氣喘是一種與氣管發炎相關的複雜疾病，已知人類嗜酸性白血球陽離子蛋白(ECP)在氣喘發病原理中扮演很重要的角色。嗜酸性白血球顆粒中的嗜酸性白血球陽離子蛋白是一種能與肝素(Heparin)結合的核醣核苷酸水解酶(Ribonuclease)，會伴隨嗜酸性白血球的活化釋放到細胞外，進而對支氣管上皮細胞造成極強的細胞毒性，肝素則能抑制嗜酸性白血球陽離子蛋白的細胞毒性。嗜酸性白血球陽離子蛋白之細胞毒性與其進入細胞的內噬作用有關，但至目前為止嗜酸性白血球陽離子蛋白進入細胞路徑的詳細機制並不清楚。本篇論文中，我們證明細胞表面的肝黏醣硫酸鹽蛋白醣 (Heparan sulfate proteoglycan/HSPG)為嗜酸性白血球陽離子蛋白與細胞結合的主要受體。利用酵素切除細胞表面的肝黏醣硫酸鹽、或降低細胞表面的醣硫酸化程度皆能有效地抑制嗜酸性白血球陽離子蛋白與支氣管上皮細胞(Beas-2B)的結合。本研究並發現醣氨多醣(Glycosaminglycan)缺失的細胞能吸收嗜酸性白血球陽離子蛋白的量大幅降低，並能抵抗該蛋白的細胞毒性。利用各種藥物與小型干擾RNA(siRNA)阻斷不同的內噬作用，發現嗜酸性白血球陽離子蛋白係利用與網格蛋白(clathrn)或內陷素(caveolin)都無關的新穎細胞內噬作用進入細胞。此作用受到小G蛋白Rac1及Arf6調控，並與細胞中的膽固醇濃度、肌動蛋白的排列及PI3K的活性相關，這些特性十分符合透過細胞膜上脂肪筏(lipid Raft)區域進行的巨胞飲作用。
利用合成的高純度肝素與嗜酸性白血球陽離子蛋白結合的實驗，我們發現五單醣長度的合成肝素即能有效抑制蛋白與細胞的結合。利用點突變方法及合成的小胜肽分子研究則顯示位於脉2螺旋與刍1褶版間的L3環區域中的R34、W35、R36、K38等胺基酸參與嗜酸性白血球陽離子蛋白與肝素分子及細胞表面的結合。本研究合成的L3環區域胜肽具有與5單元長度的合成肝素之高度親合性，並能阻斷嗜酸性白血球陽離子蛋白與細胞的結合，R34A/W35A/R36A/K38A變異蛋白與細胞結合能力及細胞毒性均幅降低。本研究對於嗜酸性白血球陽離子蛋白及肝黏醣硫酸鹽之專一性分子識別的結構及功能提出創新的見解，本研究結果將有助於開發抑制嗜酸性白血球陽離子蛋白與細胞結合以治療氣喘的新穎方法。

Eosinophil cationic protein (ECP) is currently used as a biomarker of airway inflammation. It is a heparin-binding ribonuclease (RNase) released by activated eosinophils and is toxic to bronchial epithelial cells following its endocytosis. The mechanism by which ECP is internalized into cells is poorly understood. In first part of the study, we show that cell surface-bound heparan sulfate proteoglycans (HSPGs) 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 as compared to their wild-type counterparts. Furthermore, pharmacological treatment combined with si-RNA knock-down identified a clathrin- and caveolin-independent endocytic pathway as the major route for ECP internalization. This pathway is regulated by Rac1 and Arf6 GTPases. It requires cholesterol, actin cytoskeleton rearrangement and PI3K activities, and is compatible with the characteristics of raft-dependent macropinocytosis.
In terms of specific polysaccharide binding activity, ssynthetic heparin with 5 or higher monosaccharide units showed strong inhibition of ECP binding to cell surface. The heparin binding site in ECP is determined by site-directed mutagenesis and synthetic peptide segment derived from ECP. Analysis of ECP mt1 (R34A/W35A/R36A/K38A) showed that the charged and aromatic residues were involved in ECP binding to heparin and cell surface. Such binding motif is located in the loop L3 region between helix 脉2 and strand 刍1, outside the RNA binding domain. The peptide derived from the loop L3 region displayed strong pentasaccharide binding affinities and blocked ECP binding to cells. In addition, ECP mt1 showed reduced cytotoxicity. Thus, the tight interaction between ECP and heparin act as the primary step for protein endocyosis. These results provide new insights into the structure and function of ECP regarding specific molecular interaction with heparan sulfate. Thus, our results define the early events of ECP internalization and may have implications for novel therapeutic design for ECP-associated diseases.

1 Bousquet, J., Chanez, P., Vignola, A. M., Lacoste, J. Y. and Michel, F. B. (1994) Eosinophil inflammation in asthma. Am J Respir Crit Care Med. 150, s33-38
2 Venge, P., Bystrom, J., Carlson, M., Hakansson, L., Karawacjzyk, M., Peterson, C., Seveus, L. and Trulson, A. (1999) Eosinophil cationic protein (ECP): molecular and biological properties and the use of ECP as a marker of eosinophil activation in disease. Clin Exp Allergy. 29, 1172-1186
3 Koh, G. C., Shek, L. P., Goh, D. Y., Van Bever, H. and Koh, D. S. (2007) Eosinophil cationic protein: is it useful in asthma? A systematic review. Respiratory medicine. 101, 696-705
4 Barker, R., Loegering, D., Ten, R., Hamann, K., Pease, L. and Gleich, G. (1989) Eosinophil cationic protein cDNA. Comparison with other toxic cationic proteins and ribonucleases. Journal of Immunology. 143, 952-955
5 Giembycz, M. A. and Lindsay, M. A. (1999) Pharmacology of the eosinophil. Pharmacological Reviews. 51, 213-340
6 Fredens, K., Dahl, R. and Venge, P. (1982) The Gordon phenomenon induced by the eosinophil cationic protein and eosinophil protein X. J Allergy Clin Immunol. 70, 361-366
7 Durack, D. T., Sumi, S. M. and Klebanoff, S. J. (1979) Neurotoxicity of human eosinophils. Proc Natl Acad Sci U S A. 76, 1443-1447
8 Young, J. D., Peterson, C. G., Venge, P. and Cohn, Z. A. (1986) Mechanism of membrane damage mediated by human eosinophil cationic protein. Nature. 321, 613-616
9 Rosenberg, H. F. (1995) Recombinant human eosinophil cationic protein. Ribonuclease activity is not essential for cytotoxicity. J Biol Chem. 270, 7876-7881
10 Mallorqui-Fernandez, G., Pous, J., Peracaula, R., Aymami, J., Maeda, T., Tada, H., Yamada, H., Seno, M., Llorens, R. d., Gomis-Ruth, F. X. and Coll, M. (2000) Three-dimensional crystal structure of human eosinophil cationic protein (RNase 3) at 1.75 A resolution1. Journal of Molecular Biology. 300, 1297-1307
11 Young, J., Peterson, C., Venge, P. and Cohn, Z. (1986) Mechanism of membrane damage mediated by human eosinophil cationic protein. Nature. 321, 613-616
12 Carreras, E., Boix, E., Navarro, S., Rosenberg, H. F., Cuchillo, C. M. and Nogues, M. V. (2005) Surface-exposed amino acids of eosinophil cationic protein play a critical role in the inhibition of mammalian cell proliferation. Molecular and Cellular Biochemistry. 272, 1-7
13 Libonati, M. (2004) Biological actions of the oligomers of ribonuclease A. Cell Mol Life Sci. 61, 2431-2436
14 Carreras, E., Boix, E., Rosenberg, H. F., Cuchillo, C. M. and Nogues, M. V. (2003) Both aromatic and cationic residues contribute to the membrane-lytic and bactericidal activity of eosinophil cationic protein. Biochemistry. 42, 6636-6644
15 Prydz, K. and Dalen, K. T. (2000) Synthesis and sorting of proteoglycans. Journal of Cell Science. 113, 193-205
16 Bandtlow, C. E. and Zimmermann, D. R. (2000) Proteoglycans in the developing brain: new conceptual insights for old proteins. Physiol Rev. 80, 1267-1290
17 Raman, R., Sasisekharan, V. and Sasisekharan, R. (2005) Structural insights into biological roles of protein-glycosaminoglycan interactions. Chemistry & biology. 12, 267-277
18 Tumova, S., Woods, A. and Couchman, J. R. (2000) Heparan sulfate proteoglycans on the cell surface: versatile coordinators of cellular functions. The international journal of biochemistry & cell biology. 32, 269-288
19 Whitelock, J. M. and Iozzo, R. V. (2005) Heparan sulfate: a complex polymer charged with biological activity. Chemical Reviews. 105, 2745-2754
20 Bernfield, M., Gotte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J. and Zako, M. (1999) Functions of cell surface heparan sulfate proteoglycans. Annual review of biochemistry. 68, 729-777
21 Yanagishita, M. (1998) Cellular catabolism of heparan sulfate proteoglycans. Trends in Glycoscience and Glycotechnology. 10, 57-63
22 Tkachenko, E. and Simons, M. (2002) Clustering Induces Redistribution of Syndecan-4 Core Protein into Raft Membrane Domains. Journal of Biological Chemistry. 277, 19946-19951
23 Iozzo, R. V. and San Antonio, J. D. (2001) Heparan sulfate proteoglycans: heavy hitters in the angiogenesis arena. J Clin Invest. 108, 349-355
24 Perrimon, N. and Bernfield, M. (2000) Specificities of heparan sulfate proteoglycans in developmental processes. Nature. 404, 725-728
25 Mayor, S. and Pagano, R. E. (2007) Pathways of clathrin-independent endocytosis. Nature reviews. 8, 603-612
26 Grimmer, S., van Deurs, B. and Sandvig, K. (2002) Membrane ruffling and macropinocytosis in A431 cells require cholesterol. Journal of Cell Science. 115, 2953-2962
27 Nhieu, G. T. and Sansonetti, P. J. (1999) Mechanism of Shigella entry into epithelial cells. Current Opinion in Microbiology 2, 51-55
28 Nakase, I., Niwa, M., Takeuchi, T., Sonomura, K., Kawabata, N., Koike, Y., Takehashi, M., Tanaka, S., Ueda, K., Simpson, J. C., Jones, A. T., Sugiura, Y. and Futaki, S. (2004) Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement. Molecular Therapy 10, 1011-1022
29 Wadia, J. S., Stan, R. V. and Dowdy, S. F. (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nature Medicine. 10, 310-315
30 Sieczkarski, S. B. and Whittaker, G. R. (2002) Dissecting virus entry via endocytosis. Journal of General Virology. 83, 1535-1545
31 Brown, F. D., Rozelle, A. L., Yin, H. L., Balla, T. and Donaldson, J. G. (2001) Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic. Journal of Cell Biology. 154, 1007-1017
32 Wu, C. M., Chang, H. T. and Chang, M. D. (2004) Membrane-bound carboxypeptidase E facilitates the entry of eosinophil cationic protein into neuroendocrine cells. Biochemical Journal. 382, 841-848
33 Boix, E., Nikolovski, Z., Moiseyev, G. P., Rosenberg, H. F., Cuchillo, C. M. and Nogues, M. V. (1999) Kinetic and product distribution analysis of human eosinophil cationic protein indicates a subsite arrangement that favors exonuclease-type activity. Journal of Biological Chemistry. 274, 15605-15614
34 Gauthier, N. C., Monzo, P., Kaddai, V., Doye, A., Ricci, V. and Boquet, P. (2005) Helicobacter pylori VacA cytotoxin: a probe for a clathrin-independent and Cdc42-dependent pinocytic pathway routed to late endosomes. Molecular Biology of the Cell. 16, 4852-4866
35 Wu, S. C., Chiang, J. R. and Lin, C. W. (2004) Novel cell adhesive glycosaminoglycan-binding proteins of Japanese encephalitis virus. Biomacromolecules. 5, 2160-2164
36 Richard, J. P., Melikov, K., Brooks, H., Prevot, P., Lebleu, B. and Chernomordik, L. V. (2005) Cellular Uptake of Unconjugated TAT Peptide Involves Clathrin-dependent Endocytosis and Heparan Sulfate Receptors. Journal of Biological Chemistry. 280, 15300-15306
37 Calabro, A., Benavides, M., Tammi, M., Hascall, V. C. and Midura, R. J. (2000) Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted carbohydrate electrophoresis (FACE). Glycobiology. 10, 273-281
38 Holmes, O., Pillozzi, S., Deakin, J. A., Carafoli, F., Kemp, L., Butler, P. J., Lyon, M. and Gherardi, E. (2007) Insights into the structure/function of hepatocyte growth factor/scatter factor from studies with individual domains. Journal of molecular biology. 367, 395-408
39 Venge, P. and Bystrom, J. (1998) Eosinophil cationic protein (ECP). The international journal of biochemistry & cell biology. 30, 433-437
40 Liang, C. T., Chueh, L. L., Pang, V. F., Zhuo, Y. X., Liang, S. C., Yu, C. K., Chiang, H., Lee, C. C. and Liu, C. H. (2007) A non-biotin polymerized horseradish-peroxidase method for the immunohistochemical diagnosis of canine distemper. Journal of comparative pathology. 136, 57-64
41 Makarov, A. A. and Ilinskaya, O. N. (2003) Cytotoxic ribonucleases: molecular weapons and their targets. FEBS Letters. 540, 15-20
42 Bracale, A., Spalletti-Cernia, D., Mastronicola, M., Castaldi, F., Mannucci, R., Nitsch, L. and D'Alessio, G. (2002) Essential stations in the intracellular pathway of cytotoxic bovine seminal ribonuclease. Biochemical Journal. 362, 553-560
43 Maeda, T., Kitazoe, M., Tada, H., Llorens, R. d., Salomon, D. S., Ueda, M., Yamada, H. and Seno, M. (2002) Growth inhibition of mammalian cells by eosinophil cationic protein. European Journal of Biochemistry. 269, 307-316
44 Leland, P. A., Staniszewski, K. E., Kim, B. and Raines, R. T. (2000) A synapomorphic disulfide bond is critical for the conformational stability and cytotoxicity of an amphibian ribonuclease. . FEBS Letters. 477, 203-207
45 Leland, P. A., Staniszewski, K. E., Kim, B. M. and Raines, R. T. (2001) Endowing human pancreatic ribonuclease with toxicity for cancer cells. Journal of Biological Chemistry. 276, 43095-43102
46 Rosenberg, H. (1998) The eosinophil ribonucleases. Cell Mol Life Science. 54, 795-803
47 Gleich, G. J. (2000) Mechanisms of eosinophil-associated inflammation. Journal of Allergy and Clinical Immunology 105, 651-663
48 Gleich, G. J., Loegering, D. A., Bell, M. P., Checkel, J. L., Ackerman, S. J. and McKean, D. J. (1986) Biochemical and functional similarities between human eosinophil-derived neurotoxin and eosinophil cationic protein: homology with ribonuclease. Proc Natl Acad Sci USA. 83, 3146-3150
49 Park, P. W., Reizes, O. and Bernfield, M. (2000) Cell surface heparan sulfate proteoglycans: selective regulators of ligand-receptor encounters. Journal of Biological Chemistry. 275, 29923-29926
50 Simons, A. and Ikonen, E. (1997) Functional rafts in cell membranes. Nature. 387, 569-572
51 Nichols, B. (2003) Caveosomes and endocytosis of lipid rafts. Journal of Cell Science. 116, 4707-4714
52 Parton, R. (1996) Caveolae and caveolins. Current Opinion in Cell Biology. 8, 542-548
53 D'Souza-Schorey, C. and Chavrier, P. (2006) ARF proteins: roles in membrane traffic and beyond. Nature Revies Molecular Cell Biology. 7, 347-358
54 Naslavsky, N., Weigert, R. and Donaldson, J. G. (2003) Convergence of non-clathrin- and clathrin-derived endosomes involves Arf6 inactivation and changes in phosphoinositides. Molecular Biology of the Cell. 14, 417-431
55 Farley, J., Nakayama, G., Cryns, D. and Segel, I. (1978) Adenosine triphosphate sulfurylase from Penicillium chrysogenum equilibrium binding, substrate hydrolysis, and isotope exchange studies. Archives of biochemistry and biophysics. 185, 376-390
56 Noel, G. J., Love, D. C. and Mosser, D. M. (1994) High-molecular-weight proteins of nontypeable Haemophilus influenzae mediate bacterial adhesion to cellular proteoglycans. Infection and Immunity. 62, 4028-4033
57 Wang, L. H., Rothberg, K. G. and Anderson, R. G. (1993) Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. Journal of Cell Biology. 123, 1107-1117
58 Anderson, H. A., Chen, Y. and Norkin, L. C. (1996) Bound simian virus 40 translocates to caveolin enriched membrane domains and its entry is inhibited by drugs that selectively disrupt caveolae. Molecular Biology of the Cell. 7, 1825-1834
59 Puri, V., Watanabe, R., Singh, R. D., Dominguez, M., Brown, J. C., Wheatley, C. L., Marks, D. L. and Pagano, R. E. (2001) Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways. Journal of Cell Biology. 154, 535-547
60 Benmerah, A., Bayrou, M., Cerf-Bensussan, N. and Dautry-Varsat, A. (1999) Inhibition of clathrin-coated pit assembly by an Eps15 mutant. Journal of Cell Science. 112, 1303-1311
61 Conner, S. D. and Schmid, S. L. (2003) Regulated portals of entry into the cell. Nature. 422, 37-44
62 Damke, H., Baba, T., Warnock, D. E. and Schmid, S. L. (1994) Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. Journal of Cell Biology. 127, 915-934
63 Soldati, T. and Schliwa, M. (2006) Powering membrane traffic in endocytosis and recycling. Nature Reviews Molecular Cell Biology. 7, 897-908
64 Sampath, P. and Pollard, T. D. (1991) Effects of cytochalasin, phalloidin, and pH on the elongation of actin filaments. Biochemistry. 30, 1973-1980
65 West, M. A., Bretscher, M. S. and Watts, C. (1989) Distinct endocytotic pathways in EGF-stimulated human carcinoma A431 cells. Journal of Cell Biology. 109, 2731-2739
66 Araki, N., Johnson, M. T. and Swanson, J. A. (1996) A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages. Journal of Cell Biology. 135, 1249-1260
67 Murray, J., Wilson, L. and Kellie, S. (2000) Phosphatidylinositol-3' kinase-dependent vesicle formation in macrophages in response to macrophage colony stimulating factor. Journal of Cell Biology. 113, 337-348
68 West, M. A., Prescott, A. R., Eskelinen, E., Ridley, A. J. and Watts, C. (2000) Rac is required for constitutive macropinocytosis by dendritic cells but does not control its downregulation Current Biology. 10, 839-848
69 Molina, H. A., Kierszenbaum, F., Hamann, K. J. and Gleich, G. J. (1988) Toxic effects produced or mediated by human eosinophil granule components on Trypanosoma cruzi. The American journal of tropical medicine and hygiene. 38, 327-334
70 Lehrer, R., Szklarek, D., Barton, A., Ganz, T., Hamann, K. and Gleich, G. (1989) Antibacterial properties of eosinophil major basic protein and eosinophil cationic protein. Journal of Immunology. 142, 4428-4434
71 Domachowske, J. B., Dyer, K. D., Adams, A. G., Leto, T. L. and Rosenberg, H. F. (1998) Eosinophil cationic protein/RNase 3 is another RNase A-family ribonuclease with direct antiviral activity. Nucleic acids research. 26, 3358-3363
72 Fan, T. C., Chang, H. T., Chen, I. W., Wang, H. Y. and Chang, M. D. (2007) A Heparan Sulfate-Facilitated and Raft-Dependent Macropinocytosis of Eosinophil Cationic Protein. Traffic. 8, 1778-1795
73 Capila, I. and Linhardt, R. J. (2002) Heparin-protein interactions. Angewandte Chemie International Edition. 41, 391-412
74 Cardin, A. D. and Weintraub, H. J. (1989) Molecular modeling of protein-glycosaminoglycan interactions. Arteriosclerosis (Dallas, Tex. 9, 21-32
75 Joyce, J. A., Freeman, C., Meyer-Morse, N., Parish, C. R. and Hanahan, D. (2005) A functional heparan sulfate mimetic implicates both heparanase and heparan sulfate in tumor angiogenesis and invasion in a mouse model of multistage cancer. Oncogene. 24, 4037-4051
76 Parish, C. R., Freeman, C., Brown, K. J., Francis, D. J. and Cowden, W. B. (1999) Identification of sulfated oligosaccharide-based inhibitors of tumor growth and metastasis using novel in vitro assays for angiogenesis and heparanase activity. Cancer Res. 59, 3433-3441
77 Yu, G., Gunay, N. S., Linhardt, R. J., Toida, T., Fareed, J., Hoppensteadt, D. A., Shadid, H., Ferro, V., Li, C., Fewings, K., Palermo, M. C. and Podger, D. (2002) Preparation and anticoagulant activity of the phosphosulfomannan PI-88. European journal of medicinal chemistry. 37, 783-791
78 Ferro, V., Li, C., Fewings, K., Palermo, M. C., Linhardt, R. J. and Toida, T. (2002) Determination of the composition of the oligosaccharide phosphate fraction of Pichia (Hansenula) holstii NRRL Y-2448 phosphomannan by capillary electrophoresis and HPLC. Carbohydrate research. 337, 139-146
79 Hileman, R. E., Fromm, J. R., Weiler, J. M. and Linhardt, R. J. (1998) Glycosaminoglycan-protein interactions: definition of consensus sites in glycosaminoglycan binding proteins. BioEssays. 20, 156-167
80 Lau, E. K., Paavola, C. D., Johnson, Z., Gaudry, J. P., Geretti, E., Borlat, F., Kungl, A. J., Proudfoot, A. E. and Handel, T. M. (2004) Identification of the glycosaminoglycan binding site of the CC chemokine, MCP-1: implications for structure and function in vivo. J Biol Chem. 279, 22294-22305
81 Nikolovski, Z., Buzon, V., Ribo, M., Moussaoui, M., Vilanova, M., Cuchillo, C. M., Cladera, J. and Nogues, M. V. (2006) Thermal unfolding of eosinophil cationic protein/ribonuclease 3: a nonreversible process. Protein Sci. 15, 2816-2827
82 Vives, E., Brodin, P. and Lebleu, B. (1997) A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem. 272, 16010-16017
83 Ziegler, A. and Seelig, J. (2004) Interaction of the protein transduction domain of HIV-1 TAT with heparan sulfate: binding mechanism and thermodynamic parameters. Biophysical journal. 86, 254-263
84 Peterson, C. G., Skoog, V. and Venge, P. (1986) Human eosinophil cationic proteins (ECP and EPX) and their suppressive effects on lymphocyte proliferation. Immunobiology. 171, 1-13
85 Kimata, H., Yoshida, A., Ishioka, C., Jiang, Y. and Mikawa, H. (1992) Eosinophil cationic protein inhibits immunoglobulin production and proliferation in vitro in human plasma cells. Cellular immunology. 141, 422-432
86 Maccarana, M., Sakura, Y., Tawada, A., Yoshida, K. and Lindahl, U. (1996) Domain structure of heparan sulfates from bovine organs. J Biol Chem. 271, 17804-17810
87 Ledin, J., Staatz, W., Li, J. P., Gotte, M., Selleck, S., Kjellen, L. and Spillmann, D. (2004) Heparan sulfate structure in mice with genetically modified heparan sulfate production. J Biol Chem. 279, 42732-42741
88 Guimond, S., Maccarana, M., Olwin, B. B., Lindahl, U. and Rapraeger, A. C. (1993) Activating and inhibitory heparin sequences for FGF-2 (basic FGF). Distinct requirements for FGF-1, FGF-2, and FGF-4. J Biol Chem. 268, 23906-23914
89 Tkachenko, E., Lutgens, E., Stan, R. V. and Simons, M. (2004) Fibroblast growth factor 2 endocytosis in endothelial cells proceed via syndecan-4-dependent activation of Rac1 and a Cdc42-dependent macropinocytic pathway. Journal of Cell Science. 117, 3189-3199
90 Jin, L., Abrahams, J. P., Skinner, R., Petitou, M., Pike, R. N. and Carrell, R. W. (1997) The anticoagulant activation of antithrombin by heparin. Proc Natl Acad Sci U S A. 94, 14683-14688
91 Safaiyan, F., Kolset, S. O., Prydz, K., Gottfridsson, E., Lindahl, U. and Salmivirta, M. (1999) Selective effects of sodium chlorate treatment on the sulfation of heparan sulfate. J Biol Chem. 274, 36267-36273
92 Johannes, L. and Lamaze, C. (2002) Clathrin-dependent or not: is it still the question? Traffic. 3, 443-451
93 Sabharanjak, S., Sharma, P., Parton, R. G. and Mayor, S. (2002) GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway. Developmental Cell. 2, 411-423
94 Kirkham, M., Fujita, A., Chadda, R., Nixon, S. J., Kurzchalia, T. V., Sharma, D. K., Pagano, R. E., Hancock, J. F., Mayor, S. and Parton, R. G. (2005) Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles. Journal of Cell Biology. 168, 465-476
95 Lamaze, C., Dujeancourt, A., Baba, T., Lo, C. G., Benmerah, A. and Dautry-Varsat, A. (2001) Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway. Molecular Cell. 7, 661-671
96 Manes, S., Ana Lacalle, R., Gomez-Mouton, C. and Martinez-A, C. (2003) From rafts to crafts: membrane asymmetry in moving cells. Trends in Immunology. 24, 320-326
97 Kirkham, M. and Parton, R. G. (2005) Clathrin-independent endocytosis: new insights into caveolae and non-caveolar lipid raft carriers. Biochim Biophys Acta. 1746, 349-363
98 Naslavsky, N., Weigert, R. and Donaldson, J. G. (2004) Characterization of a nonclathrin endocytic pathway: membrane cargo and lipid requirements. Molecular Biology of the Cell. 15, 3542-3552
99 Swanson, J. A. and Watts, C. (1995) Macropinosytosis. Trends in Cell Biollogy. 5, 424-428
100 Racoosin, E. L. and Swanson, J. A. (1993) Macropinosome maturation and fusion with tubular lysosomes in macrophages. Journal of Cell Biology. 121, 1011-1020
101 Hewlett, L. J., Prescott, A. R. and Watts, C. (1994) The coated pit and macropinocytic pathways serve distinct endosome populations. Journal of Cell Biology. 124, 689-703
102 Mallard, F., Antony, C., Tenza, D., Salamero, J., Goud, B. and Johannes, L. (1998) Direct pathway from early/recycling endosomes to the Golgi apparatus revealed through the study of shiga toxin B-fragment transport. Journal of Cell Biology. 143, 973-990
103 Gengrinovitch, S., Berman, B., David, G., Witte, L., Neufeld, G. and Ron, D. (1999) Glypican-1 is a VEGF165 binding proteoglycan that acts as an extracellular chaperone for VEGF165. Journal of Biological Chemistry. 274, 10816-10822
104 Cooper, A. and Shaul, Y. (2006) Clathrin-mediated endocytosis and lysosomal cleavage of hepatitis B virus capsid-like core particles. Journal of Biological Chemistry. 281, 16563-16569
105 Bosch, M., Benito, A., Ribo, M., Puig, T., Beaumelle, B. and Vilanova, M. (2004) A nuclear localization sequence endows human pancreatic ribonuclease with cytotoxic activity. Biochemistry. 43, 2167-2177
106 Haigis, M. C. and Raines, R. T. (2002) Secretory ribonucleases are internalized by a dynamin-independent endocytic pathway. Journal of Cell Science. 116, 313-324
107 Meier, O., Boucke, K., Hammer, S. V., keller, S., Stidwill, R. P., Hemmi, S. and Greber, U. F. (2002) Adenovirus tiggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake. Journal of Cell Biology. 158, 1119-1131
108 Khalil, I. A., Kogure, K., Futaki, S. and Harashima, H. (2006) High density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression. Journal of Biological Chemistry. 281, 3544-3551
109 Esko, J. D. and Lindahl, U. (2001) Molecular diversity of heparan sulfate. J Clin Invest. 108, 169-173
110 Ellyard, J. I., Simson, L., Bezos, A., Johnston, K., Freeman, C. and Parish, C. R. (2007) Eotaxin selectively binds heparin. An interaction that protects eotaxin from proteolysis and potentiates chemotactic activity in vivo. J Biol Chem. 282, 15238-15247
111 Belzar, K. J., Dafforn, T. R., Petitou, M., Carrell, R. W. and Huntington, J. A. (2000) The effect of a reducing-end extension on pentasaccharide binding by antithrombin. J Biol Chem. 275, 8733-8741
112 Sarafanov, A. G., Ananyeva, N. M., Shima, M. and Saenko, E. L. (2001) Cell surface heparan sulfate proteoglycans participate in factor VIII catabolism mediated by low density lipoprotein receptor-related protein. J Biol Chem. 276, 11970-11979
113 Ricard-Blum, S., Beraud, M., Raynal, N., Farndale, R. W. and Ruggiero, F. (2006) Structural requirements for heparin/heparan sulfate binding to type V collagen. J Biol Chem. 281, 25195-25204
114 Ricard-Blum, S., Feraud, O., Lortat-Jacob, H., Rencurosi, A., Fukai, N., Dkhissi, F., Vittet, D., Imberty, A., Olsen, B. R. and van der Rest, M. (2004) Characterization of endostatin binding to heparin and heparan sulfate by surface plasmon resonance and molecular modeling: role of divalent cations. J Biol Chem. 279, 2927-2936
115 Sallum, C. O., Kammerer, R. A. and Alexandrescu, A. T. (2007) Thermodynamic and structural studies of carbohydrate binding by the agrin-G3 domain. Biochemistry. 46, 9541-9550
116 Krilleke, D., DeErkenez, A., Schubert, W., Giri, I., Robinson, G. S., Ng, Y. S. and Shima, D. T. (2007) Molecular mapping and functional characterization of the VEGF164 heparin-binding domain. J Biol Chem. 282, 28045-28056
117 Nascimento, F. D., Hayashi, M. A., Kerkis, A., Oliveira, V., Oliveira, E. B., Radis-Baptista, G., Nader, H. B., Yamane, T., dos Santos Tersariol, I. L. and Kerkis, I. (2007) Crotamine mediates gene delivery into cells through the binding to heparan sulfate proteoglycans. J Biol Chem. 282, 21349-21360