2Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya street, 16/10, Moscow, GSP-7, 117997, Russian Federatiok
3University of Turku, Vatselankatu 2, 1st floor, Turku, 20500 Finland
Keywords: Protein: Peptide; Receptor; Cholera toxin B subunit; IEC-6 cell line
Peptide |
Purity, % |
Amino acid analysis data |
Molecular mass, D |
LKEKK |
>98 |
Glu 1.09 (1), Leu 1.00 (1), Lys 3.27 (3) |
645.2 (calculated value ‑ 644.87) |
KKEKL |
>97 |
Glu 1.12 (1), Leu 1.03 (1), Lys 3.32 (3) |
648.6 (644.87) |
To prove this hypothesis, in this study we have prepared 125I-labeled CT-B and investigated its interaction with rat IEC-6 intestinal epithelial cells in the absence and in the presence of unlabeled TM- α1, IFN-α2, peptides LKEKK and KKEKL.
The guanylate cyclase (sgc and pgc) activity was measured by monitoring the conversion of [α -322P]GTP to [32P]cgmp ;the product was isolated by precipitation with zinc carbonate and chromatography on a column of aluminum oxide [23,26]. The enzyme activity was expressed as the amount of cgmp produced in 10 min (in nano moles per 1 mg protein). The protein concentration was determined by the Lowry method [14] using bovine serum albumin as a standard. Statistical study was performed by the Student’s t-test.
To characterize the specificity of the 125I-labeled CT-B binding to riec-6 cells, unlabeled TM-α1, IFN-α2, the peptide LKEKK, and the peptide KKEKL with the reverse sequence were tested as potential competitors. The Ki values (Table 2) demonstrated strong inhibitory capacity of TM-a1, IFN-a2, and the peptide LKEKK (Ki = 1.5 ± 0.3, 1.0 ± 0.3, 2.0 ± 0.5), whereas the peptide KKEKL did not inhibit the 125I-labeled CT-B binding (Ki > 10 mm), indicating a high specificity of TM-α1, IFN-α2, and the peptide LKEKK binding. Thus, TM-α1, IFN-α2, and the peptideLKEKK bind with high affinity and specificity to the CT-B receptor on riec-6 cells.
Ligand |
IC50(nM) |
Ki(nM) |
IFN-α2 |
1.5 ± 0.2 |
1.0 ± 0.2 |
TM- α1 |
2.3 ± 0.3 |
1.5 ± 0.3 |
LKEKK |
3.0 ± 0.2 |
2.0 ± 0.3 |
KKEKL |
>100 |
>100 |
Ligand (nM) |
Level of NO (nmol of NO2¯/mL medium ± SEM) |
||
CT-B |
LKEKK |
KKEKL |
|
Control |
16 ± 3 |
||
0.1 |
14 ± 3 |
16 ± 2 |
17 ± 3 |
1 |
15 ± 2 |
18 ± 4 |
14 ± 3 |
10 |
20 ± 3 |
19 ± 3 |
16 ± 4 |
100 |
28 ± 3* |
26 ± 2* |
18 ± 3 |
1000 |
29 ± 3* |
27 ± 3* |
15 ± 3 |
Ligand (nM) |
Guanylate cyclase activity (nmoles of cGMP per 1 mg protein in 10 min± SEM) |
|||||
sGC |
pGC |
|||||
CT-B |
LKEKK |
KKEKL |
CT-B |
LKEKK |
KKEKL |
|
Control |
0.9 ± 0.1 |
1.5 ± 0.2 |
||||
1 |
0.8 ± 0.2 |
0.9 ± 0.2 |
1.0 ± 0.2 |
1.6 ± 0.2 |
1.6 ± 0.2 |
1.4 ± 0.2 |
10 |
1.2 ± 0.2* |
1.3 ± 0.2* |
0.8 ± 0.3 |
1.5 ± 0.3 |
1.6 ± 0.2 |
1.5 ± 0.3 |
50 |
1.5 ± 0.3* |
1.4 ± 0.3* |
0.9 ± 0.2 |
1.6 ± 0.2 |
1.5 ± 0.2 |
1.5 ± 0.2 |
100 |
1.7 ± 0.3* |
1.5 ± 0.3* |
0.9 ± 0.2 |
1.4 ± 0.3 |
1.6 ± 0.3 |
1.4 ± 0.3 |
1000 |
1.6 ± 0.4* |
1.5 ± 0.3* |
0.9 ± 0.3 |
1.6 ± 0.2 |
1.7 ± 0.2 |
1.8 ± 0.3 |
Concentration of compound (nM) |
Adenylate cyclase activity (nmoles of cAMP per 1 mg protein in 10 min) |
|||
CT-B |
LKEKK |
KKEKL |
Isoproterinol |
|
0 |
1.8 ± 0.2 |
|||
0.1 |
1.9 ± 0.3 |
2.0 ± 0.3 |
1.9 ± 0.2 |
1.8 ± 0.2 |
1 |
1.8 ± 0.2 |
1.9 ± 0.4 |
1.9 ± 0.3 |
2.2 ± 0.3 |
10 |
1.9 ± 0.2 |
1.9 ± 0.2 |
1.7 ± 0.2 |
2.4 ± 0.2 |
100 |
1.7 ± 0.3 |
1.8 ± 0.3 |
1.8 ± 0.2 |
2.8 ± 0.3 |
1000 |
1.9 ± 0.2 |
1.9 ± 0.2 |
1.9 ± 0.3 |
3.2 ± 0.4 |
CT-B is now viewed as a promising immune modulating and anti inflammatory agent. Recombinant CT-B has been recently found to suppress immunopathological reactions in allergy and autoimmune diseases to stimulate humoral immunity and to induce anti inflammatory responses in vivo, in particular, to mitigate the intestinal inflammation of Crohn’s disease in mice and humans [25,29,2,28]. Since CT-B can prevent infection but also autoimmune reactions, the question is how these two apparently opposite immune reactions can be achieved by the same protein. Currently, there is no answer to this question.
The results of this study show that 125I-labeled CT-B binds with high affinity to riec-6 cells (Kd = 3.7 nm, Figure 2), and unlabeled TM- a1, IFN-α2, the peptide LKEKK inhibit its binding by 100% (Figure 3, Table 2). Previously, we showed that [3H]LKEKK binds with high affinity to non-protein receptor on donor blood T lymphocytes , and on rat intestine epithelial cell membranes [16,17]. In both cases, the labeled peptide binding was competitively inhibited by unlabeled TM- α1, IFN-α2, and CT-B. These data, as well as the fact that cholera toxin has no receptors other than GM1-ganglioside, allow us to conclude that GM1-ganglioside is the receptor common for TM- α1, IFN-α2, and the peptide LKEKK. The data in (Tables 3-5) show that CT-B and the peptide: LKEKK at concentrations of 10 - 1000 nm increased in a dose-dependent manner the nitric oxide (NO) production and the soluble guanylate cyclase (sgc) activity in riec-6 cells.
Nitric oxide (NO) is the principal inhibitory neurotransmitter in the gut, endothelial-derived NO is involved in the local regulation of mucosal blood flow and inflammatory-derived NO is involved in the loss of mucosal integrity [27,1]. Increased production of NO and subsequent local cytotoxicity to mucosal epithelial cells has been proposed as one of the putative mechanisms in the development of necrotizing enterocolitis (NEC) [6]. NO is synthesized from L-arginine by NO synthase (NOS), of which there are three isoforms inducible NOS (inos), endothelial NOS (enos) and neuronal NOS (nnos) [15]. Of the three isoforms of NOS described, inos is not constitutively expressed, but induced at high
According to the results of this work, CT-B and the peptide: LKEKK increased in a dose-dependent manner the sgc activity in riec-6 cells. Sgc, a heterodimer consisting of α and β subunits, which is activated by the direct interaction of NO with the heme of the b subunit [13]. There is conclusive evidence that effect of low concentrations of NO is cgmp dependent [18, 19]. These authors measured the levels of intracellular cgmp in CD4+ T cells cultured in the presence of graded concentrations of NOC-18. Cgmp concentration was significantly elevated by 5 mm and 10mm of NOC-18; this amount declined to the control level at 100 mm of NOC-18. These results strongly indicate that the enhancing effect of low concentrations of NO is mediated by cgmp. The pattern of cgmp elevation closely correlated with the enhanced cell activation by NO. Our data also suggest that low levels of NO activate sgc.
As we mentioned above, CT-B has a high potential as an immune modulatory and anti-inflammatory agent. In this regard, the study of the activity of the peptide LKEKK capable of binding to GM1-ganglioside and activating sgc is of great interest.
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