The CUG-translated WT1, not AUG-WT1, is an oncogene.

The Wilms' tumor 1 (WT1) gene is believed to act as a canonical tumor suppressor. However, it has also been reported to function as an oncogene. Germline WT1 deletion is associated with Wilms' tumor, and exogenous WT1 cDNA introduction into cells induces the transcriptional suppression of its oncogenic target genes. In contrast, high WT1 expression is associated with poor prognosis in patients with various cancers. Why WT1 acts as a tumor suppressor under certain conditions but as an oncogene under other conditions is unknown. Here, we report that CUG initiation site for WT1 protein synthesis (CUG)-translated WT1 (cugWT1), an N-terminally extended form of canonical AUG initiation site for WT1 protein synthesis (AUG)-translated WT1 (augWT1), was overexpressed in most cancer cell lines and cancer tissues and functioned as an oncogene, whereas the classical augWT1 acted as a tumor suppressor as reported previously and inhibited the function of cugWT1. Translation of cugWT1 is initiated from a CUG codon upstream and in-frame with the coding region of augWT1. cugWT1 induced cell transformation and increased the gene expression of c-myc, bcl-2 and egfr, whereas overexpression of augWT1 repressed colony formation of cancer cells and inhibited the expression of the same target genes by recruiting histone deacetylase 1 (HDAC1). In addition, we found that protein kinase B (AKT)-phosphorylated cugWT1 on Ser62 and protected cugWT1 from proteasomal degradation induced by the F-box/WD repeat-containing protein 8 (FBXW8). These results provide an important breakthrough in the field of cancer biology and contribute significantly to the resolution of the chameleon function of WT1.

Polycomb (PcG) proteins, BMI1 and SUZ12, regulate arsenic-induced cell transformation.

Inorganic arsenic is a well-documented human carcinogen associated with cancers of the skin, lung, liver, and bladder. However, the underlying mechanisms explaining the tumorigenic role of arsenic are not well understood. The present study explored a potential mechanism of cell transformation induced by arsenic exposure. Exposure to a low dose (0.5 µm) of arsenic trioxide (As(2)O(3)) caused transformation of BALB/c 3T3 cells. In addition, in a xenograft mouse model, tumor growth of the arsenic-induced transformed cells was dramatically increased. In arsenic-induced transformed cells, polycomb group (PcG) proteins, including BMI1 and SUZ12, were activated resulting in enhanced histone H3K27 tri-methylation levels. On the other hand, tumor suppressor p16(INK4a) and p19(ARF) mRNA and protein expression were dramatically suppressed. Introduction of small hairpin (sh) RNA-BMI1 or -SUZ12 into BALB/c 3T3 cells resulted in suppression of arsenic-induced transformation. Histone H3K27 tri-methylation returned to normal in BMI1- or SUZ12-knockdown BALB/c 3T3 cells compared with BMI1- or SUZ12-wildtype cells after arsenic exposure. As a consequence, the expression of p16(INK4a) and p19(ARF) was recovered in arsenic-treated BMI1- or SUZ12-knockdown cells. Thus, arsenic-induced cell transformation was blocked by inhibition of PcG function. Taken together, these results strongly suggest that the polycomb proteins, BMI1 and SUZ12 are required for cell transformation induced by organic arsenic exposure.

Radicicol Inhibits iNOS Expression in Cytokine-Stimulated Pancreatic Beta Cells.

Here, we show that radicicol, a fungal antibiotic, resulted in marked inhibition of inducible nitric oxide synthase (iNOS) transcription by the pancreatic beta cell line MIN6N8a in response to cytokine mixture (CM: TNF-α, IFN-γ, and IL-1ß). Treatment of MIN6N8a cells with radicicol inhibited CM-stimulated activation of NF-κB/Rel, which plays a critical role in iNOS transcription, in a dose-related manner. Nitrite production in the presence of PD98059, a specific inhibitor of the extracellular signal-regulated protein kinase-1 and 2 (ERK1/2) pathway, was dramatically diminished, suggesting that the ERK1/2 pathway is involved in CM-induced iNOS expression. In contrast, SB203580, a specific inhibitor of p38, had no effect on nitrite generation. Collectively, this series of experiments indicates that radicicol inhibits iNOS gene expression by blocking ERK1/2 signaling. Due to the critical role that NO release plays in mediating destruction of pancreatic beta cells, the inhibitory effects of radicicol on iNOS expression suggest that radicicol may represent a useful anti-diabetic activity.

Synergistic Induction of iNOS by IFN-γ and Glycoprotein Isolated from Dioscorea batatas.

Dioscorea species continue to be used in traditional Chinese medicine, and represent a major source of steroid precursors for conventional medicine. In the previous study, We isolated glycoprotein (GDB) from Dioscorea batatas, characterized, and demonstrated immunostimulating activity in C57BL/6 mice. The aim of this study was to investigate the mechanism whereby GDB activates macrophages. Macrophages activation by GDB was investigated by analyzing the effects of GDB on nitric oxide (NO) production, iNOS expression, mitogen activated protein kinase (MAPK) phosphorylation, and transcription factor activation. In the presence of IFN-γ, GDB strongly stimulated macrophages to express iNOS and produce NO. Furthermore, the activation of p38 was synergistically induced by GDB plus IFN-γ , but SB203580 (a p38 inhibitor) inhibited GDB plus IFN-γ-induced p38 activation. This study indicates that GDB is an important activator of macrophages. Furthermore, due to the critical role that macrophage activation plays in innate immune response, the activation effects of GDB on macrophages suggest that GDB may be a useful immunopotentiating agent.

Role of NEK6 in tumor promoter-induced transformation in JB6 C141 mouse skin epidermal cells.

NEK6 (NIMA-related kinase 6) is a homologue of the Aspergillus nidulans protein NIMA (never in mitosis, gene A). We demonstrate that overexpression of NEK6 induces anchorage-independent transformation of JB6 Cl41 mouse epidermal cells. Tissue arrays and Western immunoblot analysis show that NEK6 is overexpressed in malignant tissues and several cancer cell lines. Our data also show that NEK6 interacts with STAT3, an oncogenic transcription factor, and phosphorylates STAT3 on Ser(727), which is important for transcriptional activation. Additional studies using NEK6 mutants suggested that the phosphorylation on both Ser(206) and Thr(210) of NEK6 is critical for STAT3 phosphorylation and anchorage-independent transformation of mouse epidermal cells. Notably, knockdown of NEK6 decreased colony formation and STAT3 Ser(727) phosphorylation. Based on our findings, the most likely mechanism that can account for this biological effect involves the activation of STAT3 through the phosphorylation on Ser(727). Because of the critical role that STAT3 plays in mediating oncogenesis, the stimulatory effects of NEK6 on STAT3 and cell transformation suggest that this family of serine/threonine kinases might represent a novel chemotherapeutic target.

Phosphorylation of Sox2 cooperates in reprogramming to pluripotent stem cells.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by transduction of reprogramming factors, including Oct4, Sox2, Klf4, and c-Myc. A coordinated network of these factors was suggested to confer a pluripotency of iPSCs. Together with Oct4, Sox2 plays a major role as a master regulator in ESCs. However, the underlying mechanisms by which Sox2 contributes to self-renewal or reprogramming processes remain to be determined. Here, we provide new evidence for a phosphorylation-based regulation of Sox2 activity. Akt directly interacts with Sox2 and promotes its stabilization through phosphorylation at Thr118, which enhances the transcriptional activity of Sox2 in ESCs. Moreover, phosphorylation of Sox2 cooperates in the reprogramming of mouse embryonic fibroblasts by enabling more efficient induction of iPSCs. Overall, our studies provide new insights into the regulatory mechanism of Sox2 in ESCs and also provide a direct link between phosphorylation events and somatic cell reprogramming.

Phenotype of a calbindin-D9k gene knockout is compensated for by the induction of other calcium transporter genes in a mouse model.

UNLABELLED: CaBP-9k may be involved in the active calcium absorption and embryo implantation. Although we generated CaBP-9k KO mice to explore its function, no distinct phenotypes were observed in these KO mice. It can be hypothesized that TRPV5 and 6 and plasma membrane calcium ATPase 1b may play a role in the regulation of calcium transport to compensate CaBP-9k deficiency in its KO model. INTRODUCTION: Active calcium transport in the duodenum and kidney is carried in three steps: calcium entry through epithelial Ca2+ channels (TRPV5 and TRPV6), buffering and/or transport by calbindin-D9k (CaBP-9k) and -D28k (CaBP-28k), and extrusion through the plasma membrane calcium ATPase 1b (PMCA1b) and sodium/calcium exchanger 1. Although the molecular mechanism of calcium absorption has been studied using knockouts (KOs) of the vitamin D receptor and CaBP-28k in animals, the process is not fully understood. MATERIALS AND METHODS: We generated CaBP-9k KO mice and assessed the phenotypic characterization and the molecular regulation of active calcium transporting genes when the mice were fed different calcium diets during growth. RESULTS: General phenotypes showed no distinct abnormalities. Thus, the active calcium transport of CaBP-9k-null mice proceeded normally in this study. Therefore, the compensatory molecular regulation of this mechanism was elucidated. Duodenal TRPV6 and CaBP-9k mRNA of wildtype (WT) mice increased gradually during preweaning. CaBP-9k is supposed to be an important factor in active calcium transport, but its role is probably compensated for by other calcium transporter genes (i.e., intestinal TRPV6 and PMCA1b) during preweaning and renal calcium transporters in adult mice. CONCLUSIONS: Depletion of the CaBP-9k gene in a KO mouse model had little phenotypic effect, suggesting that its depletion may be compensated for by calcium transporter genes in the intestine of young mice and in the kidney of adult mice.

Serine 347 Phosphorylation by JNKs Negatively Regulates OCT4 Protein Stability in Mouse Embryonic Stem Cells.

The POU transcription factor OCT4 is critical for maintaining the undifferentiated state of embryonic stem cells (ESCs) and generating induced pluripotent stem cells (iPSCs), but its precise mechanisms of action remain poorly understood. Here, we investigated the role of OCT4 phosphorylation in the biological functions of ESCs. We observed that c-Jun N-terminal kinases (JNKs) directly interacted with and phosphorylated OCT4 at serine 347, which inhibited the transcriptional activity of OCT4. Moreover, phosphorylation of OCT4 induced binding of FBXW8, which reduced OCT4 protein stability and enhanced its proteasomal degradation. We also found that the mutant OCT4 (S347A) might delay the differentiation process of mouse ESCs and enhance the efficiency of generating iPSCs. These results demonstrated that OCT4 phosphorylation on serine 347 by JNKs plays an important role in its stability, transcriptional activities, and self-renewal of mouse ESCs.

Macrophage activation by polysaccharide fraction isolated from Salicornia herbacea.

We demonstrate that polysaccharides isolated from Salicornia herbacea (Salicornia polysaccharides, SPS) significantly induces nitric oxide (NO) production and inducible NO synthase (iNOS) transcription through the activation of nuclear factor-kappaB/Rel (NF-kappaB/Rel). SPS dose-dependently induced the production of NO in isolated mouse peritoneal macrophages and RAW 264.7, a mouse macrophage-like cell line. Moreover, iNOS gene expression was strongly induced by SPS in RAW 264.7 cells. To further investigate the mechanism responsible for the induction of iNOS gene expression, we investigated the effect of SPS on the activation of transcription factors including NF-kappaB/Rel and Oct, whose binding sites were located in the promoter of iNOS gene. Treatment of RAW 264.7 cells with SPS produced strong induction of NF-kappaB/Rel-dependent reporter gene expression, whereas Oct-dependent gene expression was not affected by SPS. Nuclear translocation and DNA binding activity of NF-kappaB/Rel was significantly induced by SPS. The treatment with NF-kappaB SN50, an inhibitor of NF-kappaB/Rel nuclear translocation, effectively inhibited the activation of NF-kappaB/Rel binding complexes and NO production. In conclusion, we demonstrate that SPS stimulates macrophages to express iNOS gene through the activation of NF-kappaB/Rel.

KIOM-79 inhibits LPS-induced iNOS gene expression by blocking NF-kappaB/Rel and p38 kinase activation in murine macrophages.

We demonstrate that KIOM-79, combined extracts obtained from Magnolia officinalis, Pueraria lobata, Glycyrrhiza uralensis, and Euphorbia pekinensis, inhibits LPS-induced expression of iNOS gene in RAW 264.7 cells. Treatment of RAW 264.7 cells with KIOM-79 inhibited LPS-stimulated nitric oxide production in a dose-related manner. Immunohisto-chemical staining of iNOS and RT-PCR analysis showed that the decrease of NO was due to the inhibition of iNOS gene expression. Immunostaining of p65, EMSA, and reporter gene assay showed that KIOM-79 inhibited NF-kappa/Rel nuclear translocation, DNA binding, and transcriptional activation, respectively. Western immunoblot analysis of p38 kinase showed KIOM-79 significantly inhibited the phosphoylation of p38 kinase which is important in the regulation of iNOS gene expression. Collectively, this series of experiments indicates that KIOM inhibits iNOS gene expression by blocking NF-kappa/Rel and p38 kinase signaling. Due to the critical role that NO release plays in mediating inflammatory responses, the inhibitory effects of KIOM-79 on iNOS suggest that KIOM-79 may represent a useful anti-inflammatory agent.

A natural small molecule, catechol, induces c-Myc degradation by directly targeting ERK2 in lung cancer.

Various carcinogens induce EGFR/RAS/MAPK signaling, which is critical in the development of lung cancer. In particular, constitutive activation of extracellular signal-regulated kinase 2 (ERK2) is observed in many lung cancer patients, and therefore developing compounds capable of targeting ERK2 in lung carcinogenesis could be beneficial. We examined the therapeutic effect of catechol in lung cancer treatment. Catechol suppressed anchorage-independent growth of murine KP2 and human H460 lung cancer cell lines in a dose-dependent manner. Catechol inhibited ERK2 kinase activity in vitro, and its direct binding to the ERK2 active site was confirmed by X-ray crystallography. Phosphorylation of c-Myc, a substrate of ERK2, was decreased in catechol-treated lung cancer cells and resulted in reduced protein stability and subsequent down-regulation of total c-Myc. Treatment with catechol induced G1 phase arrest in lung cancer cells and decreased protein expression related to G1-S progression. In addition, we showed that catechol inhibited the growth of both allograft and xenograft lung cancer tumors in vivo. In summary, catechol exerted inhibitory effects on the ERK2/c-Myc signaling axis to reduce lung cancer tumor growth in vitro and in vivo, including a preclinical patient-derived xenograft (PDX) model. These findings suggest that catechol, a natural small molecule, possesses potential as a novel therapeutic agent against lung carcinogenesis in future clinical approaches.

Macrophage activation by polysaccharide isolated from Astragalus membranaceus.

We show that APS, a polysaccharide isolated from the roots of Astragalus membranaceus, significantly induces nitric oxide (NO) production and inducible NO synthase (iNOS) transcription through the activation of nuclear factor-kappaB/Rel (NF-kappaB/Rel). In vivo administration of APS induced NO production by peritoneal macrophages of B6C3F1 mice. APS also dose-dependently induced the production of NO in isolated mouse peritoneal macrophages and RAW 264.7, a mouse macrophage-like cell line. Moreover, iNOS protein and mRNA transcription were strongly induced by APS in RAW 264.7 cells. To further investigate the mechanism responsible for the induction of iNOS gene expression, we investigated the effect of APS on the activation of transcription factors including NF-kappaB/Rel and Oct, whose binding sites were located in the promoter of iNOS gene. Treatment of RAW 264.7 cells with APS produced strong induction of NF-kappaB/Rel-dependent reporter gene expression, whereas Oct-dependent gene expression was not affected by APS. Nuclear translocation and DNA binding activity of NF-kappaB/Rel was significantly induced by APS. The treatment with NF-kappaB SN50, an inhibitor of NF-kappaB/Rel nuclear translocation, effectively inhibited the activation of NF-kappaB/Rel binding complexes and NO production. In conclusion, we demonstrate that APS stimulates macrophages to express iNOS gene through the activation of NF-kappaB/Rel.

Transcriptional regulation of the mouse calbindin-D9k gene by the ovarian sex hormone.

Calbindin-D9k levels in the rat uterus are under the control of estrogen. We found that the putative estrogen response element (ERE) failed to bind to the estrogen receptor from the mouse uterus. We therefore isolated mouse genomic clones of the calbindin-D9K gene and analyzed their expression in the mouse uterus. The promoter region of the gene contained several putative steroid hormone receptor binding sites. To characterize these elements, we constructed several promoter-reporter plasmids, and transiently transfected them into T47D breast cancer cells that express both estrogen and progesterone receptors. Luciferase activity was expressed from a promoter region containing the putative progesterone response element (PRE) and expression was stimulated by progesterone. In the uterus of oophorectomized mice, the calbindin-D9k gene was up-regulated by progesterone, but not by estrogen. These results suggest that the mouse uterine calbindin-D9k gene is expressed under the control of a PRE.

Polysaccharide isolated from Poria cocos sclerotium induces NF-kappaB/Rel activation and iNOS expression in murine macrophages.

We show that PCSC, a polysaccharide isolated from the sclerotium of Poria cocos with 1% sodium carbonate, significantly induces nitric oxide (NO) production and inducible NO synthase (iNOS) transcription through the activation of nuclear factor-kappaB/Rel (NF-kappaB/Rel). In vivo administration of PCSC induced NO production by peritoneal macrophages of B6C3F1 mice. PCSC also dose-dependently induced the production of NO in isolated mouse peritoneal macrophages and RAW 264.7, a murine macrophage-like cell line. Moreover, iNOS protein and mRNA transcription were strongly induced by PCSC in RAW 264.7 cells. To further investigate the mechanism responsible for the induction of iNOS gene expression, we investigated the effect of PCSC on the activation of transcription factors including NF-kappaB/Rel and Oct, whose binding sites were located in the promoter of iNOS gene. Treatment of RAW 264.7 cells with PCSC produced strong induction of NF-kappaB/Rel-dependent reporter gene expression, whereas Oct-dependent gene expression was not affected by PCSC. DNA binding activity of NF-kappaB/Rel was significantly induced by PCSC, and this effect was mediated through the degradation of IkappaBalpha. In conclusion, we demonstrate that PCSC stimulates macrophages to express iNOS gene through the activation of NF-kappaB/Rel.

Polysaccharide isolated from Poria cocos sclerotium induces NF-kappaB/Rel activation and iNOS expression through the activation of p38 kinase in murine macrophages.

In our previous studies, we showed that PCSC, a polysaccharide isolated from Poria cocos, activated macrophages to induce the translocation of NF-kappaB/Rel into nucleus and DNA binding to its cognate site in the promoter of iNOS gene [Int. Immunopharmacol. 3 (2003) 1353]. In the present study, we investigated the role of p38 kinase pathway and membrane receptors (CD14, Toll-like receptor 4 (TLR4), and CR3) in mediating nitric oxide (NO) production and NF-kappaB/Rel activation induced by PCSC. Treament of RAW 264.7 cells with PCSC resulted in significant activation of p38. The specific p38 inhibitor SB203580 abrogated the PCSC-induced NF-kappaB/Rel activation and NO generation, whereas the selective mitogen-activated protein kinase/extracellular signal-regulated kinase 1 (MEK-1) inhibitor PD98059 did not affect the NF-kappaB/Rel and NO induction. Treatment of RAW 264.7 cells with anti-CD14 Ab, anti-TLR4 Ab, and anti-CR3 Absignificantly blocked PCSC-induced NO production activation. In conclusion, we demonstrate that PCSC induces NF-kappaB/Rel activation and iNOS expression through the CD14, TLR4, and CR3 membrane receptor and p38 kinase which is critically involved in the signal transduction leading to NF-kappaB/Rel activation in murine macrophages.

JNK1 and 2 play a negative role in reprogramming to pluripotent stem cells by suppressing Klf4 activity.

Embryonic stem (ES) cells are pluripotent cells with the capacity for unlimited self-renewal or differentiation. Inhibition of MAPK pathways enhances mouse ES cell pluripotency characteristics. Compared to wildtype ES cells, jnk2(-/-) ES cells displayed a much higher growth rate. To determine whether JNKs are required for stem cell self-renewal or differentiation, we performed a phosphorylation kinase array assay to compare mouse ES cells under LIF+ or LIF- culture conditions. The data showed that activation of JNKs was induced by LIF withdrawal. We also found that JNK1 or 2 phosphorylated Klf4 at threonines 224 and 225. Activation of JNK signaling and phosphorylation of Klf4 inhibited Klf4 transcription and transactivation activity. Importantly, jnk1(-/-) and jnk2(-/-) murine embryonic fibroblasts (MEFs) exhibited a significantly greater potency in the ability to increase the number of iPS colonies compared with jnk wildtype MEFs. Overall, our results demonstrated that JNK1 and 2 play a negative role in reprogramming to pluripotent stem cells by suppressing Klf4 activity.

Autophagy and cellular senescence mediated by Sox2 suppress malignancy of cancer cells.

Autophagy is a critical cellular process required for maintaining cellular homeostasis in health and disease states, but the molecular mechanisms and impact of autophagy on cancer is not fully understood. Here, we found that Sox2, a key transcription factor in the regulation of the "stemness" of embryonic stem cells and induced-pluripotent stem cells, strongly induced autophagic phenomena, including intracellular vacuole formation and lysosomal activation in colon cancer cells. The activation occurred through Sox2-mediated ATG10 gene expression and resulted in the inhibition of cell proliferation and anchorage-independent colony growth ex vivo and tumor growth in vivo. Further, we found that Sox2-induced-autophagy enhanced cellular senescence by up-regulating tumor suppressors or senescence factors, including p16(INK4a), p21 and phosphorylated p53 (Ser15). Notably, knockdown of ATG10 in Sox2-expressing colon cancer cells restored cancer cell properties. Taken together, our results demonstrated that regulation of autophagy mediated by Sox2 is a mechanism-driven novel strategy to treat human colon cancers.

ERK1 and ERK2 regulate embryonic stem cell self-renewal through phosphorylation of Klf4.

Understanding and controlling the mechanism by which stem cells balance self-renewal versus differentiation is of great importance for stem cell therapeutics. Klf4 promotes the self-renewal of embryonic stem cells, but the precise mechanism regulating this role of Klf4 is unclear. We found that ERK1 or ERK2 binds the activation domain of Klf4 and directly phosphorylates Klf4 at Ser123. This phosphorylation suppresses Klf4 activity, inducing embryonic stem cell differentiation. Conversely, inhibition of Klf4 phosphorylation enhances Klf4 activity and suppresses embryonic stem cell differentiation. Notably, phosphorylation of Klf4 by ERKs causes recruitment and binding of the F-box proteins ßTrCP1 or ßTrCP2 (components of an ubiquitin E3 ligase) to the Klf4 N-terminal domain, which results in Klf4 ubiquitination and degradation. Overall, our data provide a molecular basis for the role of ERK1 and ERK2 in regulating Klf4-mediated mouse embryonic stem cell self-renewal.

Magnolol Inhibits LPS-induced NF-κB/Rel Activation by Blocking p38 Kinase in Murine Macrophages.

This study demonstrates the ability of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, to inhibit LPS-induced expression of iNOS gene and activation of NF-κB/Rel in RAW 264.7 cells. Immunohisto-chemical staining of iNOS and Western blot analysis showed magnolol to inhibit iNOS gene expression. Reporter gene assay and electrophoretic mobility shift assay showed that magnolol inhibited NF-κB/Rel transcriptional activation and DNA binding, respectively. Since p38 is important in the regulation of iNOS gene expression, we investigated the possibility that magnolol to target p38 for its anti-inflammatory effects. A molecular modeling study proposed a binding position for magnolol that targets the ATP binding site of p38 kinase (3GC7). Direct interaction of magnolol and p38 was further confirmed by pull down assay using magnolol conjugated to Sepharose 4B beads. The specific p38 inhibitor SB203580 abrogated the LPS-induced NF-κB/Rel activation, whereas the selective MEK-1 inhibitor PD98059 did not affect the NF-κB/Rel. Collectively, the results of the series of experiments indicate that magnolol inhibits iNOS gene expression by blocking NF-κB/Rel and p38 kinase signaling.

Oriented immobilization of antibodies with GST-fused multiple Fc-specific B-domains on a gold surface.

We have constructed a novel platform for the oriented buildup of immunoglobulins on a gold surface for a surface plasmon resonance imaging microarray. To this end, genetically engineered glutathione S-transferase proteins bearing one, two, and three Fc-specific B-domains in protein G from Streptococci (GST-GB1, -GB2, and -GB3, respectively) were produced. In order to tether these GST-GBx proteins specifically, a novel glutathione-derivatized ligand (LA-GSH) was also synthesized from a biaminated tri(ethylene glycol) backbone. Each end of the backbone was further functionalized with a maleimide group for a glutathione modification and a lipoic acid for surface immobilization. The glutathione ligand demonstrated a negligible nonspecific protein adsorption toward other spectator proteins while showing a strong specific association toward GST-GBx proteins. This Fc-specific surface exhibited at least a 2-fold enhancement in the immunoglobulin density (from human and mouse) with its antigen capture capability totally conserved compared to a covalently tethered GBx proteins. A single antibody tethered on the GST-GB3 is estimated to capture two antigens (enhanced green fluorescent protein), and this antigen capture ratio seems to be the most efficient value ever observed.