Cell adhesion

Fibronectin contains at least two major domains that support cell adhesion. One is the central cell-binding domain that is recognized by a variety of cell types via the integrin alpha 5 beta 1. The second, originally identified by its ability to support melanoma cell adhesion, is located in the alternatively spliced type III connecting segment (IIICS). A dominant cell type-specific adhesion site within the IIICS has been localized to a peptide designated as CS1 comprising its amino-terminal 25 residues. The receptor for CS1 is the integrin alpha 4 beta 1. We have synthesized a variety of peptides with overlapping sequences in order to identify the minimum active amino acid sequence of this major cell adhesion site. A peptide comprising the carboxyl-terminal 8 amino acids of CS1, EILDVPST, was found to support melanoma cell spreading, while all peptides without this sequence had little or no activity. Two smaller overlapping pentapeptides, EILDV and LDVPS, were also active, whereas EILEV, containing a conservative substitution of Glu for Asp, was inactive. These data suggested that the minimum sequence for cell adhesion activity is Leu-Asp-Val, the tripeptide sequence common to both active peptides. This prediction was confirmed by the observed ability of the Leu-Asp-Val peptide itself to block spreading on fibronectin, whereas Leu-Glu-Val was inactive. Interspecies amino acid sequence comparison also supports the importance of the LDV sequence, since it is completely conserved in the IIICS regions of human, rat, bovine, and avian fibronectins.[1]

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Fibronectin contains at least two domains that support cell adhesion. One is the central cell-binding domain that is recognized by a variety of cell types, including fibroblasts. The second, originally identified by its ability to support melanoma cell adhesion, is located in the alternatively spliced type III connecting segment (IIICS). Using specific adhesive ligands and inhibitory probes, we have examined the role of each of these domains in fibronectin-mediated neurite extension of neurons from chick embryo dorsal root and sympathetic ganglia. In studies using explanted ganglia, both fl3, a 75-kD tryptic fragment of human plasma fibronectin containing the central cell-binding domain, and CS1-IgG, a synthetic peptide-IgG conjugate containing the principal cell adhesion site from the IIICS, supported neurite outgrowth after adsorption onto the substrate. The maximal activities of fl3 and CSl-IgG were 45-55% and 25-30% that of intact fibronectin, respectively. Co-coating of the substrate with f13 and CS1-IgG produced an additive stimulation of neurite outgrowth, the extent of which approached that obtained with fibronectin. Similar results were obtained with purified neuronal cell preparations isolated by tryptic dissociation of dorsal root ganglia. In complementary studies, blockage of the adhesive function of either the central cell-binding domain (with mAb 333, an antiadhesive monoclonal antibody) or the IIICS (with CS1 peptide), resulted in approximately 60 or 30% reduction in fibronectin-mediated neurite outgrowth, respectively. When tested in combination, the inhibitory activities of mAb 333 and CSl were additive. From these results, we conclude that neurons from the peripheral nervous system can extend neurites on both the central cell-binding domain and the IIICS region of fibronectin, and that these cells are therefore the first normal, embryonic cell type shown to adhere to the IIICS. These results suggest that spatiotemporal fluctuations in the alternative mRNA splicing of the IIICS region of fibronectin may be important in regulation of cell adhesive events during development of the peripheral nervous system [2].

[1]. The minimal essential sequence for a major cell type-specific adhesion site (CS1) within the alternatively spliced type III connecting segment domain of fibronectin is leucine-aspartic acid-valine. J Biol Chem. 1991 Aug 15;266(23):15075-9.
[2]. Neurite extension of chicken peripheral nervous system neurons on fibronectin: relative importance of specific adhesion sites in the central cell-binding domain and the alternatively spliced type III connecting segment. J Cell Biol. 1988 Apr;106(4):1289-97.

Parenthetically, the observed activity of CS1-A relative to that of CS1 in the cell-spreading promotion assay was much less than its biological activity in the cell-spreading inhibition assay (compare Figs. 3 and 4A). This apparent discrepancy may be due to the fact that the peptide is conjugated to IgG through the side chain of the added amino-terminal cysteine residue for the cell-spreading promotion assay, and the free CS1-A peptide was used in the cell-spreading inhibition assay. The biologically important residues are located spatially closer to the IgG carrier protein for CS1-A as compared with the other CS1 subpeptides. Hence, potential steric hindrance by the IgG molecule may partially interfere with the interaction between the CS1-A peptide and its receptor, the integrin a4pl complex. The minimal LDV sequence we have identified as essential for the recognition of fibronectin by the a4& integrin receptor compares with the RGD sequence necessary for recognition by several other integrin receptors including a&, a,& all&, and others (15-17). Both peptides contain an aspartyl residue, but they appear otherwise unrelated. In addition, the other minimal sequence from the human fibronectin IIICS region is REDV (l), which may be related either to LDV (both have DV) or to RGDS (the rat homologue is RGDV (18)). The use of these short recognition sequences by cells for fibronectinmediated functions suggests a common pattern of local peptide sequence recognition by specific integrin receptors. Whether the aspartyl residue in each of these peptide sequences constitutes part of an underlying mechanism of receptor recognition remains to be determined. [1]

C123H195N31O39

2732.05

2730.42

107978-77-8

16131087

H-Asp-Glu-Leu-Pro-Gln-Leu-Val-Thr-Leu-Pro-His-Pro-Asn-Leu-His-Gly-Pro-Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr-OH; Asp-Glu-Leu-Pro-Gln-Leu-Val-Thr-Leu-Pro-His-Pro-Asn-Leu-His-Gly-Pro-Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr; L-alpha-aspartyl-L-alpha-glutamyl-L-leucyl-L-prolyl-L-glutaminyl-L-leucyl-L-valyl-L-threonyl-L-leucyl-L-prolyl-L-histidyl-L-prolyl-L-asparagyl-L-leucyl-L-histidyl-glycyl-L-prolyl-L-alpha-glutamyl-L-isoleucyl-L-leucyl-L-alpha-aspartyl-L-valyl-L-prolyl-L-seryl-L-threonine

DELPQLVTLPHPNLHGPEILDVPST; H-DELPQLVTLPHPNLHGPEILDVPST-OH

Typically exists as solid at room temperature

0.507

32

42

80

193

6430

27

OC(CCC(C(NC(C(N1CCCC1C(NC(C(NC(C(NC(C(NC(C(NC(C(N1CCCC1C(NC(C(N1CCCC1C(NC(C(NC(C(NC(C(NCC(N1CCCC1C(NC(C(NC(C(NC(C(NC(C(NC(C(N1CCCC1C(NC(C(NC(C(=O)O)C(O)C)=O)CO)=O)=O)C(C)C)=O)CC(=O)O)=O)CC(C)C)=O)C(CC)C)=O)CCC(=O)O)=O)=O)=O)CC1=CN=CN1)=O)CC(C)C)=O)CC(=O)N)=O)=O)CC1=CN=CN1)=O)=O)CC(C)C)=O)C(O)C)=O)C(C)C)=O)CC(C)C)=O)CCC(=O)N)=O)=O)CC(C)C)=O)NC(C(CC(=O)O)N)=O)=O

NXAKDBCNODQXBZ-YSZCVPRFSA-N

InChI=1S/C123H195N31O39/c1-19-64(16)97(117(186)140-74(42-58(4)5)106(175)138-78(50-94(167)168)109(178)146-96(63(14)15)122(191)154-40-24-29-87(154)115(184)144-82(54-155)110(179)149-99(66(18)157)123(192)193)147-104(173)72(32-35-92(163)164)134-111(180)83-25-20-36-150(83)90(160)53-129-101(170)76(46-67-51-127-55-130-67)137-105(174)73(41-57(2)3)136-107(176)77(49-89(126)159)139-113(182)85-27-22-39-153(85)121(190)81(47-68-52-128-56-131-68)142-114(183)86-28-23-38-152(86)120(189)80(45-61(10)11)143-118(187)98(65(17)156)148-116(185)95(62(12)13)145-108(177)75(43-59(6)7)135-102(171)70(30-33-88(125)158)133-112(181)84-26-21-37-151(84)119(188)79(44-60(8)9)141-103(172)71(31-34-91(161)162)132-100(169)69(124)48-93(165)166/h51-52,55-66,69-87,95-99,155-157H,19-50,53-54,124H2,1-18H3,(H2,125,158)(H2,126,159)(H,127,130)(H,128,131)(H,129,170)(H,132,169)(H,133,181)(H,134,180)(H,135,171)(H,136,176)(H,137,174)(H,138,175)(H,139,182)(H,140,186)(H,141,172)(H,142,183)(H,143,187)(H,144,184)(H,145,177)(H,146,178)(H,147,173)(H,148,185)(H,149,179)(H,161,162)(H,163,164)(H,165,166)(H,167,168)(H,192,193)/t64-,65+,66+,69-,70-,71-,72-,73-,74-,75-,76-,77-,78-,79-,80-,81-,82-,83-,84-,85-,86-,87-,95-,96-,97-,98-,99-/m0/s1

(4S)-4-[[(2S)-1-[2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-1-[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-carboxypropanoyl]amino]-4-carboxybutanoyl]amino]-4-methylpentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-4-methylpentanoyl]amino]-3-methylbutanoyl]amino]-3-hydroxybutanoyl]amino]-4-methylpentanoyl]pyrrolidine-2-carbonyl]amino]-3-(1H-imidazol-5-yl)propanoyl]pyrrolidine-2-carbonyl]amino]-4-oxobutanoyl]amino]-4-methylpentanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]acetyl]pyrrolidine-2-carbonyl]amino]-5-[[(2S,3S)-1-[[(2S)-1-[[(2S)-3-carboxy-1-[[(2S)-1-[(2S)-2-[[(2S)-1-[[(1S,2R)-1-carboxy-2-hydroxypropyl]amino]-3-hydroxy-1-oxopropan-2-yl]carbamoyl]pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]amino]-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-5-oxopentanoic acid

107978-77-8; CS1 Peptide; CS-1 fibronectin; Connecting segment 1 fibronectin; Fibronectin connecting segment 1; L-Threonine, L-alpha-aspartyl-L-alpha-glutamyl-L-leucyl-L-prolyl-L-glutaminyl-L-leucyl-L-valyl-L-threonyl-L-leucyl-L-prolyl-L-histidyl-L-prolyl-L-asparaginyl-L-leucyl-L-histidylglycyl-L-prolyl-L-alpha-glutamyl-L-isoleucyl-L-leucyl-L-alpha-aspartyl-L-valyl-L-prolyl-L-seryl-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)