Derived substances intercellular adhesion icam-1, the dna sequence

 

(57) Abstract:

The invention can be used in biotechnology and refers to a derived substances intercellular adhesion ICAM-I. Derivative has five consecutive immunoglobulin domains of ICAM-I and the cytoplasmic domain of ICAM-I or transmembrane domain of ICAM-I. DNA encoding a derivative of ICAM-I, receive recombinant means. Derived ICAM-I may be useful as a new highly effective anti-inflammatory agent. 2 S. p. f-crystals, 1 tab., 3 Il.

The invention relates to new substances with pharmacological activity, and more particularly to a derived substances intercellular adhesion ICAM-1 and coding his DNA.

ICAM-1 ("Intercellulat adhesion molecule - molecule intercellular adhesion), which is the ligand binding to the LFA-1 (associated with lymphocyte function antigen-1) is a glycoprotein on the cell surface size 76-114 KD expressed on necrovation cells, such as endothelial cells of blood vessels and fibroblast, and hematopoietic cells, such as endothelial cells of blood vessels, timoshii epithelial cells, and some epithelial cells, fibroblast, and on such kravet the native centered B-cells and genderaware cells in the tonsils, lymph nodes and Meyerovich plaques. ICAM-1 is strongly expressed on endothelial cells of blood vessels in areas of T cells in the lymph nodes and tonsils in which there is hyperplasia. Small quantities of ICAM-1 is expressed on peripheral blood lymphocytes.

ICAM-1 contains the extracellular region of 5 immunoglobulinovogo domain (domain 5 is nearest to the cell surface, domain 1 is the most remote from the cell surface), transmembrane domain and cytoplasmic domain.

Object of the invention is the introduction of a new highly effective anti-inflammatory agent.

This task is solved proposed derived substances intercellular adhesion ICAM-1, which has anti-inflammatory properties containing five consecutive immunoglobulinovi domains of ICAM-1, or five consecutive immunoglobulinovi domain and the cytoplasmic domain of ICAM-1, or five consecutive immunoglobulinovi domain and the cytoplasmic domain of ICAM-1, or five consecutive immunoglobulinovi domains and the transmembrane domain of ICAM-1. A further object of the MUI DNA produced by constructing a recombinant methods, and thus obtained DNA Express in the body-master, with subsequent isolation and purification.

In Fig. 1 shows the nucleic and amino acid sequence of cDNA of ICAM-1. The first fragment PBX is in position 58. Underlined transmitted sequence corresponding to trypticase peptides ICAM-1. Hydrophobic estimated signal peptide and transmembrane sequence underlined with a solid line, N-linked glycosylation sites outlined. The signal sequence AATAAA in position 2976 marked feature of the above line. The sequence corresponds to the cDNA clone HL-60. The cDNA sequence for endothelial cells defined for the total length of the cDNA molecules and shows that there are only minor differences.

In Fig. 2 shows the homologous domains of ICAM-1 and their relationship with family supergene immunoglobulins. (A) the Order of five homologous domains (D 1-5). In a frame made of two or more identical residues in the structured sequences. Residues occurring twice or more times in the domains of NCAM, and residues conserved in the domain of rows C2 and C1, orderly VM are the letters above the sequence, and the known position of the structures in the domains of the immunoglobulin C marked traits and capital letters above the sequence. The location of the proposed disulfide bridge in the domains of ICAM-1 is indicated through 5-5. (B-D). The order of residues in the protein domains that are homologous to domains of ICAM-1. Protein sequences are MAG, NCAM, V-domain-subunit receptor of T-cells, a chain of Ig M and a-1-glycoprotein.

Fig. 3 is a chart comparing the secondary structures of ICAM-1 and MAG.

5 above immunoglobulinovi domains or full extracellular domain of ICAM-1 are shown (with an ordered arrangement of residues) in Fig. 2A.

Domains 1-4 contain 88, 97, 99 and 99 residues, respectively, which is typical for the size of the domain 1; domain truncated to 5 68 residues. The analysis shows that there is significant gomologichnosti with members of the immunoglobulin family supergene, including C-domains of Ig M and IgG variable domain-subunit receptor of T-cells, and alpha-1-betaglycomune (Fig. 2B-D).

There are three types of domains of the Ig superfamily: V, C1 and C2. Domains V and C consist of 2 layers associated with each other intradomain disulfide bond; the domains of the V content is at the rows of C1 and C2 on the basis of characteristic residues, it is shown in Fig. 2A. The number of C1 includes proteins involved in antigen recognition. The number of C2 includes several Ec-receptors and proteins involved in adhesion of cells, including CD2, LFA-3, MAG and NCAM. It was found that the domains of ICAM-1 have the most pronounced homologically domains of a number of C2 that can be attributed to ICAM-1 to this number, which is expressed in very similar residues with residues conserved domains in C2 than in C1 domains, as shown for structures B-F in Fig. 2. In addition, the domains of ICAM-1 significantly better match - structures A and G C2 domains than with these structures in domains V and C1, which gives a good correspondence between the order along the length of the C2 domain. Comparison of the order of residues with the order in C2 domains of NCAN. MAG and alpha-1-beta-glycoprotein is shown in Fig. 2B and 2C; the degree of identity ranges from 28% to 33%. Also shown are comparisons (Fig. 2B, 2D) with V receptor T-cells (the degree of identity 27%) and C-domain 3 IgM (degree of identity 34%).

One of the most important characteristics of the domains of immunoglobulins is the presence of associated by a disulfide bridge cysteine connecting structure B and F, which stabilizes the sandwich layer; ICAM-1 cysteine is rotated inside of the sandwich and to stabilize the contact as suggested for some other domains of series V and C2. The distance between cysteine (residues 43, 50, 52, 37) such as described for a number of C2.

Found that ICAM-1 has the most pronounced homologically with glycoproteins NCAN and MAC series C2. This is of particular interest because NCAM and MAC are mediators of cell adhesion to each other, NCAM plays an important role in the interactions of neutrons with neutron and neutron-muscle interactions, while MAG plays an important role in the interactions of neutrons with oligodendrocytes and oligodendrocyte with oligodendrocytes if minimizatio.

Example 1. Construction and expression of the derivatives of ICAM-1 cDNA ICAM-1 were cleaved with restriction endonucleases Sal 1 and Kpn 1, and the obtained DNA fragment size 1.8 thousand base pairs subcloning in plasmid vector CDM8. The obtained construct pCD178C transform strain dut-, ung-E. coli (BW313/P3). Single-stranded brazilsugarexp matrix extracted from the transformants using infection by phage-R408 helper. Mutant cDNA ICAM-1 is produced by synthesis of the second thread using the oligonucleotide containing mismatched bases, and the subsequent transformation of the host ung+(MC1061/P3) received heteroge is, introduced mutant oligonucleotide. Mutant protein ICAM-1 Express by transfection of cells COS-7 mutant DNA in a eukaryotic expression vector CDMDB.

Synthesize functional derivative of ICAM-1, containing the extracellular region, having all 5 immunoglobulinovi domains. Mutant oligonucleotide consisting of 30 base pairs (CNC TCC CCC CGG TTC TAG ATT GTC ATC ATC), is used to transform the codons of the amino acids tyrosine (Y) and glutamic acid (E) at positions 452 and 453, respectively, in the codon phenylalanine (F) and the stop codon of translation (TAG). Mutant allocate its restriction to the Xba1 site. Its mean Y452E/F, TAG.

For expression of mutant protein cells COS transfection three mutant subclones (N 2, N 7, N 8). Three days after transfection these three mutant clones supernatant culture and cell lysates analyzed by the method of immunoassay using monoclonal antibodies against ICAM-1 (RRI/I) and the method of the LTO-PAGE (electrophoresis on polyacrylamide gel using sodium dodecyl sulfate). ICAM-1 is deposited from supernatant cultures of cells transformed by mutant subclones N 2 and N 8, but not from detergent lysates of these cells. The molecular is Anna forms of ICAM-1, which is consistent with the size expected given mutant DNA. Thus, a functional derivative of ICAM-1 is secreted as a soluble protein. In contrast, ICAM-1 cannot be immunosurgery from supernatants control cultures, transfection native ICAM-1, which shows that the membrane form ICAN-1 is not secreted from COS cells. Moreover not observed immunoassay ICAM-1 nor from supernatant cultures, or cell lysates pseudotranslation cells (negative control), "Truncated" ICAM-1, isolated from transfection cells, purify immunoaffinity chromatography using specific antibody against ICAM-1 (R6-5-6), and tested for functional activity using analysis of binding to cells. After purification in the presence of detergent octylglucoside preparations containing native ICAN-1 or "truncated" ICAM-1, diluted to a final concentration of octylglucoside equal to 0.25% (concentration less than the critical concentration of micelle formation of detergent). These drugs ICAM-1 gives the opportunity to contact the surfaces of plastic cups, having 96 wells, in order to obtain ICAM-1, associated with the solid phase. After washing away unbound substances, approximately 75-80%of M-1, respectively. The data obtained indicate that you have received "truncated" soluble ICAM-1 saves as immunological reactivity, and the ability to mediate dependent ICAM-1 adhesion that is characteristic of native ICAM-1.

The functional derivative of ICAM-1 containing 5 immunoglobulin domains and the transmembrane domain, get a similar way.

Consisting of 25 base pairs oligonucletide (TC AGC ACG TAC CTC TAG AAC CGC CA) is used to replace the codon for the amino acid at position 476 (Y) to the stop codon broadcast TAG. This mutant denote Y476/TAG. Immunogenum and analysis of the LTO-PAGE COS cells, transfection mutant allocate associated with the membrane form of ICAM-1 with a molecular mass of approximately 3 KD less than that of native ICAM-1. Indirect immunofluorescence transfection mutant COS cells shows the presence of spots, similar in the case of native ICAM-1 expressed on endothelial cells stimulated by LPS. Finally, cells, transfection mutant DNA, specifically associated with purified LFA-1 on plastic surfaces in a manner analogous with the case of COS cells, transfection DNA Naty 454-476, remove from ICAM-1 by treatment consisting of 48 base pairs of the oligonucleotide. This codons Y452 and E453 respectively associated with codons N477 and R478, which is confirmed by analysis of amino acid sequence. In the result of immunoassay and electrophoresis on polyacrylamide gel using sodium dodecyl sulfate found reduced molar weight compared to ICAM-1. In addition, expression of this mutant ICAM-1 test using monoclonal antibodies RRI/I R6.5, LB-2 and CL203.

As mentioned above, the proposed derivative of ICAM-1 are highly effective anti-inflammatory agent.

1. Derived substances intercellular adhesion ICAM-I, which has anti-inflammatory properties containing five consecutive immunoglobulinovi domains of ICAM-I, or five consecutive immunoglobulinovi domain and the cytoplasmic domain of ICAM-I, or five consecutive immunoglobulinovi domains and the transmembrane domain of ICAM-I.

2. The DNA sequence encoding a derivative of a substance intercellular adhesion ICAM-I, which has anti-inflammatory properties containing five consistently raznyh domains of ICAM-I and the cytoplasmic domain of ICAM-I, or five consecutive immunoglobulinovi domains of ICAM-I and the transmembrane domain of ICAM-I.

 

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