title: Structural analysis of the CD69 early activation antigen by two monoclonal antibodies directed to different epitopes.
abstract: The biochemical structure of CD69 early activation antigen has been characterized by means of two newly isolated mAb, namely C1.18 and E16.5. Upon analysis by SDS-PAGE, C1.18-reactive molecules immunoprecipitated from 125I-surface labeled PMA activated PBL consisted of a 32 + 32 kD dimer, a 32 + 26 kD dimer, a 26 + 26 kD dimer and a 21 + 21 kD dimer. E16.5-reactive molecules consisted of a 26 + 26 kD dimer and a 21 + 21 kD dimer. Cross absorption experiments showed that E16.5 mAb reacts with an epitope of the CD69 molecule distinct from the one recognized by C1.18 mAb and present only on a subpopulation of the CD69 molecular pool. The patterns of migration of C1.18- and E16.5-reactive molecules in two-dimensional gel-electrophoresis, under reducing conditions before and after treatment with Endoglycosidase F enzyme suggest that the two mAb recognize the same glycoprotein structure, but in two distinct glycosylation forms, both expressed on the cell surface membrane. Finally, p32, p26 and p21 of CD69 complex obtained from three distinct normal donors did not show appreciable structural polymorphism, by two-dimensional peptide mapping, not only among single subunits within the same individual, but also among homologous subunits in distinct individuals. Further, it was found that CD69 complex is expressed at the cell surface of resting PBL, although at a very reduced level in comparison to PMA activated cells. C1.18 and E16.5 mAb induced comparable cell proliferation and IL-2 production in PBL in the presence of PMA. C1.18 mAb increased intracellular free calcium concn in PMA activated PBL after cross-linking with goat anti mouse Ig, while the effect induced by E16.5 mAb after cross-linking was consistently lower. Finally, it was found that Sepharose-linked C1.18 mAb, in the presence of rIL-2 or PMA, did not induce TNF release from 6 NK cell clones.
authors.affiliation: Istituto di Scienze Immunologiche, Università di Verona, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Gerosa
authors.forename: M
authors.initials: M
authors.lastname: Tommasi
authors.forename: M
authors.initials: M
authors.lastname: Scardoni
authors.forename: R S
authors.initials: RS
authors.lastname: Accolla
authors.forename: T
authors.initials: T
authors.lastname: Pozzan
authors.forename: M
authors.initials: M
authors.lastname: Libonati
authors.forename: G
authors.initials: G
authors.lastname: Tridente
authors.forename: G
authors.initials: G
authors.lastname: Carra
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology*; D015703:Antigens, CD / Q000737:chemistry / Q000276:immunology*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000737:chemistry / Q000276:immunology*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000276:immunology; D015180:Electrophoresis, Gel, Two-Dimensional; D000939:Epitopes; D006023:Glycoproteins / Q000276:immunology; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007694:Killer Cells, Natural / Q000276:immunology; D037181:Lectins, C-Type; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000276:immunology; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D000099192:CD69 Antigens
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D002118:Calcium; D000939:Epitopes; D006023:Glycoproteins; D006026:Glycoside Hydrolases; D037181:Lectins, C-Type; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D000099192:CD69 Antigens