Characterization of a novel monoclonal antibody with restricted specificity to the free beta 2 integrin alpha M CD11b subunit.
Leukocyte cell surface expression and function of beta2 integrins require the intracellular association of alpha subunits, CD11a, b, c, d, respectively, with the common CD18 beta2 subunit. We have raised and characterized a murine MAb -- ME-MDF -- directed against the low affinity form of the human integrin alphaM subunit CD11b A-domain. MAb ME-MDF is an IgG2a that has a kDa of 2,45461 +/- 0.12 x 10(-9) M. MAb ME-MDF recognizes both the low and high affinity forms of the CD11b A-domain. Flow cytometry showed that ME-MDF does not recognize the heterodimeric CD11b/CD18 molecule at the surface of polymorphonuclear cells and the human monoblast cell line U937. Western blot analysis of U937 cell line cell surface proteins demonstrated that ME-MDF reacts specifically with the CD11b subunit but does not react with the heterodimeric CD11b/CD18 complex, a feature that differentiates it from other CD11b A-dom-specific MAbs. These observations suggest that ME-MDF recognizes an epitope that is involved in the association of the two subunits and hence is not accessible within the heterodimeric form of the CD11b/CD18 molecule. These data show that the CD11b A-dom engages not only the MIDAS but also the ME-MDF-specific epitope to associate with the CD18 subunit. We have also constructed, and expressed in the yeast Pichia pastoris, the corresponding recombinant scFv form of MAb ME-MDF and characterized the CDRs. MAb ME-MDF is characterized by short VH and VL CDR3. MAb ME-MDF and/or its recombinant scFv form would be very useful to study the structural basis of the association between the alpha and beta2 integrin subunits and to investigate the possibility of modulating CR3 cell surface expression by preventing subunit association.
Tanfous
Naouel Guedel-Ben
NG
Molecular Biotechnology Group, Laboratory of Immunology, Institute Pasteur de Tunis, Tunis, Tunisia.
Essafi
Makram
M
Larguech
Beya
B
Barbouche
Ridha
R
Fathallah
Dahmani M
DM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D039481:CD11b Antigen / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D018821:CD18 Antigens / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020298:U937 Cells
D000911:Antibodies, Monoclonal; D039481:CD11b Antigen; D018821:CD18 Antigens; D021122:Protein Subunits; D011994:Recombinant Proteins