ability of TGF-,1 to its receptor results in down-regulation of Samd2 activation. This disturbed the homeostasis of ECM and MMPs. The increase in proteolytic potential leads to destruction of lung tissue and emphysema in Fut8-null mice.

group of zinc- and calcium-dependent proteinases that have an important role in the normal turnover of ECM components. Abnormal production of MMPs is implicated in the induction of emphysema. RT-PCR analysis showed that expression levels of McolB (a mouse ortholog of human MMP-1), MMP-12, and MMP-13 were greatly enhance in lung tissue from FutS~'~. Conversely, elastin expression was down-regulated in lungs from Fut8-deficient mice. On the other hand, fragmentation and a significantly reduced number of elastic fibers were observed by elastin staining in FutS~y~ mice. These results suggest that overexpression of a set of MMPs might be causally linked to the development of emphysema in FutS~'~ mice.

The TGF-^1 receptor-mediated signaling pathway is a key pathway for regulating expression of ECM proteins, including suppression of MMPs to produce a "synthetic'' phenotype (Massague et al., 2000). The enhancement of McolB and MMP12 were block by TGF-^1 treatment in FutS+'+ cells but not in FutS~'~ cells, indicating that the deletion of FutS diminishes TGF-^1 mediated signaling. Actually, abolishment of core fucosylation on TGF-,3 type II receptor in FutS~'~ cells results in reduced binding ability of TGF-^1 to its receptor compared with FutS+/+ cells. Furthermore, TGF-^1 receptor-mediated signaling was suppressed in FutS~'~ cells and lung by carrying out Smad2 phosphorylation analysis. The TGF-^1 signaling deficiency was restored by re-introduction of FutS~'~ cells with wildtype FutS gene. Importantly, administration of exogenous TGF-^1 resulted in a significant rescue of the emphysema-like phenotype, stimulated the formation of elastin fiber, and, concomitantly, reduced MMP-12 expression in Fut8_/~ lung. In additional, using antibodies specific for surfactant protein C (SP-C, a marker of differentiated type-II alveolar epithelial cells), the expression levels of SP-C protein at each stage were slightly weaker in FutS~'~ lungs than in FutS+/+ lungs, suggesting that lung development was also disturbed by the loss of core fucosylation.

Taken together, we propose that the lack of core fucosylation of TGF-^1 receptor is crucial for developmental and progressive/destructive emphysema, suggesting that perturbation of this function could underlie certain cases of human emphysema (Fig. 3).

Core Fucosylation Regulates EGF Receptor-Mediated Intracellular Signaling

Epidermal growth factor receptor (EGFR)-mediated cellular responses to EGF and transforming growth factor-a stimulation regulate several biological functions, including cell growth and cell differentiation. The binding of these ligands to the extracellular domain of EGFR induces activation of its intrinsic tyrosine kinase activity, leading to the receptor autophosphorylation and the phosphorylation of tyrosine residues in various cellular substrates, many of which serve as intracellular signal molecules (Carpenter and Cohen, 1990; Schlessinger, 1988; Ullrich and Schlessinger, 1990). The extracellular domain of EGFR contains 12 potential N-glycosylation sites (Ullrich et al., 1984), and the remodeling of N-glycans on EGFR can modulate EGFR-mediating functions (Gu et al., 2004; Hazan et al., 1995; Rebbaa et al., 1996, 1997; Soderquist and Carpenter, 1984; Zeng et al., 1995). It has been reported that the binding of EGF to EGFR was significantly reduced by treatment with some N-glycosylation inhibitors (Soderquist and Carpenter, 1984). In addition, EGF binding, as well as its tyrosine kinase activity, was reduced by addition of certain lectins (Hazan et al., 1995; Rebbaa et al., 1996; Zeng et al., 1995), indicating that N-glycans are required for ligand binding. On the other hand, the overexpression of N-acetylglucosaminyltransferase III (GnT-III), a pivotal glycosyltransferase that plays a major role in the biosynthesis of hybrid and complex types of N-linked oligosaccharides (Nishikawa et al., 1992), significantly reduces the ability of EGF to bind to its receptor, EGFR autophosphorylation, and subsequently blocks EGFR-mediated ERK phosphorylation in U373 MG glioma cells (Rebbaa et al., 1997) or PC12 cells (Gu et al., 2004). Recently it was also reported that N-glycans of EGFR, as well as other cytokine receptors modified by GnT-V, which catalyzes the formation of GlcNAc^1,6 branches, play an important role in the endocytosis of EGFR to regulate its expression levels on the cell surface (Partridge et al., 2004). Thus, N-linked oligosaccharides on EGFR seem to be important factors for receptor function. However, to date, the roles of core fucosylation in EGFR-mediating functions have not been identified yet.

The epidermal growth factor (EGF)-induced phosphorylation levels of the EGF receptor (EGFR) were substantially blocked in compared with Fut8+/+cells, whereas 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. Moreover, the tyrosine-phosphorylation levels of the EGF receptor in Fut8-null embryos were lower than that in wild-type embryos (Wang et al., 2006). Consistent with this, EGFR-mediated JNK or ERK activation was significantly suppressed in Fut8_/~ cells. The down-regulation of JNK and ERK activation in Fu8_/~ cells was rescued in the restored cells. Furthermore, these differences in responsiveness to EGF stimulation between Fut8+/+ and Fut8~/~ cells were much more obvious at low concentrations of EGF stimulation (^0.05 ng/ml) rather than higher concentrations (0.1^ ng/ml), indicating the high binding affinity of EGF to its receptor is mainly down-regulated by a lack of core fucosylation. In fact, it has been reported that EGFR kinase activation occurs exclusively through the high-affinity subclass (Bellot et al., 1990). It is noteworthy that down-regulation of phosphorylation of ERK induced by PDGF was also observed in Fu8~'~ cells. Although there are no significant changes in FGF-mediated signaling, the possibility that other growth factor receptor-mediated signaling may also affect cell growth cannot be excluded.

The binding of 125I-EGF to EGFR was reduced in Fut8_/" cells compared with Fut8+/+ or the restored cells at low doses, whereas similar levels of binding were found at relatively high concentrations (Wang et al., 2006). A Scatchard analysis revealed that both low- and high-affinity binding of EGFR were present in Fut8+/+ and the restored cells, but only low affinity EGFR was detected in Fut8_/~ cells. Thus, these results suggest that the modulation of N-glycans by core fucosylation on EGFR may regulate the high-affinity binding EGF to EGFR, which is required and sufficient for EGF-induced responses ( Gregorou and Rees, 1 984; Defize et al., 1988,1989), but not for the low affinity of EGFR.

Collectively, these results strongly suggest that core fucosylation is essential for EGFR-mediated biological functions. Down-regulation of EGFR-mediated signaling because of lack of core fucosylation, in part, may attribute to growth retardation in Fut8_/~ mice (Fig. 4).

Materials and Methods

Gene Targeting

A part of the mouse Fut8 gene spanning 13.9 kb, which includes the exon containing the translation-initiation site, was isolated by screening a mouse 129SvJ l genomic library (Stratagene, La Jolla, CA) using a SacI-SacI fragment of porcine Fut8 cDNA (nt-39 to 373) (Uozumi et al., 1996; Yanagidani et al., 1997) as a probe. A targeting vector was constructed by replacing the 184-bp SacI-Hind III fragment containing the translation-initiation site with a 4.9-kb SacI-SalI fragment of the plasmid pGT1.8IresBgeo (Mountford et al., 1994) that contains an internal ribo-some entry site (IRES)-LacZ-Neor- polyadenylation signal (pA) cassette, flanked with a 1.5-kb XhoI-NotI fragment of the plasmid pMC1DTpA (Yanagawa et al., 1999), which encodes diphtheria toxin A chain (DT-A) for negative screening (Fig. 1B). The targeting vector was transfected into D3 embryonic stem cells, and clones were selected with G418. Southern blot analysis of selected clones with 5' (A) and 3' (B) probes revealed that 1.2% (4 of 343) of the embryonic stem clones had undergone correct homologous recombination. Targeted cell clones were then injected into

Core Fucosylation

Fig. 4. Core fucosylation plays an important role in EGFR-mediating signaling pathway Absence of core fucosylation on EGFR results in down-regulation of EGFR phosphorylation. EGFR-mediated downstream signaling such as phosphorylation of JNK or ERK was also decreased in Fut8_/~ cells. Down-regulation of EGFR-mediated signaling caused by lack of core fucosylation, in part, may attribute to growth retardation in Fut8_/~ mice.

Fig. 4. Core fucosylation plays an important role in EGFR-mediating signaling pathway Absence of core fucosylation on EGFR results in down-regulation of EGFR phosphorylation. EGFR-mediated downstream signaling such as phosphorylation of JNK or ERK was also decreased in Fut8_/~ cells. Down-regulation of EGFR-mediated signaling caused by lack of core fucosylation, in part, may attribute to growth retardation in Fut8_/~ mice.

blastocysts from B6C3F1 mice, which are F1 mice resulting from the intercross of female C57BL/6 and male C3H mice. Germ-line transmission of the mutant allele was achieved from male chimeras derived from two independent embryonic stem cell clones.

Establishment of Embryonic Fibroblasts

For preparation of embryonic fibroblasts, a whole mouse embryo at 18.5 days after coitus was dissected, and the head and all internal organs were removed. The carcasses were minced, incubated in PBS (-) containing 0.05% trypsin, 0.53 mM EDTA, and 40 ^g/ml DNase at 37° for 30 min with stirring three times, and then cells were plated on a 100-mm dish in

Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and incubated at 37° in humidified air containing 5% CO2. To obtain immortal cells, Zeocine-resistant vector (pcDNA3.1) containing the SV40 gene was introduced to these primary embryonic fibroblasts. Transfectants were screened in the presence of 400 yg/ml Zeocine, and SW and SK immortal cells were established from Fut8+/+ and Fut8~'~ primary fibroblasts, respectively.

Fut8 Activity Assay

The specific activity of Fut8 was determined using a synthetic substrate, 4-(2-pyridylamio)-butyl-amine (PABA)-labeled oligosaccharide as a substrate. Cells grown to subconfluence were washed with PBS (-) once, and the cell pellet was suspended in 200 yl lysis buffer containing 10 mM Tris-HCl, pH 7.4,150 mM NaCl, and 1% Triton X-100. The cell lysate was then assayed for Fut8 activity as described before (Uozumi et al., 1996).

Lectin Blotting Analysis

Whole cell lysate or immunoprecipitated EGFR was subjected to 10% or 7.5% SDS-PAGE and transferred to PVDF membranes, the membranes were blocked with 5% BSA in TBST overnight at 4°, and then incubated with 0.5 yg/ml biotinylated aleuria aurantia lectin (AAL) (Seikagaku Corp., Japan), which preferentially recognizes Fuca1,6GlcNAc structure, in TBST for 1 h at room temperature. After washing with TBST four times, lectin reactive proteins were detected using a Vectastain ABC kit (Vector laboratories, Burlingame, CA) and ECL kit.

TGF-$1 Binding Assay

The cells (1.5 x 105/well) were cultured on 24-well plates, and washed twice with 500 yl of PBS containing 0.1% BSA, and then incubated with 200 yl of PBS containing different amounts of 125I-TGF-^1 in a concentration range of 0.1-1.0 ng and 10 ng of unlabeled TGF-^1. Nonspecific binding was determined by adding 100 ng of unlabeled TGF-^1. After incubation for 2 h at 4° with shaking, the cells were washed three times with ice-cold PBS containing 0.1% BSA, and then solubilized in 500 yl of 1 N NaOH. The radioactivity of the cell lysates was counted with a 7-counter (Wang et al., 2005).

EGF Binding Assay

The cells were seeded at a density of 1 x 105 cell/well in 24-well plates and incubated overnight. The medium was then replaced with DMEM, which contained 0.1%BSA (M-BSA) and incubated for 20 min at 37°. After replacing the medium with ice-cold M-BSA, 125I-EGF and EGF mixture was added, followed by incubation for 2 h at 4°. The cells were then washed with ice-cold PBS and hydrolyzed on 0.5 ml 1 M NaOH. The radioactivity was counted with a 7 counter (Wang et al., 2006).

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10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

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