Cloning of the rat CR3 alphaM (CD11b) subunit, expression and binding assay of recombinant isolated CD11b VA (A-domain) and ICAM-1 Ig modules.
The leukocyte beta2 integrin CR3 (CD11/CD18), is a surface heterodimeric glycoprotein that functions as a divalent cation-dependent adhesive complex. It mediates several important cell-substrate and cell-cell adhesive interactions among which the interaction with vascular endothelial cells that lead to leukocyte transmigration. We have isolated cDNA clones-coding for the rat complement receptor type 3 (CR3) alphaM subunit (CD11b) from a cDNA library. The cDNA sequence showed respectively 89.4% and 74.6% homology with its mouse and human counterpart. We have expressed the sequence coding for the VA module or Von Willebrand type domain (A-domain) and produced it in E. coli as a soluble recombinant fusion protein with GST. Simultaneously, we have cloned DNA fragments specific to the rat ICAM-1 domain 1 and domain 3 and expressed each clone in E. coli as recombinant soluble (rs) fusion proteins with GST. Recombinant CD11b A-domain was released from the fusion protein by thrombin cut. Purified ICAM-1 fusion peptides and CD11b A-domain were used to develop a direct binding assay that showed a specific binding between the rat ICAM-1 Ig like domain 3 and CD11b A-domain. These data demonstrate that the IgSF modules can be produced as a soluble recombinant fusion protein and used to study direct binding to the VA module displayed by members of the integrin superfamily.
Zerria
K
K
Molecular Biotechnology Group, Immunology Laboratory, Institut Pasteur de Tunis, Tunisia.
Bebbiche
N
N
Barbouche
M R
MR
Dellagi
K
K
Fathallah
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D039481:CD11b Antigen / Q000235:genetics*; D018821:CD18 Antigens / Q000235:genetics*; D002448:Cell Adhesion / Q000235:genetics; D002634:Chemotaxis, Leukocyte / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017385:Sequence Homology; D014841:von Willebrand Factor / Q000235:genetics*
D039481:CD11b Antigen; D018821:CD18 Antigens; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D018799:Intercellular Adhesion Molecule-1