Glycobiology Advance Access originally published online on October 2, 2007
Glycobiology 2008 18(1):114-124; doi:10.1093/glycob/cwm107
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Reduced 
4β1 Integrin/VCAM-1 Interactions Lead to Impaired Pre-B Cell Repopulation in Alpha 1,6-Fucosyltransferase Deficient Mice
2 Departments of Glycotherapeutics, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
3 Departments of Hematology and Oncology Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
4 Departments of Biochemistry, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan
5 Department of Immunology and Molecular Genetics, Kawasaki Medical School, Okayama 701-0192, Japan
6 Takara Bio Inc. Shiga 520-2193, Japan
7 Department of Functional Diagnostic Science, Division of Health Science, Osaka University School of Health Sciences, Osaka 565-0871, Japan
8 Department of Disease Glycomics Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
1 To whom correspondence should be addressed: e-mail address: kondoa{at}glycot.med.osaka-u.ac.jp
Received on August 14, 2007; revised on September 21, 2007; accepted on September 27, 2007
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Abstract |
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Mice with a targeted gene disruption of Fut8 (Fut8–/–) showed an abnormality in the transition from pro-B cell to pre-B cell, reduced peripheral B cells, and a decreased immunoglobulin production. Alpha 1,6-fucosyltransferase (FUT8) is responsible for the alpha 1,6 core fucosylation of N-glycans, which could modify the functions of glycoproteins. The loss of a core fucose in both very late antigen 4 (VLA-4,
4β1 integrin) and vascular cell adhesion molecule 1 (VCAM-1) led to a decreased binding between pre-B cells and stromal cells, which impaired pre-B cells generation in Fut8–/– mice. Moreover, the B lineage genes, such as CD79a, CD79b, Ebf1, and Tcfe2a, were downregulated in Fut8–/– pre-B cells. Indeed, the frequency of preBCR+CD79blow cells in bone marrow pre-B cells in Fut8–/– was much lower than that in Fut8+/+ cells. These results reveal a new role of core fucosylated N-glycans in mediating early B cell development and functions.
Key words:
alpha 1,6-fucosyltransferase
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B-cell development
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CD45R; N-glycans
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4β1 integrin/VCAM-1
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interestsReferences |
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B lymphocytes differentiate in the bone marrow (BM) from hematopoietic stem cells through a sequential series of intermediates, which are characterized by cell surface antigens and immunoglobulin rearrangement (Hardy and Hayakawa 2001
; Pelayo et al. 2005). Precursor B cells develop in close interaction with the BM environment. Adhesion molecules, including CD44, selectin, and integrins, control the interaction between B cell progenitors and BM stromal cells or extracellular matrix (ECM) components. The integrins comprise a large family of heterodimeric transmembrane molecules consisting of and β subunits, which mediate adhesion, migration, the survival and differentiation of the cells (Hynes 1992). Cell surface integrins are all major carriers of N-glycans, and the N-glycosylation of integrins plays an important role in their biological functions (Pochec et al. 2003; Gu and Taniguchi 2004).
GDP-L-Fuc:N-acetyl-β-D-glucosaminide 1,6-fucosyltransferase (FUT8) catalyzes the transfer of a fucose residue from GDP-fucose to the innermost GlcNAc residue of complex N-glycans via an 1,6-linkage (core fucosylation) in the golgi apparatus in mammals (Wilson et al. 1976). Fut8 deficient (Fut8–/–) mice showed a postnatal failure to thrive, and all of the survivors manifested growth retardation and emphysema-like changes (Wang et al. 2005). We recently reported that signaling through the transforming growth factor-β (TGF-β) receptor (Wang et al. 2005) and the epidermal growth factor (EGF) receptor (Li et al. 2006) was downregulated in Fut8–/– mice, due to a decreased ligand affinity for the receptor. Shinkawa et al. (2003) reported that deletion of the core fucose from C
2 of IgG1 enhanced antibody-dependent cell-mediated cytotoxity (ADCC) activity by up to 50–100 fold. Collectively, these results strongly suggest that the core fucosylation of N-glycans modifies the function of the corresponding glycoprotein.
Integrins bind to ECM components such as fibronectin and laminin as well as cellular receptors such as vascular adhesion molecule-1 (VCAM-1), thereby facilitating signal transduction from stromal cell-derived soluble factors such as IL-7, which are important for the survival of B cell precursors (Hynes 1992). Very late antigen-4 (VLA-4; CD49d/CD29; 4β1 integrin) belongs to the integrin family of cell adhesion molecules and is expressed by a wide range of leucocytes (Springer 1990). 4β1 integrin is expressed at high levels on the surface of lymphohematopoietic progenitors, and is involved in their development and proliferation (Miyake et al. 1991; Grabovsky et al. 2000). In addition, its interaction with fibronectin and/or VCAM-1 mediates leukocyte tethering, rolling, and firm adhesion on the endothelium, which is indispensable for recruiting leukocytes to inflammatory tissues (Vonderheide et al. 1994; Alon et al. 1995). VCAM-1, a ligand of 4β1 integrin, is constitutively expressed by the BM stromal cells (Miyake et al. 1991) and by the follicular dendritic cells (Koopman et al. 1991). Indeed, pre-B cells fail to transmigrate and proliferate in the absence of 4 integrins (Arroyo et al. 1999), and conditional VCAM-1 mutant mice show an impaired humoral immune response against the T cell dependent antigens (Leuker et al. 2001). The ability to facilitate leukocyte adhesion to the stromal cells implies that 4β1 integrin/VCAM-1 interactions is an important factor in the initiation of early B cell development (Miyake et al. 1992). Several studies have highlighted the capacity of 4β1 integrin/VCAM-1 interactions to mediate strong adhesion within a model of leukocyte trafficking and enhance activation (van Dinther-Janssen et al. 1991; Carrasco and Batista 2006). Pre-B cell integrins and their stromal cell ligands, together with pre-B cell receptor (preBCR) and galectin-1, form a homogeneous lattice at the contact area between pre-B and stromal cells (Rossi et al. 2006). Springer's group (Carman and Springer 2004) has shown that VCAM-1 is associated with actin-rich microvilli in the "cup-like" structure, and has suggested that this provides directional guidance to leucocytes for extravasation.
Previous reports showed that the functions of integrins were regulated by N-glycans catalyzed by N-acetylglucosaminyltransferase III (GnT-III), GnT-V, sialyltransferases etc. (Yamamoto et al. 2001; Guo et al. 2002; Pochec et al. 2003). The introduction of bisecting GlcNAc into 5β1 integrin was recently shown to down-regulate cell adhesion, ligand affinity, and cell migration (Isaji et al. 2004). More recently, impaired 3β1 integrin-mediated cell migration was found in Fut8–/– embryonic fibroblasts (Zhao et al. 2006). Based on the amino acid sequences, 4β1 integrin contains 11 and 12 potential asparagine-linked glycosylation sites on each of the 4 and β1 subunits, respectively. There are seven potential N-linked glycosylation sites in the immunoglobulin-like domains of VCAM-1 (Vonderheide et al. 1994). Given the various biological functions of N-linked glycosylation, the function of the core fucose of 4β1 integrin and VCAM-1 deserves a more detailed investigation. In particular, the function of core fucose associated with 4β1 integrin/VCAM-1 interactions in B cell development has not yet been investigated.
In the present study, we show that core fucosylated N-glycans were required for functional 4β1 integrin/VCAM-1 interactions to support B cell development. Pre-B cell colony formation was attenuated due to the lowered 4β1 integrin/VCAM-1-mediated interaction of pre-B cells and stromal cells by the loss of core fucose. These findings clearly demonstrate the crucial role of Fut8 in early B lymphopoiesis.
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Results |
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Disruption of Fut8 led to B lymphopoietic failure at the pre-B cell stage
We first focused on the differentiation of hemato-lymphopoietic cells by Fut8 deficiency. Flow cytometry analysis revealed that, while CD45R+CD43+ (pro-B enriched) population was sustained, CD45R+CD43– (pre-B enriched) and CD45R+IgM+ (immature B enriched) population were significantly reduced in Fut8–/– BM (Table II and representative data in Figure 1). CD45R+CD43– (pre-B) and CD45R+IgM+ (immature B) cells comprised 17.8 ± 4.5% and 4.7 ± 0.8% of the Fut8–/– BM, while those of control littermates were 30.2 ± 2.6% and 7.8 ± 1.7%, respectively. However, no significant difference was found in the frequency of the CD45R+CD43+ (pro-B) cell population between the Fut8+/+ and Fut8–/– mice (Table II). Flow cytometry analysis of the spleen subsequently revealed that the frequency of CD45R+IgM+ cells was significantly reduced in Fut8–/– mice, whereas those of CD11b+ myeloid cells and the TER119+ erythroid cells were increased (Table II). In contrast to the significant change of early B cell populations, development of CD4+ or CD8+ T cells, natural killer cells and natural killer T cells were relatively normal in the Fut8–/– mice (Table II). These findings implied that Fut8 expression is required for pro-B cells to properly differentiate to pre-B and later stages of B cell development.
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It is noteworthy that the FUT8 product, a core fucosylated N-glycan, is ubiquitously expressed in the BM microenvironment of Fut8+/+ BM but not in Fut8–/– mice. There was no difference in apoptosis, as evidenced by Tunel staining has been found between Fut8+/+ and Fut8–/– mice (Figure 2). A selective and profound reduction in the pre-B and immature B cell populations, and no concomitant change in the population containing pro-B cells were observed in the Fut8–/– BM (Figure 1). These results suggest that the Fut8 defect results in impaired pre-B cell expansion, which is not due to any accelerated apoptosis.
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Loss of Fut8 reduced the frequency of pre-B cells supported by stromal cells
To address the abnormality of the pro-B to pre-B transition in Fut8–/– mice, fractions containing CD45R+CD43+, CD45RlowCD43–, CD45R+IgM–, and CD45R+IgM+ cells in the BM were sorted, and the expression of Fut8 in these fractions were examined by real-time polymerase chain reaction (PCR). All of these B progenitor fractions expressed the Fut8 gene and the developmental progression from pro-B to the pre-B cell stage was accompanied by an increase in the RNA expression level of Fut8 (Fig. 3). The results further support the hypothesis that Fut8 plays important roles in the pro-B to pre-B cell transition, and that the targeting of Fut8 consequently lead to B lymphopenia beyond the pro-B cell stage.
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We then performed colony assays to compare the frequencies of the B cell progenitors defined by their growth requirement between Fut8+/+ BM and Fut8–/– BM cells. In the Fut8–/– BM, the frequency of clonable pre-B cell progenitors in the presence of stromal cells (ST2, PA6) and IL-7 declined, compared to those of control littermates (Table III). Furthermore, we found that Fut8–/– CD45R+IgM– fraction contained significantly less clonable pre-B cells compared to Fut8+/+ cells (Figure 4A). In contrast, colony formation of pre-B cells in complete methylcellulose medium in response to IL-7 alone (CFU-IL-7) was indistinguishable between Fut8+/+ pre-B cells and Fut8–/– pre-B cells (Figure 4B). These results strongly indicate that Fut8 expression in pre-B progenitor is important for their growth through pre-B cells/stromal cells interaction, but not IL-7 reactivity.
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It is noteworthy that the frequency of pre-B cell progenitors on PA6 was significantly lower than those on ST2 (Table III). We also found that the FUT8 protein was much lower in PA6 cells by Western blot, compared to ST2 cells that strongly expressed (Figure 5A). To further clarify the involvement of Fut8 of stromal cells for the growth of B progenitor cells, we stably knocked down the Fut8 gene of ST2 cells, referred to as ST2-KD. No apparent changes were found in the expressions of other glycosyltransferase genes, such as GnT III and β1,4-galactosyltransferase I (β4GalT-I) (data not shown). As shown in Figure 5B, the introduction of Fut8 siRNA successfully suppressed FUT8 expression. An Aspergillus oryzae lectin (AOL) blot analysis confirmed the effective knockdown of FUT8 expression (Figure 5C). To determine whether the core fucosylated N-glycans of ST2 cells functionally compromised pre-B cell colony formation, we cultivated Fut8+/+ BM cells on ST2 cells and on ST2-KD cells. The frequency of B cell progenitors was 1/251 on ST2-KD cells, which was less than half of that on ST2 cells (1/114) (Figure 5D). These results indicate that Fut8 in BM stromal cells is involved in pre-B supporting ability.
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Ablation of core fucose of VCAM-1 and
4β1 integrin lowered binding ability of pre-B cells to ST2 and VCAM-1 moleculeIt is well known that
4β1 integrin/VCAM-1 interactions are involved in facilitating B cell adhesion to stromal cells and in enhancing B cell activation (Carrasco et al. 1992). Indeed, colony formation of BM cells on ST2 cell with IL-7 was completely blocked by treatment with 10 µg/mL of an anti-VCAM-1 antibody or an anti-4β1 integrin antibody (Figure 5D). Thus, we analyzed the effect of core fucose on 4β1 integrin and VCAM-1 in the adhesion of pre-B cells to stromal cells. To ascertain the role of the core fucose of VCAM-1, we compared the binding ability of Fut8+/+ pre-B cells with that of ST2 and ST2-KD cells. The core fucosylated N-glycans of VCAM-1 was abolished in ST2-KD cells without any effect on the total VCAM-1 expression levels (Figure 6A). As shown in Figure 6B, the Fut8+/+ pre-B cells showed a weaker adhesion to ST2-KD, indicating that FUT8 in stroma has positive role in tethering pre-B cells. On the other hand, Fut8–/– pre-B cells showed weaker binding to ST2-KD than Fut8+/+ pre-B cells. Furthermore, when the recombinant mouse VCAM-1/Fc chimera was used, binding of Fut8–/– pre-B cells was weaker than that of Fut8+/+ pre-B cells, suggesting that the adhesion of pre-B cells themselves by 4β1 integrin was also impaired by defect of FUT8 in pre-B cells.
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To further examine the importance of the core fucosylated N-glycans on
4β1 integrin, we stably silenced Fut8 expression in a pre-B cell line, 70Z/3 cells, and named 70Z/3-KD. As shown in Figure 6C and D, FUT8 expression and the core fucosylated N-glycans of 4β1 integrin were ablated in 70Z/3-KD. Reintroduction of the Fut8 gene into 70Z/3-KD cells restored the core fucosylation of 4β1 integrin (70Z/3-KD-re). No significant differences in the expression levels of 4β1 integrin on the cell surface were found among the three cell types (Figure 6D). In a binding assay, 70Z/3-KD cells showed an impaired adhesion to VCAM-1, compared to mock cells. Preincubation of 70Z/3 cells with an anti-4β1 integrin antibody significantly blocked the binding of 70Z/3 to VCAM-1. The reintroduction of Fut8 restored the binding, as evidenced by an increase in the percentage of binding cells from 30 to 45% (Figure 6E), indicating that core fucosylated N-glycans are required for functional 4β1 integrin/VCAM-1 interactions to support B cell development. Collectively, these results demonstrated that core fucosylation plays a pivotal role in a key event in pre-B cell development; 4β1 integrin/VCAM-1-mediated interactions between pre-B cells and stromal cells.
Pre-B cells in Fut8–/– mice exhibit a reduced gene expression critical for early B cell development
Completing the productive rearrangement of immunoglobulin µ heavy chain gene in B progenitor cells Igµ chain associates with the surrogate light chain (SLC), which is composed of 
5 and VpreB (encoded by Vpreb1) proteins, and the CD79a (Ig))/CD79b (Igβ) transducing complex, to form the preBCR complex. To address the underlying molecular mechanism of the impaired pre-B cell generation, we examined the pattern of gene expression in Fut8–/– pre-B cells by real-time PCR. We found that the expression of Tcfe2a, Ebf1, Cd79a, and Cd79b in Fut8–/– pre-B cells was lower than that in Fut8+/+ pre-B cells, by a decrease of 45.9%, 80.9%, 23.3%, and 53.6% relative to Fut8+/+ cells, respectively, with no change in Pax5 and Vpreb1 expression (Figure 7A). The surface expression preBCR and CD79b on CD45RlowCD43– cells was further analyzed by flow cytometry. The percentage of Fut8–/– preBCR+CD79low cells was 2.4% of the pre-B cells, whereas that of Fut8+/+ preBCR+CD79low cells was 4.4% (Figure 7B). These results suggest that pre-B cell differentiation of Fut8–/– mice is impaired at the step where B progenitors expressing the preBCR complex on their surface is generated.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interestsReferences |
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In the present study, we found that targeting of Fut8–/– resulted in the reduction of wide range of B-lineage cells. Using techniques of flow cytometry, colony assays, and quantitative RT-PCR, we revealed that the transition from pro-B to pre-B cells is affected by the deficiency of FUT8. Moreover, a lack of core fucosylated N-glycans of
4β1 integrin and VCAM-1 led to a reduction in adhesion between pre-B cells and stromal cells. Since an important role for this interaction in early B-cell development was established, we attributed this reduced interaction to the impaired development of B lymphocytes.
The physiological importance of the fucose modification of proteins has been highlighted by a leukocyte adhesion deficiency II (LAD II) (Luhn et al. 2001), which is categorized in congenital disorder of glycosylation (CDG) genetic disease and is caused by reduced GDP-fucose. In LADII patients, a leukocyte adhesion is reduced due to a deficit in selectin-mediated adhesion through fucosylated ligands, such as sialyl Lex. As previously reported, FUT8 is able to modify multiple glycoproteins and affect their functions. Core fucosylation could affect the conformation and flexibility of the antenna of N-linked biantennary oligosaccharides (Stubbs et al. 1996). The binding affinities of the TGF-β type II receptor and/or EGF receptor for their respective ligands are reduced in Fut8–/– cells (Wang et al. 2005; Li et al. 2006; Wang et al. 2006), suggesting that the core fucose is involved in ligand binding and the subsequent activation of various receptors (Kondo et al. 2006; Taniguchi et al. 2006). It has also been reported that the increase of Fut8 activity in megakaryocytes (MKs) progenitors preceded the increase of CD41a+ cell generation (Bany-Laszewicz et al. 2004). In the present study, we found that a deficiency in core fucosylation caused an abnormality in the immune system, particularly in B cell development.
4β1 integrin binds to fibronectin and VCAM-1 expressed by stromal cells in the BM microenvironment (Miyake et al. 1991), mediates interactions of cell-extracellular matrix proteins as well as cell–cell interactions. 4β1 integrin binds to domain 1 or domain 4 of VCAM-1, involving amino acids within the linear sequence Q-I-D-S-P-L, which is expressed in each domain (Vonderheide and et al. 1994). Although integrin-mediated adhesion is based on the binding of and β subunits to a defined peptide sequence, the strength of this binding is modulated by various mechanisms such as posttranslational glycosylation (Gu 2004). There is an evidence to show that glycosylation alters β1 integrin function (Wadsworth et al. 1993). In addition, it has been reported that murine β1 integrins deficient in glycosylation show a reduced affinity for fibronectin and laminin (Oz et al. 1989). Moreover, impaired 3β1 integrin-mediated cell migration was found in Fut8–/– embryonic fibroblasts (Zhao et al. 2006). Our results showed that the ability of 70Z/3 cells to bind a VCAM-1 chimera was significantly attenuated by the knockdown of Fut8 expression, and that the reintroduction of Fut8 restored this binding ability (Figure 6E). The binding of Fut8–/– pre-B cells to ST2 and VCAM-1 was also decreased, compared with Fut8+/+ cells (Figure 6B), suggesting that core fucosylated N-glycans involved in pre-B cell and stromal cell interactions. Since both 4β1 integrin and VCAM-1 are heavily N-glycosylated (Vonderheide et al. 1994), it is very likely that core fucosylation affects 4β1 integrin/VCAM-1 interaction. The increasing affinity of integrins for their extracellular ligands, i.e., integrin activation, is controlled by conformational changes in the extracellular domains from an inactive to active form (Calderwood 2004). The lack of core fucose might cause conformational changes in the extracellular domains of integrin, subsequently affecting 4β1 integrin/VCAM-1 interactions. The phenotype of Fut8–/– mice combined with our in vitro data presented an intriguing possibility that core fucose is involved in the appropriate interactions of pre-B cells and stromal cells.
The important roles for 4β1 integrin/VCAM-1 interactions in early B cell development were demonstrated in vitro (Miyake et al. 1991). Here, the core fucose of 4β1 integrin and VCAM-1 was found to be important in 4β1 integrin/VCAM-1 interactions that could account for the decreased development of B cell progenitors in response to stromal cells and IL-7. First, the extent of B cell colony formation on ST2-KD cells in the presence of IL-7 was much less than on parent ST2 cells (Figure 5D), indicating that core-fucosylation of VCAM-1 was required for full ability of stromal cells to support pre-B cells. Second, a significant decrease was observed in the frequencies of clonable pre-B cells in Fut8–/– CD45R+IgM– cells as compared with Fut8+/+ CD45R+IgM– cells (Figure 4A), suggesting that core fucose of pre-B cells is also required for receiving the growth-supporting signals from stromal cells. The impaired signal was not IL-7-mediated, because CFU-IL-7 was not altered in Fut8–/– CD45R+IgM– cells. Finally, 70Z/3-KD showed significantly decreased adhesion to the VCAM-1/Fc chimera molecule, indicating that adhesion of 4β1 integrin on pre-B cells was also impaired by loss of core fucosylation. Collectively, it is reasonable to conclude that the low 4β1 integrin/VCAM-1 interaction caused the impaired pre-B cell development in Fut8–/– mice. Given the appropriate 4β1 integrin/VCAM-1 interaction enables pre-B cells to communicate with stromal cells, it would be interesting to see the changes of early B- related gene expressions by the communication.
In this context, our analyses provide the suggestive evidence that the gene expressions crucial for transition from pro-B cells to pre-B cells are affected by Fut8 deficiency. Early B cell development is controlled by a hierarchical regulatory network that induces several key transcription factors, such as PU.1, Ikaros, E2A (encoded by Tcfe2a), EBF (encoded by Ebf1), and Pax5 (Singh et al. 2005). Interestingly, quantitative real-time PCR analyses revealed that Fut8–/– pre-B cells express lower transcriptional genes such as Ebf1 and Tcfe2a, which promote the activation of B cell. Single and dual knockout mice showed that EBF1 and E2A proteins collaborate to activate the expression of early B cell genes such as CD79a, B29, Vpre-B, 5, RAG-1, and RAG-2. Mice lacking the E2A showed defective B lymphopoiesis similar to that of Ebf–/- mice (Zhuang Soriano et al. 1994; Lin and Grosschedl 1995). Similar to E2A- or EBF-deficient mice, Pax5–/- mice exhibited an early block of B cell differentiation, but at a slightly later stage (pre-BI) (Urbanek Wang et al. 1994). Interestingly, recent research reported that Fut8 expression was increased in pro-B cells lacking Pax5, which is an indispensable transcription factor for lineage decision to B lymphocytes (Deloguet al. 2006). Although it is difficult to determine the blocked step precisely by using materials with heterogenous populations, the step of transition from pro-B cells to pre-B cells seems to be a vulnerable point affected by Fut8 deficiency. The combined down-regulation of those genes, CD79a, CD79b, Ebf1, and Tcfe2a in Fut8–/– B progenitors is likely to be the most crucial effect resulted from the impaired 4β1 integrin/VCAM-1 interactions.
Overall, the most important finding of this study is the significant decrease in pre-B cells in the Fut8–/– BM, which provides an intriguing evidence that a core fucose moiety is critically important in pre-B cell development. FUT8 modulates 4β1 integrin/VCAM-1 interactions during B cell development and could regulate pre-B cell repopulation via the down-regulation of B cell related genes. Our study provides new insights into the biological functions of the core fucose during B cell development and provides a hint for elucidation of B cell related diseases, agammaglobulinaemia and lymphoma.
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Materials and methods |
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Mice
BALB/cA mice were purchased from the Charles River Laboratories Japan (Yokahama, Japan). Fut8–/– mice were generated as previously described (Wang et al. 2005
), and had been backcrossed eight times to the BALB/cA background. Homozygous wild (Fut8+/+) and knockout (Fut8–/–) mice were obtained by crossing heterozygous Fut8+/– mice. Fut8–/– and control wild-type (Fut8+/+) mice in the same littermate were analyzed at around two weeks of age. All animal procedures complied with the institutional animal protocol guidelines.
Antibodies
FITC-labeled anti-CD4 (GK1.5), anti-IgM (11/41), anti-erythroid (TER-119), anti-NK cells (DX5), anti-CD79b (HM79–16), anti-IgD (11–26), anti-CD43 (S7), anti-Gr-1 (RB6–8C5), APC-labeled anti-CD11b (Mac-1, M1/70), anti-CD45R (RA3–6B2), anti-CD8 (53–6.7) were obtained from e-Bioscience. Additional biotin-conjugated anti-preBCR antibody (SL156) and streptavidin-PE Cy5 were purchased from BD Bioscience (Franklin Lakes, NJ). A mouse monoclonal antibody for rat/mouse β-actin (sc-8432) was purchased from Santa Cruz (Santa Cruz, CA); a mouse monoclonal anti-FUT8 antibody (15C6) was obtained from Fujirebio Inc. (Shinjuku, Japan); a rat anti-VCAM-1 IgG1k antibody (M/K-2) was from SouthernBiotech (Birmingham, AL); a rat anti-mouse CD49 (PS-2) antibody was from Serotec Ltd. (Oxford, U.K.); a rabbit anti-mouse IgG HRP-conjugate was from ICN Pharmaceuticals, Inc. (Arora, OH); a donkey anti rat IgG HRP-conjugate was from Beckman Coulter, Inc. (Fullerton, CA).
Cell purification
The BM cells were obtained by crushing two femurs and two tibia of two-week-old mice. The crude mixture was filtered through nylon mesh, and resuspended at 1 x 107 cells per mL. Single-cell suspensions of the spleens and thymi were prepared by first grinding the tissues with frosted slides, and then by gentle passage through the nylon mesh. These cells were resuspended at 1 x 107 cells per mL. Red blood cells were lysed by incubation with 0.14 M NH4Cl and 20 mM Tris (pH 7.4) for 3 min at room temperature. In experiments using enriched pre-B cells, IgM+ cells were depleted from BM cells, and then CD45R+ cells were positively sorted with antibody-conjugated magnetic beads (Miltenyi Biotec, Bergish Gladbach, Germany). The sorted CD45R+IgM– cell populations were routinely >96% pure by fluorescence activated cell sorting (FACS) analysis.
Cell culture conditions
The 70Z/3 cells, a pre-B lymphoma line, were obtained from Dr. Paul W. Kincade. The stromal cell lines, ST2 and PA6 were from Dr. S-I Hayashi. All cell lines were grown in RPMI 1640 supplemented with 2 mM glutamine, 50 µM 2-mercaptoethanol (2-ME) (Fluka, Buchs, Switzerland), 5% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin. In some experiments, the indicated concentration of recombinant mouse IL-7 (Sigma) was added.
Flow cytometry and cell sorting
For phenotypic analysis, the BM cells were first incubated with an anti-CD16/CD32 (2.4G2) mAb to block Fc receptors, and then stained on ice for 15 min with several combinations of mAbs, as indicated in the figure legends. Flow cytometry was performed on a FACS-Calibur (Becton Dickinson, Mountain View, CA), and the data were analyzed with the CellQuest (Becton Dickinson) or Flowjo software (Treestar, San Carlos, CA).
For cell sorting, BM cells were stained with FITC-labeled anti-IgM, PE-labeled anti-CD43, APC labeled anti-CD45R, and subpopulations were sorted with a FACStar Plus (Becton Dickinson) instrument.
Assays for B cell progenitors
The frequencies of B cell progenitors growing dependent on stromal cells + IL-7 (clonable pre-B cells) and that on IL-7 alone (CFU-IL-7) were evaluated by means of clonable pre-B cell assay and CFU-IL-7 assay, respectively. For the clonable pre-B cell assay, a monolayer of BM stromal cells such as ST2 and PA6 was allowed to form in 96-well plates (Falcon, Oxnard, CA). The total BM cells and purified CD45R+IgM– cells, diluted to the cell number as indicated in the figure legends, were inoculated into the plates. Cells were cultured in RPMI 1640 supplemented with 5% FCS, 50 µM 2-ME, 2 mM glutamine in the presence of 10 ng/mL of IL-7. After seven days of culture, the outgrowth of pre-B cells was surveyed under a microscope, and the frequencies were then calculated according to the formula; frequency = {ln [T/(T - P)]}/N, where N, inoculated cell number/well; T, number of total wells; P, number of wells containing a pre-B cell colony.
For CFU-IL-7, enriched CD45R+IgM– cells were seeded into methylcellulose media containing 10 ng/mL recombinant human IL-7 (Stemcell technologies Inc). Colonies were counted after seven days of culture. Aggregates consisting of >40 cells were differentially scored as colonies.
Establishment of Fut8 knockdown cell lines
A retroviral vector carrying siRNA targeted to Fut8 was constructed as described previously (Li et al. 2006). The targeting sequence of the Fut8 siRNA used was as follows: sense: 5' UCU CAG AAU UGG CGC UAU GTT 3', antisense: 3' TTAGA GUC UUA ACC GCG AUA C 5'. The ST2 and 70Z/3 derivative cell lines, stably transfected with the plasmid expressing siRNA that targeted FUT8 are referred to hereafter as "ST2-KD" and "70Z/3-KD."
For Fut8 reintroduction, ORF of FUT8 was cloned into Hpa I site of pLHCX vector (Clontech) To prepare pLHCXsi-mU6-Fut8 expression vectors resistant to the siRNAs expressed in the Fut8-knockdown cells, we introduced multiple mutations into pLHCXsi-mU6-Fut8 (five point mutations for the Fut8–1386-1404 region) that did not alter the original amino acid residues.
Immunohistochemistry
The femurs from Fut8+/+ mice and Fut8–/– mice were dissected and fixed overnight in 10% formaldehyde containing 2% sucrose. The tissues were then decalcified in 0.37% unbuffered formaldehyde containing 5.5% ethylenediamine tetraacetic acid (EDTA) (pH 6.0–6.5) for four days. After fixation and decalcification, the specimens were embedded in paraffin, and 5 µm sections were prepared. The sections were histochemically stained with biotin-conjugated AOL (Ishida et al. 2002), which preferentially recognizes core fucosylation on N-glycans. Briefly, sections were deparaffinized twice in xylene and hydrated through a graded series of ethanol to phosphate-buffered saline (PBS). Endogenous peroxidase activity was blocked with 3% H2O2 for 5 min. After washing with PBS containing 0.1% Tween 20 (PBS-T), the slides were blocked with an avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA). The slides were incubated with biotin-conjugated AOL, and washed three times with PBS-T. The slides were covered with horseradish peroxidase (HRP)-streptavidin (Vector Laboratories, Peterborough, U.K.) for 30 min. Finally, the slides were visualized with 3, 3'-diaminobenzidine and counterstained with hematoxylin and eosin.
Western blot and lectin blot analysis
The protein samples were electrophoresed with 10% polyacrylamide gels using Mini Protean III electrophoresis tanks (Bio-Rad, Hercules, CA). After the electrophoresis, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, 0.45 µm, Millipore, Bedford, MA,) at 240 mA for 30 min. The blots were blocked for 2 h with 5% skim milk in TBS-T (TBS-T; 10 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) for immunoblot or with 3% BSA in TBS-T for lectin blot. Following the incubation with the appropriate primary antibodies or AOL overnight, and then the membranes were washed. After washing, the blots were incubated with the corresponding secondary antibodies conjugated with HPR or ABC reagent (Vector Laboratories) for AOL blot. Finally, specific proteins were visualized by using an ECL system (Amersham).
Cell surface biotinylation and immunoprecipitation
The cells were surface labeled by a sulfosuccinimidobiotin (sulfo-NHS-biotin) (Pierce Chemical Co., Rockford, IL) procedure (Miyake et al. 1991). Briefly, after three washes, the cells were suspended in PBS with 0.2 mg/mL of sulfo-NHS-biotin. After 1 h of incubation at 4°C with occasional shaking, the cells were washed three times with chilled PBS. The cell lysates were prepared in lysis buffer containing 50 mM Tris–HCI, 150 mM NaCl, 1% Triton X-100, 2 mM MgC12, and 2 mM CaC12, with a protease inhibitor (Nacalai Tesque, Kyoto, Japan) added. After centrifugation, the lysates were immunoprecipitated with the corresponding antibodies, followed by protein G-Sepharose (GE healthcare, NJ) at 4°C with rotation overnight. After washing three times in lysis buffer, the bound proteins were released by boiling for 5 min in a sample buffer with or without 2-ME. After centrifugation, the supernatants were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed a Western blot, as indicated earlier.
Cell adhesion assay
The cell adhesion assay was performed as described by van Kessel (van Kessel et al. 1994), with minor modifications. Briefly, stromal cells were plated in a 96-well plate at 2 x 104 cells/well and were allowed to grow overnight before the adhesion assay. The BM pre-B cells were then labeled by 10 µM of 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) for 15 min at 37°C. After labeling, the cells were washed three times and resuspended in RPMI 1640 medium. The labeled cells (5 x 104 cells/well) were added to the stromal cell layer and incubated for 60 min at 37°C. Unbound cells were removed by the addition and removal of prewarmed PBS three times. In each set of experiments, a separate plate containing known numbers of labeled cells was prepared for the determination of a standard curve of fluorescence units per cell. The fluorescence of the adherent cells was quantitated using a fluorescent plate reader (Molecular Device Corp., Sunnyvale, CA) at excitation and emission wavelengths of 485 and 525 nm, respectively. All determinations were in triplicate and the percent adhesion was then calculated as the percentage of bound cells in the plate after washing. Antibodies were added at the same time as the labeled BM pre-B cells. The recombinant mouse VCAM-1/Fc chimera (10 µg/mL PBS with 50 µL/well) (R&D systems, Inc., Minneapolis, MN) was coated on to a 96-well plate at 4°C overnight. After incubation, the plate was washed twice with PBS and then blocked with 1% BSA in PBS for 2 h at 37°C. BM pre-B cells labeled with CFSE were added to the 96-well plate coated VCAM-1/Fc chimera and BSA. After 2 h incubation at 37°C, nonadherent cells were removed by the addition and removal of prewarmed PBS twice. Measurement of fluorescence was done with the fluorescence plate reader as above. Antibodies were added at the same time as the labeled BM cells.
Real-time PCR
Total RNA was prepared from sorted cells using the TRIzol reagent (Invitrogen, Carlsbad, CA), following the protocol recommended by the manufacturer. The real-time PCR analyses were performed by using a Smart Cycler II System (Cephied, Sunnyvale, CA). The cDNA synthesis was performed using SYBR Green Real-time PCR Core Kit (Takara Bio. Inc.) as recommended by manufacturer. Each reaction was performed in a volume of 25 µL, with a final concentration of 1 x SYBR Premix Ex Taq, 200 nM primers, 2 µL of 1:10 dilution of the cDNA, and RNase free water. The thermal cycling conditions for the real-time PCR were 10 s at 95°C to active SYBR Ex Taq, followed by 40 cycles of denaturation for 5 s at 95°C and annealing/extension for 20 s at 60°C. The mean number of cycles for the threshold of fluorescence detection was calculated for each sample, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was quantified to normalize the amount of cDNA in each sample. The specificity of the amplified products was monitored by its melting curve. The sequences of the real-time PCR primers (Hu et al. 2006) are shown in Table I.
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Apoptosis
We used the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay performed with a DeadEndTM Colorimetric TUNEL System (Promega, Madison, WI) as per the manufacturer's recommendations.
Statistical analysis
The results are expressed as mean value ± standard deviation (SD). Statistical analyses were carried out by using the Student's t-test. A P value of less than 0.05 was considered statistically significant.
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Conflict of interests |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interestsReferences |
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None declared.
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Acknowledgements |
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This work was supported by Core Research for Evolutional Science and Technology (CREST) and the 21st Century Center of Excellence (COE) Program from the Ministry of Education, Science, Culture, Sports, and Technology of Japan, and by the Japan Society for the Promotion of Science (JSPS) Core-to-Core program.
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Abbreviations |
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4β1 integrin, very late antigen 4; AOL, Aspergillus oryzae lectin; β4GalT-I, β1,4-galactosyltransferase I; BCR, B cell receptor; BM, bone marrow; FUT8, 1,6-fucosyltransferase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GnT III, N-acetylglucosaminyltransferase III; KD, knockdown; PBS, phosphate-buffered saline; siRNA, short interfering RNA; VCAM-1, vascular cell adhesion molecule 1 |
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