5 nM/well 2.5 nM/well
47
Fig. 2-8. Schematic drawing of HDFs attachment suppression mechanisms on the EF1zz/531 (1:4)-conjugated polysaccharide matrices.
HDFs attachment to the both peptide-conjugated chitosan and alginate matrices promotes integrin activation through the FAK phosphorylation. But at EF1zz/531 (1:4), integrin activation may not be smoothly activated, because of insufficient phosphorylation of FAK. PI3K signaling pathway may be block the inside-out signaling to activate a non-active integrin, whereas PKA signaling pathway promotes the integrin activation.
49 3
- -PCM
1
Extracellular
matrix ECM 83, 84) ECM
4, 85)
- 26, 30, 65)
-26)
30, 65)
-30, 65)
N-(m-maleimidobenzoyloxy) succinimide MBS MB-
MB-Cys
30, 65)
-PCM PCM
86) PCM
ECM 86)
87)
pH
86)
88)
88)
N Cys pH4-5
89)
88, 90, 91)
-α1 A99a a modified peptide of A99 ALRGDN mouse laminin α1 chain 1145-1150 EF1XmR RLQLQEGRLHFXFD mouse laminin α1 chain 2751-2763
1,
2--
-PCM
--PCM
51 2
3-2-1
α1 FB12 α2 P1E6 α3 P1B5 αv P1B5 β1 6S6 β3 25E11 Millipore
IgG Wako
3-2-2
1
Table 3-1 C Tyr
N Cys
Gly 2 CGG Gly2
-A99a ε-aminocaproic acid 2 εACA2
ε-aminocaproic acid 4 εACA4
Table 3-1. Laminin peptides.
a For conjugation to aldehyde-alginate, a cysteine residue was added at the N-terminus and two glycine residues were used as a spacer between the cysteine and the peptide sequences. A tyrosine residue was added at the C-terminus for determination of peptide concentration.
3-2-3
human dermal fibroblasts HDFs
PC12 1
3-2-4
Sodium Alginate 80-120 cP 50 mg 0.25 mmol of sugar unit MW 60,000 Wako
1 mL 1 mL 85
µmol Wako 6 6 14.2
µL 255 nmol 3 3500 MWCO
47 mg
Peptide Sequencea Chain (Residues) Calc. Mw ESI-MS (m/z)
A99a CGGALRGDNY Mouse Laminin α1 (1145-1150) 1024.4634 1024.4634
EF1XmR CGGRLQLQEGRLHFXFDY, X = Nle Mouse Laminin α1 (2751-2763) 2151.0922 2151.0901 A99T CGGDGNLARAPGQFTGY Mouse Laminin α1 (1141-1153, scrambled) 1682.7709 1682.7709
AmpliteTM Colorimetric Aldehyde
Quantitation Kit AAT Bioquest 0.82 mmol/g
3-2-5
-80 µg 0.01M EDTA/0.2 M pH 5.3
200 µL 0.1% TFA 50 µL 2
2 10 mM Cys 0.1% TFA 50 µL 1
-3-2-6 -PCM
Chitosan 1 mg purified powder MW ~15,000 Wako 4% 5 mL
96-well well 50 µL 30 ng/mm2
24
96-well well 1% NaHCO3 100 µL 10
PBS 100 µL 2 well
-75 µL 0.133 mg/mL 1% NaHCO3 37.5 µL 15
-PCM -PCM PBS
100 µL 2
3-2-7
1 -PCM
96-well well 100 µL 2 × 104 cells/well
37 5% CO2 1
Olympus ImageJ
3-2-8 EDTA
53
2
-PCM 96-well well 100
µL 1 × 104 cells/well 37 5% CO2 1
Olympus ImageJ
3-2-10
1 -PCM
8-well Nalge Nunc well 100 µL
8 × 103 cells/well 37 5% CO2 2
3-2-11
1
-PCM 96-well well 100 µL 5 × 103
cells/well 37 5% CO2 24
Olympus ImageJ
%
3
3-3-1
-1, 2-Scheme 3-1
α1 A99a EF1XmR N
Cys Gly 2 CGG C
Tyr
Cys -Cys
55
3-3-2 -PCM
96-well well
--PCM -PCM
Fig. 3-1 A99a-PCM EF1XmR-PCM
Fig. 3-1A A99
A99T A99T-PCM
-PCM
A99a-PCM EF1XmR-PCM Fig. 3-1B
- 26, 28, 30)
Fig. 3-1. Cell attachment activity of peptide-PCMs.
Laminin-derived peptides, A99a and EF1XmR, and A99T, an A99 scramble peptide, were used. Various amount
57
3-3-3 -PCM EDTA
-PCM
EDTA Fig.
3-2 A99a-PCM EF1XmR-PCM EDTA
Fig. 3-2A A99a-PCM
EF1XmR-PCM
A99a-PCM EF1XmR-PCM
A99a-PCM αv
β3 Fig. 3-2B EF1XmR-PCM
α2 β1
Fig. 3-2C A99a-PCM αvβ3
EF1XmR-PCM α2β1
59 3-3-4 A99a-PCM
A99a A99a-PCM
Fig. 3-3 Gly 2 Gly2 ε-aminocaproic acid 2
εACA2 ε-aminocaproic acid 4 εACA4 A99a A99a no spacer
A99a-PCM
A99a-PCM Gly2 A99a-PCM
εACA4 A99a-PCM
A99a-PCM
-PCM
Fig. 3-3. Effect of spacers between peptide and alginate on the biological activity of A99a-PCM.
Various amount of A99a-PCMs with Gly2, εACA2, and εACA4, or without a spacer were examined for the cell attachment activity. HDFs (2 × 104 cells/well) were allowed to attach to the A99a-spacer-PCMs for 30 min, and then stained. The attached cells in three randomly-selected fields were counted. The graph is representative of at least three similar experiments. Each value represents the mean ± SD.
3-3-5 A99a-PCM A99a-PCM
Fig. 3-4 Gly2 εACA4
A99a-PCM
Fig. 3-4a, b A99a-PCM
Fig. 3-4c
-PCM
Fig. 3-4. Immunostaining for both actin and vinculin in the cells on A99a-PCMs.
The peptide solutions (50 µL of 40 µM), Cys-Gly2-A99a, Cys-εACA4-A99a, and Cys-A99a, were reacted with the aldehyde-alginate (20 µg/well). After quenching with cysteine, all of the A99a-alginate solution was added to chitosan-coated (30 µg/well) 8-well chamber slides. HDFs (8 × 103 cells/well) were allowed to attach to the peptides-PCMs for 2 h. HDFs were fixed and then stained with phalloidin, anti-vinculin, and DAPI for actin filament (green), focal contact (red), and nuclei (blue), respectively. Scale bar = 50 µm.
61 3-3-6 A99a-PCM
A99a-PCM Fig. 3-5 Gly2 εACA4
A99a-PCM
A99a-PCM
A99a- 92)
A99a-PCM
Fig. 3-5. Neurite outgrowth activity on A99a-PCMs.
Various amount of peptides were conjugated to the aldehyde-alginate, and the peptide-alginate solution (75 µL) was added to chitosan (10 µg/well) coated in 96-well plates. PC12 cells (5 × 103 cells/well) were added and incubated for 24 h. (A) The data are shown as a percentage of the extended cells on the A99a-spacer-PCMs, and are representative of at least three similar experiments. The cells that had neurites more than double the length of the cell body were defined as extended cells. Each value represents the mean ± SD. *P < 0.05 (Student’s t-test) against no spacer. (B) Cell morphology of A99a-spacer-PCMs (amount of peptides 10 nmol/well). Scale bar = 100 µm.
4
1,
2
-88)
-PCM
-PCM
--PCM
Cys--PCM
-PCM
93, 94)
-95)
Lys
Gly2 Gly 3.67 Å/residue 92) εACA2 εACA 8.70 Å/residue
92) εACA4 A99a A99a-PCM
εACA4 A99a-PCM
A99a-PCM
εACA4
63
30)
-PCM
-PCM -PCM -PCM
65
2, 3) Extracellular matrix ECM
ECM
11-15, 68, 69)
ECM
-ECM
26, 30, 65) -111
-ECM
-3
1 - -111
-111 -111
60
--111 26
-14
-29
-111 6
-111
-- -111
--111
--111
2
ECM FIB1 EF1zz 531
2
-α2β1 EF1zz
α3β1 531 EF1zz/531 1:4
- FIB1/EF1zz-
FIB1/531-FAK
PI3K PKA β1
β1 PI3K
PKA
-ECM 3
67
-PCM A99a-PCM
-PCM -PCM
- -111
-6
- -111
--PCM
--PCM ECM
69
National Institutes of Health
NCI NIH
71 1
Hozumi, K., Sasaki, A., Yamada, Y., Otagiri, D., Kobayashi, K., Fujimori, C., Katagiri, F., Kikkawa, Y., Nomizu, M.
Reconstitution of laminin-111 biological activity using multiple peptide coupled to chitosan scaffolds.
Biomaterials, 33, 4241-4250 (2012).
2
Hozumi, K., Fujimori, C., Katagiri, F., Kikkawa, Y., Nomizu, M.
Suppression of cell adhesion through specific integrin crosstalk on mixed peptide-polysaccharide matrices.
Biomaterials, 37, 73-81 (2015).
3
Fujimori, C., Kumai, J., Nakamura, K., Gu, Y., Katagiri, F., Hozumi, K., Kikkawa, Y., Nomizu, M.
Biological activity of peptide-conjugated polyion complex matrices consisting of alginate and chitosan.
Biopolymers, 108, e22983 (2017).
73
1) Lutolf, M. P., Hubbell, J. A., Nat. Biotechnol., 23, 47-55 (2005).
2) Badylak, S. F., Freytes, D. O., Gilbert, T. W., Acta Biomater., 5, 1-13 (2009).
3) Rozario, T., DeSimone, D. W., Dev. Biol., 341, 126-140 (2010).
4) Wiradjaja, F., DiTommaso, T., Smyth, I., Birth Defects Research Part C, 90, 8-31 (2010).
5) Rosso, F., Giordano, A., Barbarisi, M., Barbarisi, A., J. Cell Physiol., 199, 174-180 (2004).
6) Benton, G., Kleinman, H. K., George, J., Arnaoutova, I., Int. J. Cancer, 128, 1751-1757 (2011).
7) Scheele, S., Nystrom, A., Durbeej, M., Talts, J. F., Ekblom, M., Ekblom, P., J. Mol.
Med. (Berl)., 85, 825-836 (2007).
8) Miner, J. H., Microsc. Res. Techr., 71, 341-359 (2008).
9) Durbeej, M., Cell Tissue Res., 339, 259-268 (2010).
10) Miner, J. H., Yurchenco, P. D., Annu. Rev. Cell Dev. Biol., 20, 255-284 (2004).
11) Nomizu, M., Kim, W. H., Yamamura, K., Utani, A., Song, S. Y., Otaka, A., Roller, P.
P., Kleinman, H. K., Yamada, Y., J. Biol. Chem., 270, 20583-20590 (1995).
12) Nomizu, M., Kuratomi, Y., Song, S. Y., Ponce, M. L., Hoffman, M. P., Powell, S. K., Miyoshi, K., Otaka, A., Kleinman, H. K., Yamada, Y., J. Biol. Chem., 272, 32198-32205 (1997).
13) Nomizu, M., Kuratomi, Y., Malinda, K. M., Song, S. Y., Miyoshi, K., Otaka, A., Powell, S. K., Hoffman, M. P., Kleinman, H. K., Yamada, Y., J. Biol. Chem., 273, 32491-32499 (1998).
14) Nomizu, M., Kuratomi, Y., Ponce, M. L., Song, S. Y., Miyoshi, K., Otaka, A., Powell, S. K., Hoffman, M. P., Kleinman, H. K., Yamada, Y., Arch. Biochem. Biophys., 378, 311-320 (2000).
15) Hozumi, K., Akizuki, T., Yamada, Y., Hara, T., Urushibata, S., Katagiri, F., Kikkawa, Y., Nomizu, M., Arch. Biochem. Biophys., 503, 213-222 (2010).
16) Weeks, B. S., Nomizu, M., Ramchandran, R. S., Yamada, Y., Kleinman, H. K., Exp.
Cell Res., 243, 375-382 (1998).
17) Hoffman, M. P., Engbring, J. A., Nielsen, P. K., Vargas, J., Steinberg, Z., Karmand, A.
J., Nomizu, M., Yamada, Y., Kleinman, H. K., J. Biol. Chem., 276, 22077-22085 (2001).
18) Hayashi, K., Mochizuki, M., Nomizu, M., Uchinuma, E., Yamashina, S., Kadoya, Y.,
J. Invest. Dermatol., 118, 712-718 (2002).
19) Ponce, M. L., Hibino, S., Lebioda, A. M, Mochizuki, M., Nomizu, M., Kleinman, H.
K., Cancer Res., 63, 5060-5064 (2003).
20) Suzuki, N., Nakatsuka, H., Mochizuki, M., Nishi, N., Kadoya, Y., Utani, A., Oishi, S., Fujii, N., Kleinman, H. K., Nomizu, M., J. Biol. Chem., 278, 45697-45705 (2003).
21) Hynes, R., Cell, 110, 673-687 (2002).
22) Legate, K., Wickström, S., Fässler, R., Genes Dev., 23, 397-418 (2009).
23) Berrier, A., Yamada, K. M., J. Cell. Physiol., 213, 565-573 (2007).
24) Arnaout, M. A., Goodman, S. L., Xiong, J. P., Curr. Opin. Cell Biol., 19, 495-507 (2007).
25) Wiesner, S., Legate, K. R., Fässler, R., Cell. Mol. Life Sci., 62, 1081-1099 (2005).
26) Mochizuki, M., Kadoya, Y., Wakabayashi, Y., Kato, K., Okazaki, I., Yamada, M., Sato, T., Sakairi, N., Nishi, N., Nomizu, M., FASEB J., 17, 875-877 (2003).
27) Ikemoto, S., Mochizuki, M., Yamada, M., Takeda, A., Uchinuma, E., Yamashina, S., Nomizu, M., Kadoya, Y., J. Biomed. Mater. Res. Part A, 79, 716-722 (2006).
28) Mochizuki, M., Yamagata, N., Philp, D., Hozumi, K., Watanabe, T., Kikkawa, Y., Kadoya, Y., Kleinman, H. K., Nomizu, M., Biopolymers, 88, 122-130 (2007).
29) Masuda, R., Mochizuki, M., Hozumi, K., Takeda, A., Uchinuma, E., Yamashina, S., Nomizu, M., Kadoya, Y., Wound Repair Regen., 17, 127-135 (2009).
30) Yamada, Y., Hozumi, K., Katagiri, F., Kikkawa, Y., Nomizu, M., Biopolymers, 94, 711-720 (2010).
31) Chandy, T., Sharma, C. P., Biomater. Artif. Cells Artif. Organs, 18, 1-24 (1990).
32) Suh, J. K., Matthew, H. W., Biomaterials, 21, 2589-2598 (2000).
33) Dhoot, N. O., Wheatley, M. A., J. Pharm. Sci., 92, 679-689 (2003).
34) Suzuki, Y., Tanihara, M., Nishimura, Y., Suzuki, K., Yamawaki, Y., Kudo, H., Kakimaru, Y., Shimizu, Y., J. Biomed. Mater. Res., 48, 522-527 (1999).
35) Chiu, C. T., Lee, J. S., Chu, C. S., Chang, Y. P., Wang, Y. J., J. Mater. Sci. Mater. Med,
75
42) Di Martino, A., Sittinger, M., Risbud, M. V., Biomaterials, 26, 5983-5990 (2005).
43) Mochizuki, M., Philp, D., Hozumi, K., Suzuki, N., Yamada, Y., Kleinman, H. K., Nomizu, M., Arch. Biochem. Biophys., 459, 249-255 (2007).
44) Grant, D. S., Lelkes, P. I., Fukuda, K., Kleinman, H. K., In Vitro Cell Dev. Biol., 27A, 327-336 (1991).
45) Philp, D., Chen, S. S., Fitzgerald, W., Orenstein, J., Margolis, L., Kleinman, H. K., Stem Cells, 23, 288-296 (2005).
46) Castell, J. V., Gomez-Lechon, M. J., Methods Mol. Biol., 481, 35-46 (2009).
47) Maria, O. M., Maria, O., Liu, Y., Komarova, S. V., Tran, S. D., Int. J. Biochem. Cell Biol., 43, 622-631 (2011).
48) Sato, N., Meijer, L., Skaltsounis. L., Greengard, P., Brivanlou, A. H., Nat. Med., 10, 55-63 (2004).
49) Khvorostov, I., Zhang, J., Teitell, M., J. Vis. Exp., 16, (2008).
50) Zhang, J., Khvorostov, I., Teitell, M., J. Vis. Exp., 16, (2008).
51) Yamada, M., Kadoya, Y., Kasai, S., Kato, K., Mochizuki, M., Nishi, N., Watanabe, N., Kleinman, H. K., Yamada, Y., Nomizu, M., FEBS Lett., 530, 48-52 (2002).
52) Hozumi, K., Kobayashi, K., Katagiri, F., Kikkawa, Y., Kadoya, Y., Nomizu, M., FEBS Lett., 584, 3381-3385 (2010).
53) Gonzalez, A., Bhattacharya, R., deHart, G., Jones, J., Cellular Signalling, 22, 578-583 (2010).
54) Porter, J., Hogg, N., Trends Cell Biol., 8, 390-396 (1998).
55) Huveneers, S., Danen, E., J. Cell Sci., 122, 1059-1069 (2009).
56) Streuli, C. H., Akhtar, N., Biochem. J., 418, 491-509 (2009).
57) Hood, J. D., Cheresh, D. A., Nat. Rev. Cancer., 2, 91-100 (2002).
58) Brakebusch, C., Fässler, R., Cancer Metastasis Rev., 24, 403-411 (2005).
59) Fässler, R., Meyer, M., Genes Dev., 9, 1896-1908 (1995).
60) Yamada, K. M., J. Biol. Chem., 266, 12809-12812 (1991).
61) Sreejalekshmi, K. G., Nair, P. D., J. Biomed. Mater. Res. Part A, 96, 477-491 (2011).
62) Kikkawa, Y., Hozumi, K., Katagiri, F., Nomizu, M., Kleinman, H. K., Koblinski, J.
E., Cell Adh. Migr., 7, 150-1159 (2013).
63) Ruoslahti, E., Pierschbacher, M., Science, 238, 491-497 (1987).
64) Pankov, R., Yamada, K. M., J. Cell Sci., 115, 3861-3863 (2002).
65) Yamada, Y., Hozumi, K., Katagiri, F., Kikkawa, Y., Nomizu, M., Biomaterials, 34, 6539-6547 (2013).
66) Miles, A., Knutson, J., Skubitz, A., Furcht, L., McCarthy, J., Fields, G., J. Biol.
Chem., 270, 29047-29050 (1995).
67) Hozumi, K., Sasaki, A., Yamada, Y., Otagiri, D., Kobayashi, K., Fujimori, C., Katagiri, F., Kikkawa, Y., Nomizu, M. Biomaterials, 33, 4241-4250 (2012).
68) Urushibata, S., Hozumi, K., Ishikawa, M., Katagiri, F., Kikkawa, Y., Nomizu, M., Arch. Biochem. Biophys., 497, 43-54 (2010).
69) Urushibata, S., Katagiri, F., Takaki, S., Yamada, Y., Fujimori, C., Hozumi, K., Kikkawa, Y., Kadoya, Y., Nomizu, M., Biochemistry, 48, 10522-10532 (2009).
70) Beauvais, D. M., Ell, B. J., McWhorter, A. R., Rapraeger, A. C., J. Exp. Med., 206, 691-705 (2009).
71) McQuade, K. J., Rapraeger, A. C., J. Biol. Chem., 278, 46607-46615 (2003).
72) Kusano, Y., Oguri, K., Nagayasu, Y., Munesue, S., Ishihara, M., Saiki, I., Yonekura, H., Yamamoto, H., Okayama, M., Exp. Cell Res., 256, 434-444 (2000).
73) Otagiri, D., Yamada, Y., Hozumi, K., Katagiri, F., Kikkawa, Y., Nomizu, M., Biopolymers, 100, 751-759 (2013).
74) Hozumi, K., Suzuki, N., Nielsen, P. K., Nomizu, M., Yamada, Y., J. Biol. Chem., 281, 32929-32940 (2006).
75) Yamada, K. M., Pankov, R., Cukierman, E., Braz. J. Med. Biol. Res., 36, 959-966 (2003).
76) Schiller, H., Hermann, M-R., Polleux, J., Vignaud, T., Zanivan, S., Friedel, C., Sun.
Z., Raducanu, A., Gottschalk, K., Théry, M., Fässler, R., Nat. Cell. Biol., 15, 625-636 (2013).
77) Reyes, C., Petrie, T., García, A., J. Cell Physio., 217, 450-458 (2008).
78) Gonzalez, A., Gonzales, M., Herron, G., Nagavarapu, U., Hopkinson, S., Tsuruta, D., Jones, J., Proc. Natl. Acad. Sci. USA., 99, 16075-16080 (2002).
79) Abair, T., Sundaramoorthy, M., Chen, D., Heino, J., Ivaska, J., Hudson, B., Sanders, C., Pozzi, M., Zent, R., Exp. Cell Res., 314, 3593-3604 (2008).
80) Gonzalez, A., Claiborne, J., Jones, J., J. Biol. Chem., 283, 31849-31860 (2008).