醣胺素是具多負電性的線性聚合醣。它們是由不同化學結構及不同硫酸化修飾的雙醣所聚合而成。它們以蛋白醣的形式，藉由與不同類型蛋白質的連接，廣泛的分布在不同組織或是細胞的表面。肝素與肝素硫酸是醣胺素的一種。它們在葡萄糖胺的第二氫氧基上有特殊的硫酸化。它們的結構異質性、結合專注性、以及分佈特異性，使得它們在調節許多醣胺素結合蛋白的功能上，扮演重要的角色。許多的生物現象，像是細胞分化、增長、癌細胞轉移、以及病毒入侵都與醣胺素有關係。
心臟毒蛋白是眼鏡蛇毒液中的主要成分，大約佔有50%的含量。它們是高正電性，類似三指環形狀、β摺疊結構的蛋白質。它們具有廣泛的細胞毒性、定點組織的壞死活性、以及使心臟收縮停止的毒理現象。藥理學上的觀察顯示：受到眼鏡蛇咬傷的傷者，咬傷區域的組織有嚴重的發炎反應。這與組織氨大量釋放、血管管璧通透性增加、以及免疫細胞大量湧入，進而引發大量化學誘導物的釋放，有密切的關係。相較於其他蛇毒毒液中的組成蛋白，心臟毒蛋白在定點組織的強留滯性質以及在血液中的快速清除現象，可能暗示了它跟細胞表面某個未知的成分有作用。
在本研究工作中，將會利用到一些生物物理、生物化學與細胞實驗，來探討肝素分子與心臟毒蛋白的交互作用。在目前所有研究分子辨識結合的技術中，利用表面電漿共振的及時偵測裝置能同時在動力學及熱力學上得到靈敏且準確的結果。配合上一些天然取得且不同組成的心臟毒蛋白、以及不同化學或酵素修飾的肝素衍生物，可以用來探討心臟毒蛋白與肝素之間的結合專注性、結合模式，以及結合強度。
心臟毒蛋白在細胞上的留滯能力跟細胞上的肝素硫酸有關。以表面電漿共振即時結合測量的實驗來看，此留滯能力在單純的肝素表面上也有同樣的結果。在重要的第二指環區尖端的結構變異性，跟所觀察到的結合專注性、結合模式、以及結合強度有關。N-硫酸根的強烈需求性與賴氨酸31的存在有關，顯示這是一組專注性的結合。其肝素結合模式與三指環尖端是否能形成連續性的疏水區，似乎有關係。
檸檬酸分子在台灣眼鏡蛇蛇毒毒液內有近50毫莫爾濃度的含量。在有它存在的情形下，心臟毒蛋白A3與肝素的結合會被進一步穩定而呈現很強的留滯能力。表面電漿共振即時結合實驗和留滯細胞實驗都顯示此一現象跟醣胺素有關。心臟毒蛋白A3與肝素六醣的共同結晶顯示有一個檸檬酸分子位於心臟毒蛋白雙體的介面。此組合型的凹洞是由雙體的四個賴氨酸23及賴氨酸31所構成。此結晶結構解釋了檸檬酸分子如何穩定心臟毒蛋白A3雙體形成、以及如何藉由第二指環尖端帶電荷或不帶電荷的胺基酸決定其肝素結合模式。
藉由了解心臟毒蛋白與肝素結合時之結構決定因子更可進一步確認其專注結合性。就與心臟毒蛋白A3結合的長度來看，肝素六醣是所需要的最小結合長度；而就抑制心臟毒蛋白A3細胞毒性的性質來看，肝素鏈長度則需要到十二或十四醣才能發揮作用。這顯示心臟毒蛋白A3雙體的形成與毒性被抑制有關係。熱力學的數據也顯示：高分子量的肝素與心臟毒蛋白A3的結合是一種熱熵變化所主導的反應，這可能與蛋白質的多體聚集有關。雖然表面電漿共振即時結合測量的實驗上並沒看到穩定的蛋白質多體形成，但是化學交叉連接以及螢光自我焠熄實驗都顯示心臟毒蛋白A3可以聚合在肝素上。
由於肝素分子是異質性的聚合物，心臟毒蛋白A3與肝素結合時之動力學將會顯現出異質性的結合。此結合異質性，可由解離速率常數的分析來看：它呈現了兩階段式「先慢後快」的解離速率，這與毒蛋白結合在肝素表面的多少量有關係。當毒蛋白結合量高於3~4%，就呈現了兩階段的解離速率，這暗示了心臟毒蛋白A3可能有兩類不同的結合區域或是不同的結合模式。在不同蛋白結合量下之親和性保護做酵素分解，其毒蛋白保護區的肝素雙醣組成分析顯示：在低毒蛋白結合量下，傾向專注結合於肝素的氮乙醯化區；在高毒蛋白結合量下，傾向專注結合於肝素的氮硫酸化區。這表示：肝素的異質性也決定了心臟毒蛋白A3的不同結合序列。在低毒蛋白結合量下的毒蛋白保護區是具有低氮硫酸化/高氮乙醯化的十二或十四醣片段。以化學修飾法移除氮乙醯基，除了改變原先兩階段的解離動力學現象，更降低了結合強度及結合協同性、以及ANS螢光的強度。這表示：藉由氮乙醯基的疏水性結合可能促使心臟毒蛋白A3在特定區域形成雙體。
在本研究中，心臟毒蛋白與肝素分子之結合專注性，分別在蛋白質與醣類的角度上被決定出來。肝素的異質性以及可被調控的肝素結合模式將可決定心臟毒蛋白的組織辨識特異性及其生物意義。

Glycosaminoglycans (GAGs) are negatively charged carbohydrate of linear polymer. They are composed of irregular repeat disaccharide units with various sulfation patterns. They are widely distributed at different tissues and cells and are attached on cell membranes through various protein cores in the form of proteoglycans. Heparin or heparan sulfate are one of glycosaminoglycans with unique sulfation at 2-hydroxy position of glucosamine. Their heterogeneity in chemical structures and locations make them extremely important as multifunctional regulator of many GAG-binding proteins. They are responsive many biological processes, such as cell differentiation / proliferation, tumor metastasis and viral infection, through structural specific mechanism.
Cardiotoxins (CTXs) are major components of cobra snake venoms, which account for approximate 50% in weight. They are highly basic □-sheet polypeptide resembling three finger loops. They exhibit strong tendency to cause cytotoxicity, necrosis at local tissue and systolic heart arrest. Pathological observation is the bitten victim of cobra suffered from severe inflammation in local tissue, a phenomena known to be mediated by the release of histamine, increasing capillary permeability, infiltration of immune cells and induction of many GAG-binding chemokines. Strong retention of CTXs, but not other venom proteins, at local tissues or rapid clearance from plasma implies unknown cell surface components might be responsive for CTXs retention.
In this work, the interactions between heparin and CTXs are studies by many biophysical, biochemical and cell methods. Of all the binding assays, surface plasmon resonance (SPR) gives sensitive and accurate results in both kinetic and thermodynamic point of views. Accompanied with various CTX homologues and various heparin derivatives in different chemical structure, it is shown that the interactions between heparins and CTXs are structural specific.
The retention capability of CTX is demonstrated to be heparan sulfate-dependent by retention test on immobilized CHO cells. Such retention capability is consistent with the results at immobilized heparin surface by SPR. Upon comparison of all studied CTX homologues, it is suggested that the structural important loop2 region determines the heparin binding affinity, binding mode and binding specificity. The N-sulfate on heparin is exclusively important and is correlated with the presence of Lys31 as compared by various CTX homologues. The possible role of loop2 region in heparin binding mode might associate with the connectivity of hydrophobic patch by three tips of finger loops.
The CTXA3-heparin complex is further stabilized and gain retention capability by the presence of citrate ion, which is also major constituent (~50mM) in Taiwan cobra venoms. Both SPR studies and cell retention test support that the citrate-mediated retention of CTXA3 is heparin-dependent. The co-crystal structure of CTXA3 with heparin hexasaccharide shows dimeric packing of CTXs with one citrate ion at putative charged pocket composed of Lys23 and Lys31. This crystal structure explains the retention of CTXs on heparin through CTX dimerization, and the role of charged/non-charged residue at tip of loop2 in different heparin binding modes.
The structural determinants of CTXA3-heparin interaction are further studied to confirm their binding specificities. Heparin length in hexasaccharide is the least size for CTXA3 binding, while the length up to dp12-14 is functional important as investigated by its inhibition ability on CTX cytotoxicity. The thermodynamic investigation of CTXA3 binding to high molecular weight heparin shows extremely entropy-driven interaction, which implies the strong tendency to protein aggregation. Although SPR studies shows no significant retention on heparin surface, the chemical cross-linking and fluorescent self-quenching experiments show potential oligomerization of CTXA3 upon heparin binding.
Due to the heterogeneity of heparin, the kinetic of CTXA3-heparin interaction also reveals heterogeneous binding. This heterogeneous binding is reflected by biphasic / sequential dissociation rates with CTX-occupancy dependent manner. The biphasic dissociation of CTXA3 from heparin surface is observed over 3~4% CTX-occupancy, which implies heterogeneous domains with different binding modes on heparin are responsive for this behavior. Enzymatic degradations under affinity-protection with different CTX/heparin ratios generate various CTX-protected domains. Heparin disaccharide compositions of CTXA3-protected domains at different occupancies show different specificities toward N-acetylate at low occupancy but N-sulfate at high occupancy. They indicated diverse heparin binding sequences for CTXA3 binding. The affinity-protected domain at low CTX-occupancy shows the size in dp12-14 and low charge/mass characteristics. The removal of N-acetylate eliminate biphasic dissociation with reduced binding affinity / cooperativity and reduced ANS-binding fluorescence, which implies the possible role of hydrophobic interaction in N-acetylate groups to promote CTXA3 dimerization locally.
In summary, the structural factors of CTX-heparin interaction are determined both on CTXs and heparin. The heterogeneity of heparin and regulated heparin binding mode might determine different tissue specificity for different CTX homologues and their biological significance.

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43. Faham, S., Hileman, R.E., Fromm, J.R., Linhardt, R.J., and Ree, D.C. (1996) Heparin structure and interactions with basic fibroblast growth factor. Science 271, 1116-1120.
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48. Almond, A., Brass, A., and Sheehan, J.K. (1998) Dynamic exchange between stabilized conformations predicted for hyaluronan tetrasaccharides: comparison of molecular dynamics simulations with available NMR data. Glycobiology 8, 973-980.
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53. Haxaire, K., Braccini, I., Milas, M., Rinaudo, M., and Perez, S. (2000) Conformational behavior of hyaluronan in relation to its physical properties as probed by molecular modeling. Glycobiology 10, 587-594.
54. Gribbon, P., Heng, B.C., and Hardingham, T.E. (2000) The analysis of intermolecular interactions in concentrated hyaluronan solutions suggest no evidence for chain-chain association. Biochem. J. 350, 329-335.
55. Lindahl, U., Kusche-Gullberg, M, and Kjellén, L. (1998) Regulated diversity of heparan sulfate. J. Biol. Chem. 273, 24979-24982.
56. Esko, J.D., and Lindahl, U. (2001) Molecular diversity of heparan sulfate. J.Clin. Invest. 108, 169-173.
57. Esko, J.D., and Selleck, S.B. (2002) Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu. Rev. Biochem. 71, 435-471.
58. Sugahara, K., and Kitagawa, H. (2002) Heparin and heparan sulfate biosynthesis. IUBMB Life 54, 163-175.
59. Ihrcke, N.S., and Platt, J.L. (1996) Shedding of heparan sulfate proteoglycan by stimulated endothelial cells: evidence for proteolysis of cell-surface molecules. J. Cell Physiol. 168, 625-637.
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62. Park, P.W., Pier, G.B., Hinkes, M.T., and Bernfield, M. (2001) Exploitation of syndecan-1 shedding by Pseudomonas aeruginosa enhances virulence. Nature 411, 98-102.
63. Menozzi, F.D., Pethe, K., Bifani, P., Soncin, F., Brennan, M.J., and Locht, C. (2002) Enhanced bacterial virulence through exploitation of host glycosaminoglycans. Mol. Microbiol. 43, 1379-1386.
64. Lüke, H., and Prehm, P. (1999) Synthesis and shedding of hyaluronan from plasma membrane of human fibroblasts and metastatic and non-metastatic melanoma cells. Biochem. J. 343, 71-75.
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66. Turnbull, J., Powell, A., and Guimond, S. (2001) Heparan sulfate: decoding a dynamic multifunctional cell regulator. Trends Cell Biol. 11, 75-82.
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68. Gallagher, J.T. (2001) Heparan sulfate: growth control with a restricted sequence menu. J. Clin. Invest. 108, 357-361.
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71. Faham, S. Linhardt, R.J., and Rees, D.C. (1998) Diversity does make a difference: fibroblast growth factor-heparin interaction. Curr. Opin. Struct. Biol. 8, 578-586.
72. 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.
73. Spillmann, D., and Lindahl, U. (1994) Glycosaminoglycan-protein interactions: a question of specificity. Curr. Opin. Struct. Biol. 4, 677-682
74. Lindahl, U (2000) ‘Heparin’ – from anticoagulant drug into the new biology. Glycoconjugate J. 17, 597-605.
75. Mulloy, B., and Linhardt, R.J. (2001) Order out of complexity – protein structures that interact with heparin. Curr. Opin. Struct. Biol. 11, 623-628.
76. Linhardt, R.J. (2003) Heparin: structure and activity. J. Med. Chem. 46, 2551-2564.

Chapter 1 - part C: Introduction of surface plasmon resonance

1. Schuck, P. (1997) Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of interactions between biological macromolecules. Annu. Rev. Biophys. Biomol. Struct. 26, 541-566.
2. Nagata, K., and Handa, H. (2000) Real-time analysis of biomolecular interactions. Application of BIACORE. Springer-Verlag, Tokyo, Japan. pp13-22.
3. Green, R.J., Frazier, R.A., Shakesheff, K.M., Davies, M.C., Roberts, C.J., and Tendler, S.J.B. (2000) Surface plasmon resonance analysis of dynamic biological interactions with biomaterials. Biomaterials 21, 1823-1835.
4. Salamon, Z., Brown, M.F., and Tollin, G. (1999) Plasmon resonance spectroscopy: probing molecular interactions within membranes. Trends Biochem. Sci. 24, 213-219.
5. Rich, R.L., and Myszka, D.G. (2002) Survey of the year 2001 commercial optical biosensor literature. J. Mol. Recognit. 15, 352-376.
6. Day, Y.S., Baird, C.L., Rich, R.L., Myszka, D.G. (2002) Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface- and solution-based biophysical methods. Protein Sci. 11, 1017-1025.
7. Myszka, D.G. (1997) Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors. Curr. Opin. Biotech. 8, 50-57.
8. Myszka, D.G. (1999) Impoving biosensor analysis. J. Mol. Recog. 12, 279-284.
9. Canziani, G., Zhang, W., Cines, D., Rux, A., Willis, S., Cohen, G., Eisenberg, R., and Chaiken, I. (1999) Exploring Biomolecular recognition, using optical biosensors. Methods 19, 253-269.
10. Lipschultz, C.A., Li, Y. and Smith-Gill, S. (2000) Experimental design for analysis of complex kinetics using surface plasmon resonance. Methods 20, 310-318.
11. van Regenmortel, M.H.V. (2001) Analysing structure-function relationships with biosensors. Cell. Mol. Life Sci. 58, 794-800.
12. O’Shannessy, D.J., Brigham-Burke, M., Soneson, K.K., Hensley, P., and Brooks, I. (1993) Determination of rate and equilibrium binding constants for macromolecular interactions usinf surface plasmon resonance: use of nonlinear least squares analysis methods. Anal. Biochem. 212, 457-468.
13. Roden, L.D., and Myszka, D.G. (1996) Global analysis of a macromolecular interaction measured on BIAcore. Biochem. Biophys. Res. Comm. 225, 1073-1077.
14. Quinn, J.G., and O’Kennedy, R. (2001) Biosensor-based estimation of kinetics and equilibrium constants. Anal. Biochem. 290, 36-46.
15. Karlsson, R., and Fält, A. (1997) Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors. J. Immunol Methods 200, 121-133.
16. Marquart, A. http://home.hccnet.nl/ja.marquart/
17. MacKenzie, C.R., and Hirama, T., Deng, S-J., Bundle, D.R., Narang, S.A., and Young, N.M. (1996) Analysis by surface plasmon resonance of the influence of valence on the ligand binding affinity and kinetics of an anti-carbohydrate antibody. J. Biol. Chem. 271, 1527-1533.
18. Nieba, L., Neiba-Axmann, S.E., Persson, A., Hämäläinen, M., Edebratt, F., Hansson, A., Lidholm, J., Magnusson, K., Karlsson, A.F., and Plückthun, A. (1997) BIACORE analysis oh histidine-tagged proteins using a chelating NTA sensor chip. Anal. Biochem. 252, 217-228.
19. Schuck, P. (1997) Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of interactions between biological macromolecules. Annu. Rev. Biophys. Biomol. Struct. 26, 541-566.
20. Nagata, K., and Handa, H. (2000) Real-time analysis of biomolecular interactions. Application of BIACORE. Springer-Verlag, Tokyo, Japan. pp13-22.
21. Green, R.J., Frazier, R.A., Shakesheff, K.M., Davies, M.C., Roberts, C.J., and Tendler, S.J.B. (2000) Surface plasmon resonance analysis of dynamic biological interactions with biomaterials. Biomaterials 21, 1823-1835.
22. Salamon, Z., Brown, M.F., and Tollin, G. (1999) Plasmon resonance spectroscopy: probing molecular interactions within membranes. Trends Biochem. Sci. 24, 213-219.
23. Rich, R.L., and Myszka, D.G. (2002) Survey of the year 2001 commercial optical biosensor literature. J. Mol. Recognit. 15, 352-376.
24. Day, Y.S., Baird, C.L., Rich, R.L., Myszka, D.G. (2002) Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface- and solution-based biophysical methods. Protein Sci. 11, 1017-1025.
25. Myszka, D.G. (1997) Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors. Curr. Opin. Biotech. 8, 50-57.
26. Myszka, D.G. (1999) Impoving biosensor analysis. J. Mol. Recog. 12, 279-284.
27. Canziani, G., Zhang, W., Cines, D., Rux, A., Willis, S., Cohen, G., Eisenberg, R., and Chaiken, I. (1999) Exploring Biomolecular recognition, using optical biosensors. Methods 19, 253-269.
28. Lipschultz, C.A., Li, Y. and Smith-Gill, S. (2000) Experimental design for analysis of complex kinetics using surface plasmon resonance. Methods 20, 310-318.
29. van Regenmortel, M.H.V. (2001) Analysing structure-function relationships with biosensors. Cell. Mol. Life Sci. 58, 794-800.
30. O’Shannessy, D.J., Brigham-Burke, M., Soneson, K.K., Hensley, P., and Brooks, I. (1993) Determination of rate and equilibrium binding constants for macromolecular interactions usinf surface plasmon resonance: use of nonlinear least squares analysis methods. Anal. Biochem. 212, 457-468.
31. Roden, L.D., and Myszka, D.G. (1996) Global analysis of a macromolecular interaction measured on BIAcore. Biochem. Biophys. Res. Comm. 225, 1073-1077.
32. Quinn, J.G., and O’Kennedy, R. (2001) Biosensor-based estimation of kinetics and equilibrium constants. Anal. Biochem. 290, 36-46.
33. Karlsson, R., and Fält, A. (1997) Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors. J. Immunol Methods 200, 121-133.
34. Marquart, A. http://home.hccnet.nl/ja.marquart/
35. MacKenzie, C.R., and Hirama, T., Deng, S-J., Bundle, D.R., Narang, S.A., and Young, N.M. (1996) Analysis by surface plasmon resonance of the influence of valence on the ligand binding affinity and kinetics of an anti-carbohydrate antibody. J. Biol. Chem. 271, 1527-1533.
36. Nieba, L., Neiba-Axmann, S.E., Persson, A., Hämäläinen, M., Edebratt, F., Hansson, A., Lidholm, J., Magnusson, K., Karlsson, A.F., and Plückthun, A. (1997) BIACORE analysis oh histidine-tagged proteins using a chelating NTA sensor chip. Anal. Biochem. 252, 217-228.

Chapter 2: Heparin derivatives as tools to study protein-heparin interaction
– preparation and characterization of heparin derivatives

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2. Conrad, H.E. (1998) in Heparin-Binding Proteins, Academic Press, San Diego, CA., pp203-238
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16. Desai, U.R., Wang, H., Ampofo, S.A., and Linhardt, R.J. (1993) Oligosaccharide composition of heparin and low-molecular-weight heparins by capillary electrophoresis. Anal. Biochem. 213, 120-127.
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18. Godavarti, R., and Sasisekharan, R. (1998) Heparinase I from Flavobacterium heparinum. Role of positive charge in enzymatic activity. J. Biol. Chem. 273, 248-255.
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20. Shriver, Z., Hu, Y., Pojasek, K., and Sasisekharan, R. (1998) Heparinase II from Flavobacterium heparinum. Role of cysteine in enzymatic activity as probed by chemical modification and site- directed mutagenesis. J. Biol. Chem. 273, 22904-22912.
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22. Metcalfe, D.D., Thompson, H.L., Klebanoff, S.J., and Henderson-Jr, W.R. (1990) Oxidative degradation of rat mast-cell heparin proteoglycan. Biochem. J. 272, 51-57.
23. Lahiri, B., Lai, P.S., Pousada, M., Stanton, D., and Danishefsky, I. (1992) Depolymerization of heparin by complexed ferrous ions. Arch. Biochem. Biophys. 293, 54-60.
24. Nagasawa, K., Uchiyama, H., Sato, N., and Hatano, A. (1992) Chemical change involved in the oxidative-reductive depolymerization of heparin. Carbohydr. Res. 236, 165-180.
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28. Chuang, W.L., McAllister, H., and Rabenstein, D.L. (2002) Hexasaccharides from the histamine-modified depolymerization of porcine intestinal mucosal heparin. Carbohydr Res. 337, 935-945.
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30. Inoue, Y., and Nagasawa, K. Selective N-desulfation of heparin with dimethyl sulfoxide containing water or methanol. (1976) Carbohydr. Res. 46, 87-95.
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32. Matsuo, M., Takano, R., Kamei-Hayashi, K., and Hara, S. (1993) A novel regioselective desulfation of polysaccharide sulfates: Specific 6-O-desulfation with N,O–bis (trimethylsilyl) acetamide. Carbohydr. Res. 241, 209-215.
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34. Uchiyama, H., and Nagasawa, K. (1991) Changes in the structure and biological property of N-O sulfate-transferred, N-resulfated heparin. J. Biol. Chem. 266, 6756-6760.
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36. Shaklee, P.N., and Conrad, H.E. (1984) Hydrazinolysis of heparin and other glycosaminoglycans. Biochem. J. 217, 187-197.
37. Höök, M., Riesenfeld, J., and Lindahl, U. (1982) N-[3H]acetyl-labeling, a convenient method for radiolabeling of glycosaminoglycans. Anal. Biochem. 119, 236-245.
38. Taylor, R.L., and Conrad, H.E. (1972) Stoichiometric depolymerization of polyuronides and glycosaminoglycuronans to monosaccharides following reduction of their carbodiimide-activated carboxyl groups. Biochemistry 11, 1383-1388.
39. Nadkarni, V.D., Pervin, A., and Linhardt, R.J. (1994) Directional immobilization of heparin onto beaded supports. Anal. Biochem. 222, 59-67.
40. Melrose, J., Numata, Y., and Ghosh, P. (1996) Biotinylated hyaluronan: a versatile and highly sensitive probe capable of detecting nanogram levels of hyaluronan binding proteins (hyaladherins) on electroblots by a novel affinity detection procedure. Electrophoresis 17, 205-212.
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43. Zou, S., Magura, C.E., and Hurley, W.L. (1992) Heparin-binding properties of lactoferrin and lysozyme. Comp. Biochem. Physiol. B 103, 889-895.
44. Foxall, C., Holme, K.R., Liang, W., and Wei, Z. (1995) An enzyme-linked immunosorbent assay using biotinylated heparan sulfate to evaluate the interactions of heparin-like molecules and basic fibroblast growth factor. Anal. Biochem. 231, 366-373.
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48. Yang, B., Yang, B.L., and Goetinck, P.F. (1995) Biotinylated hyaluronic acid as a probe for identifying hyaluronic acid-binding proteins. Anal. Biochem. 228, 299-306.
49. Melrose, J., Little, C.B., and Ghosh, P. (1998) Detection of aggregatable proteoglycan populations by affinity blotting using biotinylated hyaluronan. Anal Biochem 256, 149-157.
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51. Malsch, R., Guerrini, M., Torri, G., Lohr, G., Casu, B., and Harenberg, J. (1994) Synthesis of a N'-alkylamine anticoagulant active low-molecular-mass heparin for radioactive and fluorescent labeling. Anal. Biochem. 217, 255-264.
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63. Casu, B., and Torri, G. (1999) Structural characterization of low molecular weight heparins. Semin. Thromb. Hemost. 25 (Suppl 3), 17-25.

Chapter 3: Structural diversity determines heparin binding modes, affinities and specificities of CTX homologues

1. Esko, J.D., and Selleck, S.B. (2002) Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu. Rev. Biochem. 71, 435-471.
2. Lindahl, U., Kusche-Gullberg, M., and Kjellén, L. (1998) Regulated diversity of heparan sulfate. J. Biol. Chem. 273, 24979-24982.
3. Esko, J.D., and Lindahl, U. (2001) Molecular diversity of heparan sulfate. J. Clin. Invest. 108, 169-173.
4. Turnbull, J., Powell, A., and Guimond, S. (2001) Heparan sulfate: decoding a dynamic multifunctional cell regulator. Trends Cell Biol. 11, 75-82.
5. Conrad, H.E. in Heparin-Binding Proteins, (1998) Academic Press, San Diego, CA. pp203-238.
6. 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.
7. Whisstock, J.C., Pike, P.N., Jin, L., Skinner, R., Pei, X.Y., Carrell, R. W., and Lesk, A. M. (2000) Conformational changes in serpins: II. The mechanism of activation of antithrombin by heparindagger. J. Mol. Biol. 301, 1287-1305.
8. 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.
9. Rusnati, M., Coltrini, D., Caccia, P., Dell'Era, P., Zoppetti, G., Oreste, P., Valsasina, B., Presta, M. (1994) Distinct role of 2-O-, N-, and 6-O-sulfate groups of heparin in the formation of the ternary complex with basic fibroblast growth factor and soluble FGF receptor-1. Biochem Biophys Res Commun.203, 450-458.
10. Lundin, L., Larsson, H., Kreuger, J., Kanda, S., Lindahl, U., Salmivirta, M., and Claesson-Welsh, L. (2000) Selectively desulfated heparin inhibits fibroblast growth factor-induced mitogenicity and angiogenesis. J. Biol. Chem. 275, 24653-24660.
11. Kreuger, J., Salmivirta, M., Sturiale, L., Giménez-Gallego, G., and Lindahl, U. (2001) Sequence analysis of heparan sulfate epitopes with graded affinities for fibroblast growth factors 1 and 2. J. Biol. Chem. 276, 30744-30752.
12. Ishihara, M., Takano, R., Kanda, T., Hayashi, K., Hara, S., Kikuchi, H., and Yoshida, K. (1995) Importance of 6-O-sulfate groups of glucosamine residues in heparin for activation of FGF-1 and FGF-2. J. Biochem. 118, 1255-1260.
13. Pye, D.A., Vives, R.R., Hyde, P., Gallagher, J.T. (2000) Regulation of FGF-1 mitogenic activity by heparan sulfate oligosaccharides is dependent on specific structural features: differential requirements for the modulation of FGF-1 and FGF-2. Glycobiology 10, 1183-1192.
14. Kuschert, G.S.V., Coulin, F., Power, C.A., Proudfoot, A.E.I., Hubbard, R.E., Hoogewerf, A.J., and Well, T.N.C. (1999) Glycosaminoglycans interact selectively with chemokines and modulate receptor binding and cellular responses. Biochemistry 38, 12959-12968.
15. Goger, B., Halden, Y., Rek, A., Mösl, R., Pye, D., Gallagher, J.T., and Kungl, A.J. (2002) Different Affinities of Glycosaminoglycan Oligosaccharides for Monomeric and Dimeric Interleukin-8: A Model for Chemokine Regulation at Inflammatory Sites. Biochemistry 41, 1640-1646.
16. Vivès, R.R., Sadir, R., Imberty, A., Rencurosi, A., and Lortat-Jacob, H. (2002) A kinetics and modeling study of RANTES (9-68) binding to heparin reveals a mechanism of cooperative oligomerization. Biochemistry 41, 14779-14789.
17. Proudfoot, A.E., Handel, T.M., Johnson, Z., Lau, E.K., LiWang, P., Clark-Lewis, I., Borlat, F., Wells, T.N., and Kosco-Vilbois, M.H. (2003) Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. Proc. Natl. Acad. Sci. U.S.A. 100, 1885-1890.
18. Li, Q., Park, P.W., Wilson, C.L., and Parks, W.C. (2002) Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury. Cell 111, 635-646.
19. Cadigan, K.M. (2002) Regulating morphogen gradients in the Drosophila wing. Semin. Cell Dev. Biol. 13, 83-90.
20. Paine-Saunders, S., Viviano, B.L., Economides, A.N., and Saunders, S. (2002) Heparan sulfate proteoglycans retain Noggin at the cell surface. a potential mechanism for shaping bone morphogenetic protein gradients. J. Biol. Chem. 277, 2089-2096.
21. Viviano, B.L., Paine-Saunders, S., Gasiunas, N., Gallagher, J., and Saunders, S. (2004) Domain-specific modification of heparan sulfate by Qsulf1 modulates the binding of the bone morphogenetic protein antagonist Noggin. J Biol Chem. 279, 5604-5611.
22. Giráldez, A.J., Copley, R.R., Cohen, S.M. (2002) HSPG modification by the secreted enzyme Notum shapes the Wingless morphogen gradient. Dev Cell. 2, 667-676.
23. Gustafsson, M, Flood, C., Jirholt, P., and Borén, J. (2004) Retention of atherogenic lipoproteins in atherogenesis. Cell. Mol. Life Sci. 61, 4-9.
24. Belting, M. (2003) Heparan sulfate proteoglycan as a plasma membrane carrier. Trend Biochem. Sci., 28, 145-151.
25. Liu J, and Thorp SC. (2002) Cell surface heparan sulfate and its roles in assisting viral infections. Med Res Rev. 22, 1-25.
26. Spillmann, D. (2001) Heparan sulfate: anchor for viral intruders? Biochimie, 83, 811-817.
27. Mettenleiter, T.C. (2002) Brief overview on cellular virus receptors. Virus Res. 82, 3-8.
28. Wu, W. (1998) Cobra Cardiotoxin and Phospholipase A2 as GAG-binding Toxins; On the Path from Structure to Cardiotoxicity and Inflammation. Trends. Cardiovas. Med. 8, 270-278.
29. Endo, T., and Tamiya, N. (1991) in Snake Toxins, Pergamon Press, New Yorks , pp165-222.
30. Lambeau, G., and Lazdunski, M. (1999) Receptors for a growing family of secreted phospholipases A2. Trends Pharmacol Sci. 20, 162-170.
31. Lee, C.Y., Lin, J.S., and Wei, J.W. (1970) Identification of cardiotoxin with Cobramine B, DLF, Toxin □ and cobra venom cytotoxin. 2nd Intern. Symp. Animal and Plant Toxins, Tel-Aviv, Israel. 307-318.
32. Chen, Y.H., Hu, C.T., and Yang, J.T. (1984) Membrane disintegration and hemolysis of human erythrocytes by snake venom cardiotoxin (a membrane-disruptive polypeptide). Biochem. Int., 8, 329-338.
33. Lee, C.Y., Chang, C.C., Chiu, T.H., Chiu, P.J.S., Tseng, T.C., and Lee, S.Y. (1968) Pharmacological properties of cardiotoxin isolated from Formosan cobra venom. Arch. Pharmak. Exp. Path. 259, 360-374.
34. Chien, K.Y., Chiang, C.M., Hseu, Y.C., Vyas, A.A., Rule, G.S. and Wu, W. (1994) Two distinct types of cardiotoxin as revealed by the structure and activity relationship of their interaction with zwitterionic phospholipid dispersions. J. Biol. Chem. 269, 14473-14483.
35. Chien, K.Y., Huang, W.N., Jean, J.H. and Wu, W. (1991) Fusion of sphingomyelin vesicles induced by proteins from Taiwan cobra (Naja naja atra) venom. Interactions of zwitterionic phospholipids with cardiotoxin analogues. J. Biol. Chem. 266, 3252-3259.
36. Vyas, A.A., Pan, J., Patel, H.V., Vyas, K.A., Chiang, Sheu, Y. Hwang, J., and Wu, W. (1997) Analysis of binding of cobra cardiotoxins to heparin reveals a new beta-sheet heparin-binding structural motif. J. Biol. Chem. 272, 9661-9670.
37. Patel, H.V., Vyas, A.A., Vyas, K.A., Liu, Y., Chiang, C., Chi, L., and Wu, W. (1997) Heparin and heparan sulfate bind to snake cardiotoxin. Sulfated oligosaccharides as a potential target for cardiotoxin action. J. Biol. Chem. 272, 1484-1492.
38. Vyas, K.A., Patel, H.V., Vyas, A.A., and Wu, W. (1998) Glycosaminoglycans bind to homologous cardiotoxins with different specificity. Biochemistry 37, 4527-4534.
39. Forouhar,F., Huang, W.N., Lin, J.H., Chien, K.Y. Wu, W., and Hsiao, C.D. (2003) Structural basis of membrane-induced cardiotoxin A3 oligomerization. J. Biol. Chem. 278, 21980-21988.
40. Huang, W.N., Sue, S.C., Wang, D.S., Wu, P.L., and Wu W. (2003) Peripheral binding mode and penetration depth of cobra cardiotoxin on phospholipid membranes as studied by a combined FTIR and computer simulation approach. Biochemistry. 42, 7457-7466.
41. Sue, S., Chien, K., Huang, W., Abraham, J.K., Chen, K., and Wu, W. (2002) Heparin binding stabilizes the membrane-bound form of cobra cardiotoxin. J. Biol. Chem. 277, 2666-2673.
42. Tseng, L.F., Chiu, T.H., and Lee, C.Y. (1968) Absorption and distribution of 131I-labeled cobra venom and its purified toxins. Toxicol. Appl. Pharmaco., 12, 526-535.
43. Lai, M.K., Wen, C.Y., and Lee, C.Y. (1972) Local lesions caused by cardiotoxin isolated from Formosan cobra venom. J. Formosan Med. Assoc., 71, 328-332.
44. Francis, B, Seebart, C, and Kaiser, I.I. (1992) Citrate is an endogeneous inhibitor of snake venom enzymes by metal-ion chelation. Toxicon 30, 1239-1246.
45. Odell, G.V., Fenton, A.W., Ownby, C.L., Doss, M.P., and Schmidt, J.O. (1999) The role of venom citrate. Toxicon 37, 407-409.
46. Phillips, C.L., Ullman, B., Brennan, R.G., and Hill, C.P. (1999) Crystal structures of adenine phosphoribosyltransferase from Leishmania donovani. EMBO J. 18, 3533-3545.
47. Mao, Y., Nickitenko, A., Duan, X., Lloyd, T.E., Wu, M.N., Bellen, H., and Quiocho, F.A. (2000) Crystal structure of the VHS and FYVE tandem domains of Hrs, a protein involved in membrane trafficking and signal transduction. Cell 100, 447-456.
48. Schmidt, U., and Darke, P.L. (1997) Dimerization and activation of the hepes simplex virus type 1 protease. J. Biol. Chem. 272, 7732-7735.
49. Buisson, M., Valette, E., Hernandez, J.F., Baudin, F., Ebel, C., Morand, P., Seigneurin, J.M., Arlaud, G.J., and Ruigrok, R.W. (2001) Functional determinants of the Epstein-Barr virus protease. J. Mol. Biol. 311, 217-228.
50. Lee, S-H., Guan, H-H., Wang, C-H., Huang, W-N., Chen, C-J. and Wu, W. (2004) Structural basis of venom citrate-dependent heparan sulfate-mediated cell surface retention of cobra cardiotoxin A3. J. Biol. Chem. (submitted)
51. Inoue, Y., and Nagasawa, K. (1976) Selective N-desulfation of heparin with dimethyl sulfoxide containing water or methanol. Carbohydr. Res. 46, 87-95.
52. Jaseja, M., Rej, R.N., Sauriol, F., and Perlin, A.S. (1989) Novel region- and stereo-selective modifications of heparin in alkaline solution: nuclear magnetic resonance spectroscopic evidence. Can. J. Chem. 67, 1449-1456.
53. Matsuo, M., Takano, R., Kamei-Hayashi, K., and Hara, S. (1993) A novel regioselective desulfation of polysaccharide sulfates: Specific 6-O-desulfation with N,O–bis (trimethylsilyl) acetamide. Carbohydr. Res. 241, 209-215.
54. Shaklee, P.N., and Conrad, H.E. (1984) Hydrazinolysis of heparin and other glycosaminoglycans. Biochem. J. 217, 187-197.
55. Esko, J.D., Elgavish, A., Prasthofer, T., Taylor, W. H., and Weinke, J.L. (1986) Sulfate transport-deficient mutants of Chinese hamster ovary cells. Sulfation of glycosaminoglycans dependent on cysteine. J. Biol. Chem. 261, 15725-15733.
56. Berkowitz, O., Wirtz, M., Wolf, A., Kuhlmann, J., and Hell, R. (2002) Use of biomolecular interaction analysis to elucidate the regulatory mechanism of the cysteine synthase complex from Arabidopsis thaliana. J. Biol. Chem. 277, 30629-30634.
57. Bilwes, A., Rees, B., Moras, D., Menez, R., and Menez, A. (1994) X-ray structure at 1.55 Å of toxin gamma, a cardiotoxin from Naja nigricollis venom. Crystal packing reveals a model for insertion into membranes. J. Mol. Biol. 239, 122-136.
58. Capila, I., Hernaiz, M.J., Mo, Y.D., Mealy, T.R., Campos, B., Dedman, J.R., Linhardt, R.J., and Seaton, B.A. (2001) Annexin V--heparin oligosaccharide complex suggests heparan sulfate--mediated assembly on cell surfaces. Structure 9, 57-64.

Chapter 4: Molecular mechanism of CTXA3-heparin interaction (I)
- Effect of venom citrate
-
1. Francis, B, Seebart, C, and Kaiser, I.I. (1992) Citrate is an endogeneous inhibitor of snake venom enzymes by metal-ion chelation. Toxicon 30, 1239-1246.
2. Odell, G.V., Fenton, A.W., Ownby, C.L., Doss, M.P., and Schmidt, J.O. (1999) The role of venom citrate. Toxicon 37, 407-409.
3. Batra, R., Khayat, R., and Tong, L. (2001) Molecular mechanism for dimerization to regulate the catalytic activity of human cytomegalovirus protease. Nat. Struct. Biol. 8, 810-817.
4. Mao, Y., Nickitenko, A., Duan, X., Lloyd, T.E., Wu, M.N., Bellen, H., and Quiocho, F.A. (2000) Crystal structure of the VHS and FYVE tandem domains of Hrs, a protein involved in membrane trafficking and signal transduction. Cell 100, 447-456.
5. Esko, J.D., and Selleck, S.B. (2002) Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu. Rev. Biochem. 71, 435-471.
6. Lindahl, U., Kusche-Gullberg, M., and Kjellén, L. (1998) Regulated diversity of heparan sulfate. J. Biol. Chem. 273, 24979-24982.
7. Turnbull, J., Powell, A., and Guimond, S. (2001) Heparan sulfate: decoding a dynamic multifunctional cell regulator. Trends Cell Biol. 11, 75-82.
8. Conrad, H.E. (1998) in Heparin-Binding Proteins, Academic Press, San Diego, CA, pp203-238.
9. Kreuger, J., Salmivirta, M., Sturiale, L., Giménez-Gallego, G., and Lindahl, U. (2001) Sequence analysis of heparan sulfate epitopes with graded affinities for fibroblast growth factors 1 and 2. J. Biol. Chem. 276, 30744-30752.
10. Ishihara, M., Takano, R., Kanda, T., Hayashi, K., Hara, S., Kikuchi, H., and Yoshida, K. (1995) Importance of 6-O-sulfate groups of glucosamine residues in heparin for activation of FGF-1 and FGF-2. J. Biochem. 118, 1255-1260.
11. Pellegrini, L., Burke, D.F., von Delft, F., Mulloy, B., and Blundell, T.L. (2000) Crystal structure of fibroblast growth factor receptor ectodomain bound to ligand and heparin. Nature 407, 1029-1034.
12. Vivès, R.R., Sadir, R., Imberty, A., Rencurosi, A., and Lortat-Jacob, H. (2002) A kinetics and modeling study of RANTES (9-68) binding to heparin reveals a mechanism of cooperative oligomerization. Biochemistry 41, 14779-14789.
13. Proudfoot, A.E., Handel, T.M., Johnson, Z., Lau, E.K., LiWang, P., Clark-Lewis, I., Borlat, F., Wells, T.N., and Kosco-Vilbois, M.H. (2003) Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. Proc. Natl. Acad. Sci. U.S.A. 100, 1885-1890.
14. Li, Q., Park, P.W., Wilson, C.L., and Parks, W.C. (2002) Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury. Cell 111, 635-646.
15. Cadigan, K.M. (2002) Regulating morphogen gradients in the Drosophila wing. Semin. Cell Dev. Biol. 13, 83-90.
16. Paine-Saunders, S., Viviano, B.L., Economides, A.N., and Saunders, S. (2002) Heparan sulfate proteoglycans retain Noggin at the cell surface. A potential mechanism for shaping bone morphogenetic protein gradients. J. Biol. Chem. 277, 2089-2096.
17. Gustafsson, M, Flood, C., Jirholt, P., and Borén, J. (2004) Retention of atherogenic lipoproteins in atherogenesis. Cell. Mol. Life Sci. 61, 4-9.
18. Belting, M. (2003) Heparan sulfate proteoglycan as a plasma membrane carrier. Trend Biochem. Sci., 28, 145-151.
19. Liu J, and Thorp S.C. (2002) Cell surface heparan sulfate and its roles in assisting viral infections. Med Res Rev. 22, 1-25.
20. Lo, T.B., Chen, Y.H., and Lee, C.Y. (1966) Chemical studies of Formosan cobra (Naja naja atra) venom. I. Chromatographic separation of crude venom on CM-sephadex and preliminary characterization of its components. J. Chinese Chem. Soc. 13, 25-37.
21. Wu, W. (1998) Cobra cardiotoxin and phospholipase A2 as GAG-binding toxins; on the path from structure to cardiotoxicity and inflammation. Trends. Cardiovas. Med. 8, 270-278.
22. Sue, S., Chien, K., Huang, W., Abraham, J.K., Chen, K., and Wu, W. (2002) Heparin binding stabilizes the membrane-bound form of cobra cardiotoxin. J. Biol. Chem. 277, 2666-2673.
23. Vyas, A.A., Pan, J., Patel, H.V., Vyas, K.A., Chiang, Sheu, Y. Hwang, J., and Wu, W. (1997) Analysis of binding of cobra cardiotoxins to heparin reveals a new beta-sheet heparin-binding structural motif. J. Biol. Chem. 272, 9661-9670.
24. Patel, H.V., Vyas, A.A., Vyas, K.A., Liu, Y., Chiang, C., Chi, L., and Wu, W. (1997) Heparin and heparan sulfate bind to snake cardiotoxin. Sulfated oligosaccharides as a potential target for cardiotoxin action. J. Biol. Chem. 272, 1484-1492.
25. Tseng, L.F., Chiu, T.H., and Lee, C.Y. (1968) Absorption and distribution of 131I-labeled cobra venom and its purified toxins. Toxicol. Appl. Pharmaco., 12, 526-535.
26. Lai, M.K., Wen, C.Y., and Lee, C.Y. (1972) Local lesions caused by cardiotoxin isolated from Formosan cobra venom. J. Formosan Med. Assoc., 71, 328-332.
27. Chien, K.Y., Chiang, C.M., Hseu, Y.C., Vyas, A.A., Rule, G.S. and Wu, W. (1994) Two distinct types of cardiotoxin as revealed by the structure and activity relationship of their interaction with zwitterionic phospholipid dispersions. J. Biol. Chem. 269, 14473-14483.
28. Yamada, S., Murakami, T., Tsuda, H., Yoshida, K., and Sugahara, K. (1995) Isolation of the porcine heparin tetrasaccharides with glucuronate 2-O-sulfate. Heparinase cleaves glucuronate 2-O-sulfate-containing disaccharides in highly sulfated blocks in heparin. J. Biol. Chem. 270, 8696-8705.
29. Inoue, Y., and Nagasawa, K. (1976) Selective N-desulfation of heparin with dimethyl sulfoxide containing water or methanol. Carbohydr. Res. 46, 87-95.
30. Jaseja, M., Rej, R.N., Sauriol, F., and Perlin, A.S. (1989) Novel region- and stereo-selective modifications of heparin in alkaline solution: nuclear magnetic resonance spectroscopic evidence. Can. J. Chem. 67, 1449-1456.
31. Matsuo, M., Takano, R., Kamei-Hayashi, K., and Hara, S. (1993) A novel regioselective desulfation of polysaccharide sulfates: Specific 6-O-desulfation with N,O–bis (trimethylsilyl) acetamide. Carbohydr. Res. 241, 209-215.
32. Berkowitz, O., Wirtz, M., Wolf, A., Kuhlmann, J., and Hell, R. (2002) Use of biomolecular interaction analysis to elucidate the regulatory mechanism of the cysteine synthase complex from Arabidopsis thaliana. J. Biol. Chem. 277, 30629-30634.
33. Schmidt, U., and Darke, P.L. (1997) Dimerization and activation of the herpes simplex virus type 1 protease. J. Biol. Chem. 272, 7732-7735.
34. Sue, S.C., Brisson, J.R., Chang, S.C., Huang, W.N., Lee, S.C., Jarrell, H.C., and Wu, W. (2001) Structures of heparin-derived disaccharide bound to cobra cardiotoxins: context-dependent conformational change of heparin upon binding to the rigid core of the three-fingered toxin. Biochemistry. 40, 10436-10446.
35. Bilwes, A., Rees, B., Moras, D., Menez, R., and Menez, A. (1994) X-ray structure at 1.55 Å of toxin gamma, a cardiotoxin from Naja nigricollis venom. Crystal packing reveals a model for insertion into membranes. J. Mol. Biol. 239, 122-136.
36. Satoh, A., Toida, T., Yoshida, K., Kojima, K., and Matsumoto, I. (2000) New role of glycosaminoglycans on the plasma membrane proposed by their interaction with phosphatidylcholine. FEBS Lett. 477, 249-252.

Chapter 5: Molecular mechanism of CTXA3-heparin interaction (II)
– Structural determinants of chain-length and specific ligand on heparin

1. Esko, J.D., and Selleck, S.B. Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu. Rev. Biochem. 71, 435-471 (2002).
2. Lindahl, U., Kusche-Gullberg, M., and Kjellén, L. Regulated diversity of heparan sulfate. J. Biol. Chem. 273, 24979-24982 (1998).
3. Turnbull, J., Powell, A., and Guimond, S. Heparan sulfate: decoding a dynamic multifunctional cell regulator. Trends Cell Biol. 11, 75-82 (2001).
4. 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.
5. Kuschert, G.S.V., Coulin, F., Power, C.A., Proudfoot, A.E.I., Hubbard, R.E., Hoogewerf, A.J., and Well, T.N.C. (1999) Glycosaminoglycans interact selectively with chemokines and modulate receptor binding and cellular responses. Biochemistry 38, 12959-12968.
6. Goger, B., Halden, Y., Rek, A., Mösl, R., Pye, D., Gallagher, J.T., and Kungl, A.J. (2002) Different affinities of glycosaminoglycan oligosaccharides for monomeric and dimeric interleukin-8: a model for chemokine regulation at inflammatory sites. Biochemistry 41, 1640-1646.
7. Vivès, R.R., Sadir, R., Imberty, A., Rencurosi, A., and Lortat-Jacob, H. (2002) A kinetics and modeling study of RANTES (9-68) binding to heparin reveals a mechanism of cooperative oligomerization. Biochemistry 41, 14779-14789.
8. Sadir, R., Baleux, F., Grosdidier, A., Imberty, A., and Lortat-Jacob, H. (2001) Characterization of the stromal cell-derived factor-1alpha-heparin complex. J. Biol. Chem. 276, 8288-8296.
9. Aviezer, D., Levy, E., Safran, M., Svahn, C., Buddecke, E., Schmidt, A., David, G., Vlodavsky, I., and Yayon, A. (1994) Differential structural requirements of heparin and heparan sulfate proteoglycans that promote binding of basic fibroblast growth factor to its receptor. J. Biol. Chem. 269, 114-121.
10. Petersen, F., Brandt, E., Lindahl, U., and Spillmann, D. (1999) Characterization of a neutrophil cell surface glycosaminoglycan that mediates binding of platelet factor 4. J. Biol. Chem. 274, 12376-12382.
11. Conrad, H.E. (1998) in Heparin-Binding Proteins, Academic Press, San Diego, CA, pp203-238.
12. 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.
13. Whisstock, J.C., Pike, P.N., Jin, L., Skinner, R., Pei, X.Y., Carrell, R. W., and Lesk, A. M. (2000) Conformational changes in serpins: II. The mechanism of activation of antithrombin by heparin dagger. J. Mol. Biol. 301, 1287-1305.
14. Rusnati, M., Coltrini, D., Caccia, P., Dell'Era, P., Zoppetti, G., Oreste, P., Valsasina, B., Presta, M. (1994) Distinct role of 2-O-, N-, and 6-O-sulfate groups of heparin in the formation of the ternary complex with basic fibroblast growth factor and soluble FGF receptor-1. Biochem Biophys Res Commun.203, 450-458.
15. Lundin, L., Larsson, H., Kreuger, J., Kanda, S., Lindahl, U., Salmivirta, M., and Claesson-Welsh, L. (2000) Selectively desulfated heparin inhibits fibroblast growth factor-induced mitogenicity and angiogenesis. J. Biol. Chem. 275, 24653-24660.
16. Kreuger, J., Salmivirta, M., Sturiale, L., Giménez-Gallego, G., and Lindahl, U. (2001) Sequence analysis of heparan sulfate epitopes with graded affinities for fibroblast growth factors 1 and 2. J. Biol. Chem. 276, 30744-30752.
17. Ishihara, M., Takano, R., Kanda, T., Hayashi, K., Hara, S., Kikuchi, H., Yoshida, K. (1995) Importance of 6-O-sulfate groups of glucosamine residues in heparin for activation of FGF-1 and FGF-2. J. Biochem. 118, 1255-1260.
18. Pye, D.A., Vives, R.R., Hyde, P., Gallagher, J.T. (2000) Regulation of FGF-1 mitogenic activity by heparan sulfate oligosaccharides is dependent on specific structural features: differential requirements for the modulation of FGF-1 and FGF-2. Glycobiology 10, 1183-1192.
19. Proudfoot, A.E., Handel, T.M., Johnson, Z., Lau, E.K., LiWang, P., Clark-Lewis, I., Borlat, F., Wells, T.N., and Kosco-Vilbois, M.H. (2003) Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. Proc. Natl. Acad. Sci. U.S.A. 100, 1885-1890.
20. 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.
21. Manning, G.S. (1978) The molecular theory of polyelectrolyte solutions with applications to the electrostatic properties of polynucleotides. Q. Rev. Biophys. 11, 179-246.
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Chapter 6: Molecular mechanism of CTXA3-heparin interaction (III)
– The role of N-acetylation on heparin

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