Cellular adhesion mediated by factor J, a complement inhibitor. Evidence for nucleolin involvement.
Factor J (FJ) is a complement inhibitor that acts on the classical and the alternative pathways. We demonstrated FJ-cell interactions in fluid phase by flow cytometry experiments using the cell lines Jurkat, K562, JY, and peripheral blood lymphocytes. FJ bound to plastic plates was able to induce in vitro adhesion of these cells with potency equivalent to fibronectin. As evidence for the specificity of this reaction, the adhesion was blocked by MAJ2, an anti-FJ monoclonal antibody, and by soluble FJ. Attachment of the cells required active metabolism and cytoskeletal integrity. The glycosaminoglycans heparin, heparan sulfate, or chondroitin sulfates A, B, and C inhibited to varying degrees the binding of FJ to cells, as did treatment with chondroitinase ABC. In the search for a putative receptor, a protein of 110 kDa was isolated by affinity chromatography, and microsequence analysis identified this protein as nucleolin. Confocal microscopy evidenced the presence of nucleolin in cell membrane by immunofluorescence with monoclonal (D3) and polyclonal anti-nucleolin antibodies in Jurkat cells. The interaction FJ-nucleolin was evidenced by Western blot and enzyme-linked immunosorbent assay. Furthermore, purified nucleolin and D3 inhibited adhesion of Jurkat cells to immobilized FJ, suggesting that the interaction was specific and that nucleolin mediated the binding.
Larrucea
S
S
Unidad de Inmunología, Hospital La Paz, Paseo de la Castellana, 261-28046 Madrid, Spain.
González-Rubio
C
C
Cambronero
R
R
Ballou
B
B
Bonay
P
P
López-Granados
E
E
Bouvet
P
P
Fontán
G
G
Fresno
M
M
López-Trascasa
M
M
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001402:B-Lymphocytes / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002809:Chondroitin Sulfates / Q000494:pharmacology; D002846:Chromatography, Affinity; D003169:Complement Inactivator Proteins / Q000502:physiology*; D005434:Flow Cytometry; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D006025:Glycosaminoglycans / Q000494:pharmacology* / Q000502:physiology; D006493:Heparin / Q000494:pharmacology; D006497:Heparan Sulfate / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D020014:K562 Cells; D007700:Kinetics; D008214:Lymphocytes / Q000502:physiology*; D009687:Nuclear Proteins / Q000502:physiology; D010750:Phosphoproteins / Q000502:physiology*; D016601:RNA-Binding Proteins / Q000502:physiology*; D020298:U937 Cells; D000097796:Nucleolin
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D002809:Chondroitin Sulfates; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D006025:Glycosaminoglycans; D006493:Heparin; D006497:Heparan Sulfate; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D000097796:Nucleolin; C116021:factor J protein, human