A Heparan Sulfate‐Facilitated and Raft‐Dependent Macropinocytosis of Eosinophil Cationic Protein

Fan, Timothy M.; Chang, Hsiang-Kuang; Chen, I‐Wen; Wang, Hsiu-Yiu; Chang, Margaret Dah‐Tsyr

doi:10.1111/j.1600-0854.2007.00650.x

Cited by 50 publications

(74 citation statements)

References 72 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cell ELISA-The ability of MBP-ECP to bind cells in the presence of serial dilutions of oligosaccharides or peptides was determined as described (9). Briefly, confluent monolayers of Beas-2B cells in 96-well plates were pretreated with various concentrations of oligosaccharides or peptides in serum-free RPMI 1640 medium at 4°C for 30 min before incubation with 5 g/ml MBP-ECP at 4°C for 1 h. The cells were then washed with ice-cold PBS and fixed with 2% paraformaldehyde at room temperature for 15 min prior to blocking with 2% bovine serum albumin/PBS at room temperature for 90 min.…”

Section: Methodsmentioning

confidence: 99%

“…The binding of ECP to target cells has been attributed to its high arginine content (estimated pI ϭ 10.8), which facilitates the interaction between ECP and negatively charged molecules on the cell surface (7,8). Recently, we found that binding and endocytosis of ECP into bronchial epithelial cells were greatly dependent on the cell surface glycosaminoglycan, specifically heparan sulfate proteoglycans (HSPG) (9). The cytotoxicity of ECP was severely reduced toward cell lines with heparan sulfate (HS) deficiency.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Characterization of Molecular Interactions between Eosinophil Cationic Protein and Heparin

Fan

Fang

Hwang

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

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 sitedirected mutagenesis and construction of a synthetic peptide derived from this region. Synthetic heparin with >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 ␣2 and strand ␤1, 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.Eosinophil cationic protein (ECP), 2 a member of the ribonuclease A (RNase A) superfamily, is found in the specific granules of eosinophilic leukocytes. It is a single polypeptide with a molecular mass ranging from 16 to 21.4 kDa due to varying degrees of glycosylation. It shows a 67% amino acid sequence identity with eosinophil-derived neurotoxin (EDN), another eosinophil-secreted RNase. Although ECP shares the overall three-dimensional structure of RNase A, it has relatively lower RNase activity (1). ECP released by activated eosinophils contributes to the toxicity against helminth parasites, bacteria, and single strand RNA viruses (2-4). Together with other proteins secreted from eosinophils, ECP is thought to cause damage to epithelial cells, a common feature of airway inflammation in asthma (5).The mechanism underlying the cytotoxic property of ECP is unclear. It has been hypothesized that ECP cytotoxicity is due to destabilization of lipid membranes of target cells (6), and the degree of cytotoxicity is dependent on the cellular concentration (7). The binding of ECP to target cells has been attributed to its high arginine content (estimated pI ϭ 10.8), which facilitates the interaction between ECP and negatively charged molecules on the cell surface (7,8). Recently, we found that binding and endocytosis of ECP into bronchial epithelial cells were greatly dependent on the cell surface glycosaminoglycan, specifically heparan sulfate proteoglycans (HSPG) (9). The cytotoxicity of ECP was severely reduced toward cell lines with heparan sulfate (HS) deficiency.Heparin and HS are complex polysaccharides composed of alternating units of hexuronic acid and glucosamine. The uronic acid residues of heparin typically consist of 90% L-idopyranosyluronic acid and 10% D-glucopyranosyluronic acid (10). The N position of glucosamine may be substituted with an acetyl or sulfate grou...

show abstract

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Characterization of Molecular Interactions between Eosinophil Cationic Protein and Heparin

Fan

Fang

Hwang

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The timing of EGFR co-localization with EEA1-containing endosomes was delayed, which could be due to the altered ubiquitylation and subsequent endocytosis of the receptors associated with HSPG in TERM. Syndecans are known to mediate endocytosis of heparin-binding ligands and proteins (e.g., FGF2 or eosinophil cationic protein) by macropinocytosis (42,43). This pathway is slow and involves formation of uncoated vesicles that eventually fuse with sorting endosomes.…”

Section: Discussionmentioning

confidence: 99%

Metastasis Suppressor Tetraspanin CD82/KAI1 Regulates Ubiquitylation of Epidermal Growth Factor Receptor

Odintsova

Niel

Conjeaud

et al. 2013

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: Tetraspanin CD82/KAI1 is associated with EGF receptor and regulates its signaling. Results: CD82 controls ubiquitylation of EGF receptor after stimulation with heparin-binding ligands and alters receptor trafficking. Conclusion: CD82 regulates communication between heparan sulfate proteoglycans and ligand-bound EGFR, thus affecting the activity of c-Cbl. Significance: Lateral cross-talk initiated by CD82-dependent interactions is critical for modulation of EGFR function.

show abstract

“…In particular, the 33 -38 stretch is involved in ECP binding to GAGs ( Figure 2) (Fan et al , 2008 ;Torrent et al , 2011a ). The protein interaction with heparan sulphate at the matrix of epithelial cells would facilitate the protein endocytosis (Fan et al , 2007 ). Further insight on the protein residues potentially involved in heparin binding has been deduced by molecular docking as detailed below.…”

Section: Dissecting Ecp Antimicrobial Mechanism Of Actionmentioning

confidence: 99%

Structural determinants of the eosinophil cationic protein antimicrobial activity

Boix

Salazar

Torrent

et al. 2012

Biological Chemistry

View full text Add to dashboard Cite

Antimicrobial RNases are small cationic proteins belonging to the vertebrate RNase A superfamily and endowed with a wide range of antipathogen activities. Vertebrate RNases, while sharing the active site architecture, are found to display a variety of noncatalytical biological properties, providing an excellent example of multitask proteins. The antibacterial activity of distant related RNases suggested that the family evolved from an ancestral host-defence function. The review provides a structural insight into antimicrobial RNases, taking as a reference the human RNase 3, also named eosinophil cationic protein (ECP). A particular high binding affi nity against bacterial wall structures mediates the protein action. In particular, the interaction with the lipopolysaccharides at the Gram-negative outer membrane correlates with the protein antimicrobial and specifi c cell agglutinating activity. Although a direct mechanical action at the bacteria wall seems to be suffi cient to trigger bacterial death, a potential intracellular target cannot be discarded. Indeed, the cationic clusters at the protein surface may serve both to interact with nucleic acids and cell surface heterosaccharides. Sequence determinants for ECP activity were screened by prediction tools, proteolysis and peptide synthesis. Docking results are complementing the structural analysis to delineate the protein anchoring sites for anionic targets of biological signifi cance.

show abstract

A Heparan Sulfate‐Facilitated and Raft‐Dependent Macropinocytosis of Eosinophil Cationic Protein

Cited by 50 publications

References 72 publications

Characterization of Molecular Interactions between Eosinophil Cationic Protein and Heparin

Characterization of Molecular Interactions between Eosinophil Cationic Protein and Heparin

Metastasis Suppressor Tetraspanin CD82/KAI1 Regulates Ubiquitylation of Epidermal Growth Factor Receptor

Structural determinants of the eosinophil cationic protein antimicrobial activity

Contact Info

Product

Resources

About