Publications by authors named "Tan-chi Fan"

17 Publications

  • Page 1 of 1

Sialylation of CD55 by ST3GAL1 Facilitates Immune Evasion in Cancer.

Cancer Immunol Res 2021 01 11;9(1):113-122. Epub 2020 Nov 11.

Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan.

Altered glycosylations, which are associated with expression and activities of glycosyltransferases, can dramatically affect the function of glycoproteins and modify the behavior of tumor cells. ST3GAL1 is a sialyltransferase that adds sialic acid to core 1 glycans, thereby terminating glycan chain extension. In breast carcinomas, overexpression of ST3GAL1 promotes tumorigenesis and correlates with increased tumor grade. In pursuing the role of ST3GAL1 in breast cancer using -siRNA to knockdown , we identified CD55 to be one of the potential target proteins of ST3GAL1. CD55 is an important complement regulatory protein, preventing cells from complement-mediated cytotoxicity. CD55 had one -linked glycosylation site in addition to a Ser/Thr-rich domain, which was expected to be heavily -glycosylated. Detailed analyses of - and -linked oligosaccharides of CD55 released from scramble or siRNA-treated breast cancer cells by tandem mass spectrometry revealed that the -glycan profile was not affected by silencing. The -glycan profile of CD55 demonstrated a shift in abundance to nonsialylated core 1 and monosialylated core 2 at the expense of the disialylated core 2 structure after silencing. We also demonstrated that -linked desialylation of CD55 by silencing resulted in increased C3 deposition and complement-mediated lysis of breast cancer cells and enhanced sensitivity to antibody-dependent cell-mediated cytotoxicity. These data demonstrated that ST3GAL1-mediated -linked sialylation of CD55 acts like an immune checkpoint molecule for cancer cells to evade immune attack and that inhibition of ST3GAL1 is a potential strategy to block CD55-mediated immune evasion.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0203DOI Listing
January 2021

Comprehensive Cohort Analysis of Mutational Spectrum in Early Onset Breast Cancer Patients.

Cancers (Basel) 2020 Jul 28;12(8). Epub 2020 Jul 28.

Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan.

Early onset breast cancer (EOBC), diagnosed at age ~40 or younger, is associated with a poorer prognosis and higher mortality rate compared to breast cancer diagnosed at age 50 or older. EOBC poses a serious threat to public health and requires in-depth investigation. We studied a cohort comprising 90 Taiwanese female patients, aiming to unravel the underlying mechanisms of EOBC etiopathogenesis. Sequence data generated by whole-exome sequencing (WES) and whole-genome sequencing (WGS) from white blood cell (WBC)-tumor pairs were analyzed to identify somatic missense mutations, copy number variations (CNVs) and germline missense mutations. Similar to regular breast cancer, the key somatic mutation-susceptibility genes of EOBC include (40% prevalence), (37%), (17%) and (17%), which are frequently reported in breast cancer; however, the structural protein-coding genes (19%), (16%) and (11%) show a significantly higher prevalence in EOBC. Furthermore, the top 2 genes harboring EOBC germline mutations, (19%) and (19%), encode structural proteins. Compared to conventional breast cancer, an unexpectedly higher number of EOBC susceptibility genes encode structural proteins. We suspect that mutations in structural proteins may increase physical permeability to environmental hormones and carcinogens and cause breast cancer to occur at a young age.
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http://dx.doi.org/10.3390/cancers12082089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464007PMC
July 2020

Sialylation of vasorin by ST3Gal1 facilitates TGF-β1-mediated tumor angiogenesis and progression.

Int J Cancer 2019 04 3;144(8):1996-2007. Epub 2019 Jan 3.

Institute of Stem Cell and Translational Cancer Research, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Taoyuan, Taiwan.

ST3Gal1 is a key sialyltransferase which adds α2,3-linked sialic acid to substrates and generates core 1 O-glycan structure. Upregulation of ST3Gal1 has been associated with worse prognosis of breast cancer patients. However, the protein substrates of ST3Gal1 implicated in tumor progression remain elusive. In our study, we demonstrated that ST3GAL1-silencing significantly reduced tumor growth along with a notable decrease in vascularity of MCF7 xenograft tumors. We identified vasorin (VASN) which was shown to bind TGF-β1, as a potential candidate that links ST3Gal1 to angiogenesis. LC-MS/MS analysis of VASN secreted from MCF7, revealed that more than 80% of its O-glycans are sialyl-3T and disialyl-T. ST3GAL1-silencing or desialylation of VASN by neuraminidase enhanced its binding to TGF-β1 by 2- to 3-fold and thereby dampening TGF-β1 signaling and angiogenesis, as indicated by impaired tube formation of HUVECs, suppressed angiogenesis gene expression and reduced activation of Smad2 and Smad3 in HUVEC cells. Examination of 114 fresh primary breast cancer and their adjacent normal tissues showed that the expression levels of ST3Gal1 and TGFB1 were high in tumor part and the expression of two genes was positively correlated. Kaplan Meier survival analysis showed a significantly shorter relapse-free survival for those with lower expression VASN, notably, the combination of low VASN with high ST3GAL1 yielded even higher risk of recurrence (p = 0.025, HR = 2.967, 95% CI = 1.14-7.67). Since TGF-β1 is known to transcriptionally activate ST3Gal1, our findings illustrated a feedback regulatory loop in which TGF-β1 upregulates ST3Gal1 to circumvent the negative impact of VASN.
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http://dx.doi.org/10.1002/ijc.31891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590135PMC
April 2019

Reciprocal feedback regulation of ST3GAL1 and GFRA1 signaling in breast cancer cells.

Cancer Lett 2018 10 21;434:184-195. Epub 2018 Jul 21.

Institute of Stem Cell and Translational Cancer Research, Chang Gung Memorial Hospital at Linkou, Taoyuan, Taiwan; Genomics Research Center, Academia Sinica, Taipei, Taiwan; Department of Pediatrics/Hematology Oncology, University of California in San Diego, San Diego, CA, USA. Electronic address:

GFRA1 and RET are overexpressed in estrogen receptor (ER)-positive breast cancers. Binding of GDNF to GFRA1 triggers RET signaling leading to ER phosphorylation and estrogen-independent transcriptional activation of ER-dependent genes. Both GFRA1 and RET are membrane proteins which are N-glycosylated but no O-linked sialylation site on GFRA1 or RET has been reported. We found GFRA1 to be a substrate of ST3GAL1-mediated O-linked sialylation, which is crucial to GDNF-induced signaling in ER-positive breast cancer cells. Silencing ST3GAL1 in breast cancer cells reduced GDNF-induced phosphorylation of RET, AKT and ERα, as well as GDNF-mediated cell proliferation. Moreover, GDNF induced transcription of ST3GAL1, revealing a positive feedback loop regulating ST3GAL1 and GDNF/GFRA1/RET signaling in breast cancers. Finally, we demonstrated ST3GAL1 knockdown augments anti-cancer efficacy of inhibitors of RET and/or ER. Moreover, high expression of ST3GAL1 was associated with poor clinical outcome in patients with late stage breast cancer and high expression of both ST3GAL1 and GFRA1 adversely impacted outcome in those with high grade tumors.
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http://dx.doi.org/10.1016/j.canlet.2018.07.026DOI Listing
October 2018

Analysis of microbial sequences in plasma cell-free DNA for early-onset breast cancer patients and healthy females.

BMC Med Genomics 2018 02 13;11(Suppl 1):16. Epub 2018 Feb 13.

Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Nankang District, Taipei, 115, Taiwan.

Background: Cell-free circulating DNA (cfDNA) is becoming a useful biopsy for noninvasive diagnosis of diseases. Microbial sequences in plasma cfDNA may provide important information to improve prognosis and treatment. We have developed a stringent method to identify microbial species via microbial cfDNA in the blood plasma of early-onset breast cancer (EOBC) patients and healthy females. Empirically, microbe-originated sequence reads were identified by mapping non-human PE reads in cfDNA libraries to microbial databases. Those mapped concordantly to unique microbial species were assembled into contigs, which were subsequently aligned to the same databases. Microbial species uniquely aligned were identified and compared across all individuals on MCRPM (Microbial CfDNA Reads Per Million quality PE reads) basis.

Results: The predominant microbial cfDNAs in all plasma samples examined are originated from bacteria and these bacteria were limited to only a few genera. Among those, Acinetobacter johnsonii XBB1 and low levels of Mycobacterium spp. were commonly found in all healthy females, but also present in an EOBC patient. Compared to those in healthy counterparts, bacterial species in EOBC patients are more diverse and more likely to present at high levels. Among these three EOBC patients tested, a patient who has record high titer (2,724 MCRPM) of Pseudomonas mendocina together with 8.82 MCRPM of Pannonibacter phragmitetus has passed away; another patient infected by multiple Sphingomonas species remains alive; while the third patient who has similar microbial species (Acinetobacter johnsonii XBB1) commonly seen in normal controls is having a normal life.

Conclusions: Our preliminary data on the profiles of microbial cfDNA sequences suggested that it may have some prognostic value in cancer patients. Validation in larger number of patients is warranted.
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http://dx.doi.org/10.1186/s12920-018-0329-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836824PMC
February 2018

The prognostic significance of RUNX2 and miR-10a/10b and their inter-relationship in breast cancer.

J Transl Med 2014 Sep 30;12:257. Epub 2014 Sep 30.

Background: The major cancer related mortality is caused by metastasis and invasion. It is important to identify genes regulating metastasis and invasion in order to curtail metastatic spread of cancer cells.

Methods: This study investigated the association between RUNX2 and miR-10a/miR-10b and the risk of breast cancer relapse. Expression levels of RUNX2 and miR-10a/b in 108 pairs of tumor and non-tumor tissue of breast cancer were assayed by quantitative PCR analysis and evaluated for their prognostic implications.

Results: The median expression levels of RUNX2 and miR-10b in tumor tissue normalized using adjacent non-tumor tissue were significantly higher in relapsed patients than in relapse-free patients. Higher expression of these three genes were significantly correlated with the hazard ratio for breast cancer recurrence (RUNX2: 3.02, 95% CI = 1.50 ~ 6.07; miR-10a: 2.31, 95% CI = 1.00 ~ 5.32; miR-10b: 3.96, 95% CI = 1.21 ~ 12.98). The joint effect of higher expression of all three genes was associated with a hazard ratio of 12.37 (95% CI = 1.62 ~ 94.55) for relapse. In a breast cancer cell line, RUNX2 silencing reduced the expression of miR-10a/b and also impaired cell motility, while RUNX2 overexpression elicited opposite effects.

Conclusions: These findings indicate that higher expression of RUNX2 and miR-10a/b was associated with adverse outcome of breast cancer. Expression levels of RUNX2 and miR-10a/b individually or jointly are potential prognostic factors for predicting breast cancer recurrence. Data from in vitro studies support the notion that RUNX2 promoted cell motility by upregulating miR-10a/b.
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http://dx.doi.org/10.1186/s12967-014-0257-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4189660PMC
September 2014

Basic amino acid residues of human eosinophil derived neurotoxin essential for glycosaminoglycan binding.

Int J Mol Sci 2013 Sep 16;14(9):19067-85. Epub 2013 Sep 16.

Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 300, Taiwan.

Human eosinophil derived neurotoxin (EDN), a granule protein secreted by activated eosinophils, is a biomarker for asthma in children. EDN belongs to the human RNase A superfamily possessing both ribonucleolytic and antiviral activities. EDN interacts with heparin oligosaccharides and heparin sulfate proteoglycans on bronchial epithelial Beas-2B cells. In this study, we demonstrate that the binding of EDN to cells requires cell surface glycosaminoglycans (GAGs), and the binding strength between EDN and GAGs depends on the sulfation levels of GAGs. Furthermore, in silico computer modeling and in vitro binding assays suggest critical roles for the following basic amino acids located within heparin binding regions (HBRs) of EDN 34QRRCKN39 (HBR1), 65NKTRKN70 (HBR2), and 113NRDQRRD119 (HBR3) and in particular Arg35, Arg36, and Arg38 within HBR1, and Arg114 and Arg117 within HBR3. Our data suggest that sulfated GAGs play a major role in EDN binding, which in turn may be related to the cellular effects of EDN.
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http://dx.doi.org/10.3390/ijms140919067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794821PMC
September 2013

A novel cell-penetrating peptide derived from human eosinophil cationic protein.

PLoS One 2013 4;8(3):e57318. Epub 2013 Mar 4.

Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.

Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP(32-41), derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP(32-41) entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP(32-41) to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP(32-41) internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP(32-41) attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP(32-41) internalization routes. ECP(32-41) was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0057318PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587609PMC
August 2013

Chemoattraction of macrophages by secretory molecules derived from cells expressing the signal peptide of eosinophil cationic protein.

BMC Syst Biol 2012 Aug 20;6:105. Epub 2012 Aug 20.

College of Medicine, China Medical University, Taichung, Taiwan.

Background: Eosinophil cationic protein is a clinical asthma biomarker that would be released into blood, especially gathered in bronchia. The signal peptide of eosinophil cationic protein (ECPsp) plays an important role in translocating ECP to the extracellular space. We previously reported that ECPsp inhibits microbial growth and regulates the expression of mammalian genes encoding tumor growth factor-α (TGF-α) and epidermal growth factor receptor (EGFR).

Results: In the present study, we first generated a DNA microarray dataset, which showed that ECPsp upregulated proinflammatory molecules, including chemokines, interferon-induced molecules, and Toll-like receptors. The levels of mRNAs encoding CCL5, CXCL10, CXCL11, CXCL16, STAT1, and STAT2 were increased in the presence of ECPsp by 2.07-, 4.21-, 7.52-, 2.6-, 3.58-, and 1.67-fold, respectively. We then constructed a functional linkage network by integrating the microarray dataset with the pathway database of Kyoto Encyclopedia of Genes and Genomes (KEGG). Follow-up analysis revealed that STAT1 and STAT2, important transcriptional factors that regulate cytokine expression and release, served as hubs to connect the pathways of cytokine stimulation (TGF-α and EGFR pathways) and inflammatory responses. Furthermore, integrating TGF-α and EGFR with the functional linkage network indicated that STAT1 and STAT2 served as hubs that connect two functional clusters, including (1) cell proliferation and survival, and (2) inflammation. Finally, we found that conditioned medium in which cells that express ECPsp had been cultured could chemoattract macrophages. Experimentally, we also demonstrated that the migration of macrophage could be inhibited by the individual treatment of siRNAs of STAT1 or STAT2. Therefore, we hypothesize that ECPsp may function as a regulator for enhancing the migration of macrophages through the upregulation of the transcriptional factors STAT1 and STAT2.

Conclusion: The increased expression and release of various cytokines triggered by ECPsp may attract macrophages to bronchia to purge damaged cells. Our approach, involving experimental and computational systems biology, predicts pathways and potential biological functions for further characterization of this novel function of ECPsp under inflammatory conditions.
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http://dx.doi.org/10.1186/1752-0509-6-105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3478170PMC
August 2012

Synthesis of heparin oligosaccharides and their interaction with eosinophil-derived neurotoxin.

Org Biomol Chem 2012 Jan 6;10(4):760-72. Epub 2011 Dec 6.

Genomics Research Center, Academia Sinica, Taipei, Taiwan.

A convenient route for the synthesis of heparin oligosaccharides involving regioselective protection of D-glucosamine and a concise preparation of rare L-ido sugars from diacetone α-D-glucose is described. Stereoselective coupling of a D-glucosamine-derived trichloroacetimidate with a 1,6-anhydro-β-L-idopyranosyl 4-alcohol gave the desired α-linked disaccharide, which was used as repeating unit for dual chain elongation and termination. Stepwise assembly from the reducing to the non-reducing end with a D-glucosamine-derived monosaccharide as starting unit furnished the oligosaccharide skeletons having different chain lengths. A series of functional group transformations afforded the expected heparin oligosaccharides with 3, 5 and 7 sugar units. Interaction of these oligosaccharides with eosinophil-derived neurotoxin (EDN), a cationic ribonuclease and a mediator produced by human eosinophils, was further investigated. The results revealed that at 5 μg mL(-1), the heptasaccharide has sufficiently strong interference to block EDN binding to Beas-2B cells. The tri- and pentasaccharides have moderate inhibitory properties at 50 μg mL(-1) concentration, but no inhibition has been observed at 10 μg mL(-1). The IC(50) values of the tri-, penta- and heptasaccharides are 69.4, 47.2 and 0.225 μg mL(-1), respectively.
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http://dx.doi.org/10.1039/c1ob06415kDOI Listing
January 2012

Inhibition of the interactions between eosinophil cationic protein and airway epithelial cells by traditional Chinese herbs.

BMC Syst Biol 2010 Sep 13;4 Suppl 2:S8. Epub 2010 Sep 13.

Graduate Institute of Molecular Systems Biomedicine, China Medical University, Taichung, Taiwan.

Background: The eosinophil cationic protein (ECP) is cytotoxic to bacteria, viruses, parasites and mammalian cells. Cells are damaged via processes of pore formation, permeability alteration and membrane leaking. Some clinical studies indicate that ECP gathers in the bronchial tract of asthma sufferers, damages bronchial and airway epithelial cells, and leads to in breathing tract inflammation; therefore, prevention of the cytotoxicity caused by ECP may serve as an approach to treat airway inflammation. To achieve the purpose, reduction of the ECP-cell interactions is rational. In this work, the Chinese herbal combinative network was generated to predict and identify the functional herbs from the pools of prescriptions. It was useful to select the node herbs and to demonstrate the relative binding ability between ECP and Beas-2B cells with or withour herb treatments.

Results: Eighty three Chinese herbs and prescriptions were tested and five effective herbs and six prescription candidates were selected. On the basis of effective single-herbal drugs and prescriptions, a combinative network was generated. We found that a single herb, Gan-cao, served as a node connecting five prescriptions. In addition, Sheng-di-huang, Dang-guei and Mu-tong also appeared in five, four and three kinds of prescriptions, respectively. The extracts of these three herbs indeed effectively inhibited the interactions between ECP and Beas-2B cells. According to the Chinese herbal combinative network, eight of the effective herbal extracts showed inhibitory effects for ECP internalizing into Beas-2B cells. The major components of Gang-cao and Sheng-di-huang, glycyrrhizic acid and verbascose, respectively, reduced the binding affinity between ECP and cells effectively.

Conclusions: Since these Chinese herbs reduced the binding affinity between ECP and cells and inhibited subsequent ECP entrance into cells, they were potential for mitigating the airway inflammation symptoms. Additionally, we mentioned a new concept to study the Chinese herbs using combinative network in the field of systems biology. The functional single herbs could be identified from the set of prescriptions.
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http://dx.doi.org/10.1186/1752-0509-4-S2-S8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2982695PMC
September 2010

TNF-alpha mediates eosinophil cationic protein-induced apoptosis in BEAS-2B cells.

BMC Cell Biol 2010 Jan 20;11. Epub 2010 Jan 20.

Department of Life Science, Institute of Biotechnology, National Tsing Hua University, Hsinchu 30013, Taiwan.

Background: Eosinophilic granulocytes are important for the human immune system. Many cationic proteins with cytotoxic activities, such as eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), are released from activated eosinophils. ECP, with low RNase activity, is widely used as a biomarker for asthma. ECP inhibits cell viability and induces apoptosis to cells. However, the specific pathway underlying the mechanisms of ECP-induced cytotoxicity remains unclear. This study investigated ECP-induced apoptosis in bronchial epithelial BEAS-2B cells and elucidated the specific pathway during apoptosis.

Results: To address the mechanisms involved in ECP-induced apoptosis in human BEAS-2B cells, investigation was carried out using chromatin condensation, cleavage of poly (ADP-ribose) polymerase (PARP), sub-G1 distribution in cell cycle, annexin V labeling, and general or specific caspase inhibitors. Caspase-8-dependent apoptosis was demonstrated by cleavage of caspase-8 after recombinant ECP treatment, accompanied with elevated level of tumor necrosis factor alpha (TNF-alpha). Moreover, ECP-induced apoptosis was effectively inhibited in the presence of neutralizing anti-TNF-alpha antibody.

Conclusion: In conclusion, our results have demonstrated that ECP increased TNF-alpha production in BEAS-2B cells and triggered apoptosis by caspase-8 activation through mitochondria-independent pathway.
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http://dx.doi.org/10.1186/1471-2121-11-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2819994PMC
January 2010

Characterization of molecular interactions between eosinophil cationic protein and heparin.

J Biol Chem 2008 Sep 30;283(37):25468-25474. Epub 2008 Jun 30.

Institute of Molecular and Cellular Biology and Department of Life Science, National Tsing Hua University, 101, Section 2, Kuang Fu Road, Hsinchu 30013. Electronic address:

Eosinophil cationic protein (ECP) is currently used as a biomarker for airway inflammation. It is a heparin-binding ribonuclease released by activated eosinophils. Its cytotoxicity toward cancer cell lines is blocked by heparin. The objective of this study was to locate the heparin binding site of ECP by site-directed mutagenesis and construction of a synthetic peptide derived from this region. Synthetic heparin with > or =5 monosaccharide units showed strong inhibition of ECP binding to the cell surface. Analysis of ECP mt1 (R34A/W35A/R36A/K38A) showed that these charged and aromatic residues were involved in ECP binding to heparin and the cell surface. A potential binding motif is located in the loop L3 region between helix alpha2 and strand beta1, outside the RNA binding domain. The synthetic peptide derived from the loop L3 region displayed strong pentasaccharide binding affinity and blocked ECP binding to cells. In addition, ECP mt1 showed reduced cytotoxicity. Thus, the tight interaction between ECP and heparin acts as the primary step for ECP endocytosis. These results provide new insights into the structure and function of ECP for anti-asthma therapy.
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http://dx.doi.org/10.1074/jbc.M803516200DOI Listing
September 2008

A heparan sulfate-facilitated and raft-dependent macropinocytosis of eosinophil cationic protein.

Traffic 2007 Dec 15;8(12):1778-95. Epub 2007 Oct 15.

Department of Life Science, Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan 30013, China.

Eosinophil cationic protein (ECP), a human RNAseA superfamily member, highly implicated in asthma pathology, is toxic to bronchial epithelial cells following its endocytosis. The mechanism by which ECP is internalized into cells is poorly understood. In this study, we show that cell surface-bound heparan sulfate proteoglycans serve as the major receptor for ECP internalization. Removal of cell surface heparan sulfate by heparinases or reducing glycan sulfation by chlorate markedly decreased ECP binding to human bronchial epithelial Beas-2B cells. In addition, ECP uptake and associated cytotoxicity were reduced in glycosaminoglycan-defective cells compared with their wild-type counterparts. Furthermore, pharmacological treatment combined with siRNA knockdown identified a clathrin- and caveolin-independent endocytic pathway as the major route for ECP internalization. This pathway is regulated by Rac1 and ADP-ribosylating factor 6 GTPases. It requires cholesterol, actin cytoskeleton rearrangement and phosphatidylinositol-3-kinase activities, and is compatible with the characteristics of raft-dependent macropinocytosis. Thus, our results define the early events of ECP internalization and may have implications for novel therapeutic design for ECP-associated diseases.
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http://dx.doi.org/10.1111/j.1600-0854.2007.00650.xDOI Listing
December 2007

Signal peptide of eosinophil cationic protein upregulates transforming growth factor-alpha expression in human cells.

J Cell Biochem 2007 Apr;100(5):1266-75

Institute of Molecular and Cellular Biology & Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.

Eosinophil cationic protein (ECP) is a major component of eosinophil granule protein that is used as a clinical bio-marker for asthma and allergic inflammatory diseases. Previously, it has been reported that the signal peptide of human ECP (ECPsp) inhibits the cell growth of Escherichia coli (E. coli) and Pichia pastoris (P. pastoris), but not mammalian A431 cells. The inhibitory effect is due to the lack of human signal peptide peptidase (hSPP), a protease located on the endoplasmic reticulum (ER) membrane, in the lower organisms. In this study, we show that the epidermal growth factor receptor (EGFR) is upregulated by the exogenous ECPsp-eGFP as a result of the increased expression of the transforming growth factor-alpha (TGF-alpha) at both transcriptional and translational levels in A431 and HL-60 clone 15 cell lines. Furthermore, the N-terminus of ECPsp fragment generated by the cleavage of hSPP (ECPspM1-G17) gives rise to over threefold increase of TGF-alpha protein expression, whereas another ECPsp fragment (ECPspL18-A27) and the hSPP-resistant ECPsp (ECPspG17L) do not show similar effect. Our results indicate that the ECPspM1-G17 plays a crucial role in the upregulation of TGF-alpha, suggesting that the ECPsp not only directs the secretion of mature ECP, but also involves in the autocrine system.
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http://dx.doi.org/10.1002/jcb.21120DOI Listing
April 2007

Unique peptide identification of RNase A superfamily sequences based on reinforced merging algorithms.

J Bioinform Comput Biol 2006 Feb;4(1):75-92

Department of Computer Science, National Taiwan Ocean University, No. 2, Pei-Ning Road, Keelung, Taiwan 20224, ROC.

Human ribonuclease A (RNaseA) superfamily consists of eight RNases with high similarity in which RNase2 and RNase3 share 76.7% identity. The evolutionary variation of RNases results in differential structures and functions of the enzymes. To distinguish the characteristics of each RNase, we developed reinforced merging algorithms (RMA) to rapidly identify the unique peptide motifs for each member of the highly conserved human RNaseA superfamily. Many motifs in RNase3 identified by RMA correlated well with the antigenic regions predicted by DNAStar. Two unique peptide motifs were experimentally confirmed to contain epitopes for monoclonal antibodies (mAbs) specifically against RNase3. Further analysis of homologous RNases in different species revealed that the unique peptide motifs were located at the correspondent positions, and one of these motifs indeed matched the epitope for a specific anti-bovine pancreatic RNaseA (bpRNaseA) antibody. Our method provides a useful tool for identification of unique peptide motifs for further experimental design. The RMA system is available and free for academic use at http://bioinfo.life.nthu.edu.tw/rma/ and http://spider.cs.ntou.edu.tw/bioinformatics/RMA.html.
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http://dx.doi.org/10.1142/s0219720006001710DOI Listing
February 2006

A reinforced merging methodology for mapping unique peptide motifs in members of protein families.

BMC Bioinformatics 2006 Jan 25;7:38. Epub 2006 Jan 25.

Institute of Molecular and Cellular Biology, Department of Life Science, National Tsing Hua University, Hsinchu, 30013, ROC, Taiwan.

Background: Members of a protein family often have highly conserved sequences; most of these sequences carry identical biological functions and possess similar three-dimensional (3-D) structures. However, enzymes with high sequence identity may acquire differential functions other than the common catalytic ability. It is probable that each of their variable regions consists of a unique peptide motif (UPM), which selectively interacts with other cellular proteins, rendering additional biological activities. The ability to identify and localize such UPMs is paramount in recognizing the characteristic role of each member of a protein family.

Results: We have developed a reinforced merging algorithm (RMA) with which non-gapped UPMs were identified in a variety of query protein sequences including members of human ribonuclease A (RNaseA), epidermal growth factor receptor (EGFR), matrix metalloproteinase (MMP), and Sma-and-Mad related protein families (Smad). The UPMs generally occupy specific positions in the resolved 3-D structures, especially the loop regions on the structural surfaces. These motifs coincide with the recognition sites for antibodies, as the epitopes of four monoclonal antibodies and two polyclonal antibodies were shown to overlap with the UPMs. Most of the UPMs were found to correlate well with the potential antigenic regions predicted by PROTEAN. Furthermore, an accuracy of 70% can be achieved in terms of mapping a UPM to an epitope.

Conclusion: Our study provides a bioinformatic approach for searching and predicting potential epitopes and interacting motifs that distinguish different members of a protein family.
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http://dx.doi.org/10.1186/1471-2105-7-38DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1369005PMC
January 2006