ADAM disintegrin-like domain recognition by the lymphocyte integrins alpha4beta1 and alpha4beta7

Abstract

The ADAM (a disintegrin and metalloprotease) family of proteins possess both proteolytic and adhesive domains. We have established previously that the disintegrin domain of ADAM28, an ADAM expressed by human lymphocytes, is recognized by the integrin alpha4beta1. The present study characterizes the integrin binding properties of the disintegrin-like domains of human ADAM7, ADAM28 and ADAM33 with the integrins alpha4beta1, alpha4beta7 and alpha9beta1. Cell-adhesion assays demonstrated that, similar to ADAM28, the ADAM7 disintegrin domain supported alpha4beta1-dependent Jurkat cell adhesion, whereas the ADAM33 disintegrin domain did not. The lymphocyte integrin alpha4beta7 was also found to recognize both disintegrin domains of ADAM7 and ADAM28, but not of ADAM33. This is the first demonstration that mammalian disintegrins are capable of interacting with alpha4beta7. All three disintegrin domains supported alpha9beta1-dependent cell adhesion. Recognition by both alpha4beta1 and alpha4beta7 of ADAM7 and ADAM28 was activation-dependent, requiring either the presence of Mn2+ or an activating monoclonal antibody for cell attachment. Charge-to-alanine mutagenesis experiments revealed that the same residues within an individual ADAM disintegrin domain function in recognizing multiple integrins. However, the residues within a specific region of each ADAM disintegrin-like domain required for integrin binding were distinct. These results establish that ADAM7 and ADAM28 are recognized by the leucocyte integrins alpha4beta1, alpha4beta7 and alpha9beta1. ADAM33 exclusively supported only alpha9beta1-dependent adhesion.

Figures

Figure 1. The disintegrin domain of ADAM7 facilitates integrin α4β1-dependent cell adhesion

(a) Alignment of the primary amino acid sequences of the disintegrin domains of human ADAM7, ADAM28 and ADAM33. Identities of ADAM28 with ADAM7 and ADAM33 disintegrin domains are shaded, with the respective homology of ADAM7 and ADAM33 to ADAM28 displayed at the far left. The predicted integrin α9β1 recognition motif is indicated by thick underlining and contains the putative disintegrin loops. Arrows indicate charged residues selected for alanine substitution in the ADAM7 and ADAM28 disintegrin domains. (b) Microtitre wells were coated with the indicated concentrations of ADAM7 (■), ADAM28 (○) or ADAM33 Dis–Fc (▲) recombinant proteins. ADAM7 and ADAM33 Dis–Fc contained the complete disintegrin domains Tyr397-Gly497 and Ala418-Gly510 respectively and ADAM28 Dis–Fc is as described previously [15,20]. The T-lymphoma cell line Jurkat (2×105 cells/well) was added to wells in Hepes/Tyrode's buffer containing 1 mM CaCl2, 0.5 mM MgCl2 and 10 μg/ml QE2E5 mAb for exogenous activation of the β1 integrins. The number of adherent cells per well was quantified based on a standard curve generated by assessing the endogenous acid phosphatase activity as described in the Experimental section. Adherent cells/well=adherent cells(recombinant protein)−adherent cells(BSA). Results shown are the means±S.D. for a representative experiment performed in triplicate. (c) Microtitre wells were coated with 10 μg/ml of purified, recombinant ADAM28 (white bars) or ADAM7 Dis–Fc (black bars). Jurkat cells were added to wells as described previously in a buffer containing 1 mM MnCl2. Cells were incubated with the recombinant ligands in the presence of 5 μg/ml of various integrin subunit mAbs or with 100 μg/ml of the α4β1 ligand mimetic CS-1 peptide. Results are expressed as the average percentage inhibition±S.D. for a representative experiment performed in triplicate. Percentage inhibition={1−[adherent cells(inhibitor)−adherent cells(BSA)]/[adherent cells(no inhibitor)−adherent cells(BSA)]}×100. (d) α4-K562 (●) or K562 (○) cells were added to microtitre wells (2×105 cells/well) coated with various concentrations of ADAM7 Dis–Fc in the presence of 10 μg/ml QE2E5. Adherent cells/well=adherent cells(recombinant protein)−adherent cells(BSA). Results are expressed as the mean adhesion±S.D. for a representative experiment performed in triplicate.

Figure 2. The disintegrin domains of ADAM28 and ADAM7 are recognized by the immunological integrin α4β7

(a) Flow-cytometric analysis of integrin subunit expression on RPMI 8866 cells. RPMI 8866 cells were incubated with the primary mAbs 44H6 (anti-α4), Y9A2 (anti-α9β1), 6S6 (anti-β1), YCFII8.3 (anti-β2) and FIB27 (anti-β7). After washing, the bound primary antibody was disclosed with appropriate secondary FITC-conjugated antibody (unfilled curve) as described in the Experimental section. Experiments containing only secondary conjugated antibody served as negative controls (filled curve). (b) Adhesion assays were performed as described previously by adding RPMI 8866 cells (2×105 cells/well) to microtitre wells coated with various concentrations of purified, recombinant ADAM7 (■), ADAM28 (○) or ADAM33 Dis–Fc (▲). Cells were suspended in Hepes-Tyrode's buffer containing 1 mM CaCl2, 0.5 mM MgCl2 and 10 μg/ml of the anti-β7 activating mAb 2G3. Adherent cells/well=adherent cells(recombinant protein)−adherent cells(BSA). Results shown are the average±S.D. for a representative experiment performed in triplicate. (c) RPMI 8866 cells (2×105 cells/well) suspended in a buffer containing 1 mM MnCl2 were added to microtitre wells coated with 10 μg/ml of the indicated recombinant ADAM28 (white bars) or ADAM7 Dis–Fc (black bars) in the presence of 5 μg/ml of either function-blocking or non-function-blocking mAbs that recognize distinct integrin subunits. Results are expressed as the average percentage inhibition±S.D. for a representative experiment performed in triplicate. Percentage inhibition={1−[adherent cells(inhibitor)−adherent cells(BSA)]/[adherent cells(no inhibitor)−adherent cells(BSA)]}×100.

Figure 3. Recognition of the disintegrin domains of ADAM7 and ADAM28 by α4β1 and α4β7 integrins is activation-dependent

(a) Jurkat cells (2×105/well) suspended in Hepes-Tyrode's buffer containing either 1 mM MnCl2, 10 μg/ml of the anti-β1-activating antibody QE2E5 or only 1 mM CaCl2 and 0.5 mM MgCl2 were added to wells coated with 10 μg/ml each of ADAM7 (black bars) or ADAM28 Dis–Fc (white bars). (b) RPMI 8866 cell adhesion to 10 μg/ml each of ADAM7 (black bars) or ADAM28 (white bars) Dis–Fc or 2.5 μg/ml MAdCAM-Ig (hatched bars) was investigated in Hepes-Tyrode's buffer containing 1 mM MnCl2, 10 μg/ml of the anti-β7-activating antibody 2G3 or only 1 mM CaCl2 and 0.5 mM MgCl2. α4β7 specificity was demonstrated for ADAM7 and ADAM28 by including 10 μg/ml MAdCAM–Ig in solution with the 2G3-activating antibody. Results shown in (a, b) are the average±S.D. for a representative experiment conducted in triplicate. Adherent cells/well=adherent cells(recombinant protein)−adherent cells(BSA).

Figure 4. Residues required for integrin α4β1- and α4β7-mediated cell adhesion to two homologous disintegrin domains

(a) Microtitre wells were coated with 5 μg/ml of either native (white bar) or the indicated charge-to-alanine mutant of (black bars) ADAM7 or ADAM28 Dis–Fc. Jurkat cells were added as described previously in the presence of 10 μg/ml QE2E5. Adhesion assays were performed as described in the Experimental section, and results shown are the average±S.D. for three independent experiments each performed in triplicate. Percentage adhesion=[adherent cells(native or mutant rDis–Fc)−adherent cells(BSA)]/[adherent cells(native rDis–Fc)−adherent cells(BSA)]×100. (b) RPMI 8866 cell adhesion to 5 μg/ml of native ADAM7 or ADAM28 Dis–Fc (white bar) or charge-to-alanine mutants of ADAM7 or ADAM28 Dis–Fc (black bars) was evaluated in the presence of 10 μg/ml of 2G3. Results shown are the average±S.D. for three independent experiments each performed in triplicate. Percentage adhesion=[adherent cells(native or mutant rDis–Fc)−adherent cells(BSA)]/[adherent cells(native rDis–Fc)−adherent cells(BSA)]×100.

Figure 5. The integrin α9β1 functions as a receptor for multiple mammalian disintegrin domains

Adhesion of CHO cells (1×105 cells/well) expressing the human α9 integrin subunit (■) or mock-transfected cells (○) to various recombinant mammalian disintegrin domains was examined in a concentration-dependent manner. The α9β1 function-blocking mAb Y9A2 was included at 5 μg/ml at the highest ligand concentration (▲). Adhesion assays were performed as described previously in Hepes-Tyrode's buffer containing 1 mM CaCl2 and 0.5 mM MgCl2 with no exogenous integrin activators. ADAM12 Dis–Fc possessed the entire ADAM12 disintegrin domain spanning residues Asn414–Gly519. Results shown are the average±S.D. for a representative experiment performed in triplicate.

Figure 6. Mutagenesis analysis of ADAM7 and ADAM28 disintegrin domains as ligands for the integrin α9β1

Microtitre wells were coated with 10 μg/ml of native ADAM7 or ADAM28 Dis–Fc (white bars) or an appropriate charge-to-alanine mutant (black bars). α9-CHO cells were added (1×105 cells/well) to wells and the extent of adhesion was determined as previously mentioned. Results shown are the average±S.D. for three independent experiments each performed in triplicate. Percentage adhesion=[adherent cells(native or mutant rDis–Fc)−adherent cells(BSA)]/[adherent cells(native rDis–Fc)−adherent cells(BSA)]×100.