L-selectin is constitutively expressed at high levels on the surface of resting neutrophils, and these molecules are shed upon neutrophil stimulation in a distinctly robust manner (18
). The primary sheddase of L-selectin in vivo
is ADAM17 (12
). The activity of ADAM17 is regulated and intracellular signals lead to the induction of its enzymatic activity (26
). For instance, the MAPK signaling pathway participates in the activation of ADAM17 (20
). The target of this inducer mechanism, however, remains unclear. We demonstrate in this study that the cytoplasmic region of ADAM17 is not critical for L-selectin shedding, which is consistent with other ADAM17 substrates as well (32
). A caveat for these finding, however, is that a cell-based, ADAM17 reconstitution assay was not performed with primary neutrophils, nor did it involve their physiological stimuli. Moreover, the cytoplasmic region of ADAM17 may contribute to the regulation of its sheddase activity in other manners, such as by controlling ADAM17's distribution and proximity to its substrates in the plasma membrane during neutrophil polarization (69
). We also demonstrate that sulfhydryl-modifying agents can directly regulate the enzymatic activity of mature ADAM17 through its extracellular region. Two vicinal cysteine sulfhydryl motifs that provide redox-sensitive sites in various other proteins occur in the disintegrin/cysteine-rich region of ADAM17, and we show that theses motifs are critical for ADAM17 activity. Hence, our findings support thiol-disulfide conversion within the extracellular portion of ADAM17 as a mechanism that couples intracellular signaling and ADAM17 activation.
It has been reported that redox processes are of mechanistic significance in regulating the catalytic activity of metalloproteases. A prevailing hypothesis is that oxidation of a cysteine in the pro-domain of metalloproteases can dissociate its interaction with the Zn atom in the catalytic domain to activate the latent metalloprotease (i.e. redox-mediated cysteine switch) (70
). Such a mechanism has also been proposed to regulate ADAM17's activity, as oxidation of a synthesized peptide mimicking its pro-domain restored the enzymatic activity of ADAM17 (75
). However, in various studies only mature ADAM17 and not its pro-form has been identified on the surface of resting and activated cells (26
), which is the location at which L-selectin shedding primarily occurs (18
). Others have reported that the treatment of neutrophils with reducing agents inhibit L-selectin shedding, whereas oxidizing agents induce its shedding (45
), and we found that H2
-treated neutrophils shed L-selectin in an ADAM17-dependent manner (). However, we cannot exclude the possibility that these agents indirectly affected the activity of ADAM17. Therefore, we directly examined the effects of redox agents on ADAM17 activity in a cell-free assay. We found that the enzymatic activity of a purified form of human ADAM17 lacking its pro-domain and intracellular region was enhanced in the presence of H2
and diminished under very mild reducing conditions by DTT. Of interest is that the low activity state of ADAM17 under reducing conditions was converted to a significantly higher activity under oxidizing conditions (). We observed that DTT and H2
had the same effects on a recombinant, soluble form of mouse ADAM17 from the same commercial source (data not shown); however, this reagent contained a mixture of mature and pro-ADAM17, which confounds interpretation of the results. Taken together, our findings point to the involvement of cysteinyl sulfhydryl groups in the extracellular portion of mature ADAM17 that are sensitive to thiol-disulfide conversion and may serve to regulate its catalytic activity.
Accumulating evidence implicates thiol-disulfide rearrangements in the extracellular portion of cell surface proteins as a key mechanism of stimulus-response coupling. For instance, a variety of studies have reported a role for this process in the regulation of integrin binding activity (44
). Within the cysteine-rich regions of the β subunit for all integrins occurs a vicinal cysteine sulfhydryl motif referred to as CXXC. This motif was originally determined to be a redox-sensitive site in various oxidoreductases, such as members of the PDI family (47
). Moreover, CXXC motifs in the integrin αIIb
have been directly shown to be active sites for thiol-disulfide exchange and conformational change (44
). An analysis of ADAM17 revealed two highly conserved CXXC motifs occurring in its disintegrin/cysteine-rich region (C522
XXC and C600
XXC). We found that exchanging the cysteines in either motif with alanines (AXXA) abrogated L-selectin shedding (), demonstrating that these residues are critical for ADAM17 activity. In further support of this, a nonfunctional ADAM17 variant identified in a mutagenized cell line was found to contain a point mutation at residue Cys-600 (79
). Replacement of this residue with any other amino acid prevented the activity of the expressed ADAM17 construct (80
). The above findings implicate a role for the CXXC motifs in ADAM17 in its activation, perhaps by serving as active sites for redox modifications. However, we cannot formally rule out the possibility that redox agents may affect other cysteine residues in mature ADAM17. Strategies involving mass spectrometry peptide mapping, for instance, will be useful in determining whether the conversion of inactive to active ADAM17 is accompanied by selective thiol-disulfide conversion at the CXXC locations.
The sequence of the XX dipeptide of CXXC active sites in oxidoreductases has been reported to be important in controlling their thiol-disulfide redox properties (64
). Moreover, the specificity of cysteine oxidation can be influenced by flanking basic residues (65
). Both XX dipeptides of the CXXC sequences in ADAM17 possess a basic charge and contain a highly conserved lysine residue (). Interestingly, we found that exchanging the dipeptide region of the C522
XXC or C600
XXC motif of ADAM17 with glutamic acid residues (CEEC-1 and CEEC-2, respectively) had differential effects on L-selectin shedding. Only cells reconstituted with the CEEC-2 construct demonstrated impaired L-selectin shedding. These findings suggest that the two CXXC motifs in the disintegrin/cysteine-rich region of ADAM17 may vary in their biological function and/or redox properties. Of interest is that the C600
XXC motif comprises the four amino acids preceding the predicted hyper-variable region of ADAM17. The hyper-variable region occurs in all ADAM family members and consists of amino acid sequences that are the most divergent and variable in length (43
). The hyper-variable region may constitute a potential protein-protein interface, and structural analyses indicate that it is spatially juxtaposed to the catalytic domain of the ADAMs (43
). Hence, alterations in sulfhydryl redox events at the C600
XXC location would likely influence the conformation and activity of ADAM17. An alignment of 39 ADAM sequences including 23 human ADAMs reveals that the CXXC sequence preceding the hyper-variable region only occurs in ADAM10 and ADAM17 (43
), two sheddases that have several substrates in common (17
). It will be interesting to further investigate the effects of varied types and number of amino acids occurring between the cysteines of the CXXC motifs on ADAM17's activity.
Considering that H2
can induce ADAM17-dependent L-selectin shedding upon neutrophil treatment, we explored whether NADPH oxidase-derived ROS could serve as an activating agent for L-selectin shedding by neutrophils. This was addressed in two ways. First, human neutrophils were treated with the potent NADPH oxidase inhibitor DPI in combination with ROS scavengers. Second, we examined peripheral blood neutrophils isolated from CGD patients lacking NADPH oxidase activity. In either case, L-selectin shedding was not prevented, suggesting that NADPH oxidase-derived ROS is not essential for the activation of ADAM17. At this time we cannot rule out a role for separate sources of ROS production. Alternatively, ADAM17 could be a substrate of a transmembrane oxidase activity. Members of the PDI family are oxidoreductases, which are multifunctional proteins that catalyze thiol-disulfide reactions between its own CXXC active sites and sulfhydryl groups of its substrates (47
). Of interest is that PDI has been implicated in regulating ectodomain shedding (46
). PDI can be released from neutrophils upon their degranulation and it has also been detected on the cell surface of resting neutrophils (46
). Bennett et al. have reported that the treatment of neutrophils with oxidoreductase inhibitors promoted rapid and efficient L-selectin shedding independent of cell activation, and this occurred to a degree upon their treatment with anti-PDI mAbs as well (46
). The authors speculated that PDI might constitutively act on L-selectin. Consistent with our findings, however, is the possibility that an oxidoreductase activity targets ADAM17, which could maintain the sheddase in a low or high activity state through the reduction or oxidation, respectively, of active sites for thiol-disulfide conversion.
Integrins have also been reported to possess an intrinsic oxidoreductase activity. This has been best demonstrated for αIIb
in which its CXXC sites undergo intramolecular thiol-disulfide exchange (44
). Considering that the CXXC motifs occur in the cysteine-rich regions of all integrins, a similar oxidoreductase activity may occur by other members of this family as well. Several ADAMs have been reported to interact with integrins (83
), which includes ADAM17 and α5
). Thus, an intriguing hypothesis is that such an interaction may be of mechanistic significance in regulating the catalytic activity of ADAM17 through a thiol-disulfide exchange process. As with αIIb
), ADAM17 may also possess an endogenous thiol-isomerase activity, and upon receiving a cue from an intracellular signal, ADAM17 may catalyze a thiol-disulfide exchange within its own CXXC motifs, resulting in its switch to an activated sheddase.
In conclusion, our data demonstrates for the first time that the activity of mature ADAM17 can be altered by redox conditions, in which a reducing environment diminishes its activity and an oxidizing environment increases its activity. Moreover, ADAM17 contains two highly conserved CXXC motifs within its disintegrin/cysteine-rich region that are essential for L-selectin shedding. Taken together, these findings suggest a possible mechanism for ADAM17 activation that involves the targeting of critical sulfhydryl groups by redox agents or an oxidoreductase activity, resulting in reversible thiol-disulfide conversions, switching ADAM17 from a less active to a fully active conformation, or perhaps lowering the threshold for other agonists to induce an active conformation. This process may be essential for the very robust shedding of L-selectin upon neutrophil activation. Alternatively, redox modification of ADAM17 may serve as a general process for its activation. Indeed, others have reported that the treatment of cells with sulfhydryl-modifying agents induces the shedding of TNFα's receptors (76
). We as well as others have reported that ADAM17's sheddase activity is also induced during neutrophil apoptosis (52
), and this process appears to involve an up-regulation in cell surface levels of processed ADAM17 (86
). It will be interesting to determine whether redox modification of ADAM17 may regulate its activity during this cellular event as well.