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Regulation of the -secretase ADAM10 by its prodomain and proprotein convertases¹

Institute of Biochemistry, Johannes Gutenberg-University Mainz, D-55128 Mainz, Germany; and
^* Institute of Virology, Philipps-University Marburg, D-35037 Marburg, Germany

²Correspondence: Institute of Biochemistry, Johannes Gutenberg-University Mainz, Becherweg 30, D-55128 Mainz, Germany. E-mail: bio.chemie{at}uni-mainz.de

SPECIFIC AIMS

To identify the proprotein convertases responsible for maturation of the -secretase ADAM10, we investigated the influence of PC7 and furin on ADAM10 processing and the resulting effect on amyloid precursor protein cleavage. We also examined the functional role of the ADAM10 prodomain by coexpression of a prodomain-deleted ADAM10 mutant together with its prodomain in trans.

PRINCIPAL FINDINGS

1. ADAM10 is proteolytically processed by PC7 and furin
The disintegrin metalloproteinase ADAM10 possesses -secretase activity as well as a potential proprotein convertase recognition sequence (RKKR) after its prodomain. By amino-terminal sequencing of ADAM10 purified from bovine kidney plasma membranes, we recently showed that the proprotein convertase recognition sequence RKKR is used to generate the mature 62–64 kDa catalytically active form of ADAM10. Therefore, we investigated whether PC7 or furin might be proteinases that cleave the ADAM10 zymogen. To provide evidence for prodomain removal by these proprotein convertases, HEK cells and HEK cells overexpressing HA-tagged ADAM10 (HEK ADAM10) were transfected with either PC7 or furin cDNAs, leading to stable overexpression of these convertases (Fig. 1C , D ). To further investigate the sequence requirements for ADAM10 processing, the proprotein convertase consensus sequence RKKR was mutated to NAQA, thereby eliminating the putative PC7/furin cleavage site of ADAM10 (named ADAM10RKKR).

View larger version (34K): [in this window] [in a new window]   Figure 1. Proteolytic processing of ADAM10. A) Western blot of stably transfected HEK cells. Blotted samples were probed with the rabbit polyclonal antibody Y-11 against the carboxyl-terminal HA tag of ADAM10; therefore, no signals can be detected in HEK cells. B) Densitometric analysis. The proform of ADAM10 in each cell line was set to 100%. The mature forms are expressed as percentage of the proform and as means ± SE of three independent experiments. Bar 1 represents the mature form of ADAM10 (control), bar 2 the mature form in PC7-overexpressing cells, and bar 3 the mature form in furin-overexpressing cells. C) Verification of PC7 overexpression and D) of furin overexpression in transfected cells.

The effect of PC7 and furin overexpression as well as the effect of the mutated proprotein convertase consensus sequence on the proteolytic processing of ADAM10 were analyzed by Western blotting (Fig. 1A ). Densitometric analysis revealed that increased amounts (180%) of the mature form of ADAM10 relative to the proform were detectable in ADAM10-PC7 and in ADAM10-furin cells compared with control HEK ADAM10 cells, indicating that both PC7 and furin are able to cleave the prodomain of ADAM10 (Fig. 1B ). In contrast, in ADAM10RKKR cells the mature form of ADAM10 could not be detected, thus suggesting that the RKKR recognition sequence is essential for correct processing of the zymogen (Fig. 1A ).

2. ADAM10 processing is inhibited by the proprotein convertase inhibitor dec-RVKR-cmk, but takes place in furin-deficient LoVo cells
To confirm that proprotein convertases are responsible for maturation of endogenous ADAM10, we investigated whether the synthetic proprotein convertase inhibitor decanoyl-RVKR-chloromethylketone is able to suppress the production of mature ADAM10. To determine the effect of the inhibitor on ADAM10 maturation, untransfected HEK cells and an antibody specifically interacting with endogenous ADAM10 were used. After 48 h treatment with 30 µM inhibitor, the cells were lysed and subjected to immunoblot analysis. The latter revealed that endogenous ADAM10 exists mainly as the mature enzyme and that formation of mature ADAM10 is significantly lowered due to the added inhibitor.

Next we examined the effect of furin deficiency on the proteolytic processing of endogenous ADAM10. Because both the immature and the mature form of ADAM10 could be detected by Western blot analysis in the furin-deficient cell line LoVo, furin is not essential or is at least dispensable for proteolysis. This finding strongly suggests that PC7 is able to cleave ADAM10 without participation of furin.

3. Prodomain removal leads to activation of ADAM10 and increased APPs secretion
Overexpression of PC7 in HEK cells led to enhanced APPs secretion. Furthermore, ADAM10-PC7 and ADAM10-furin cells that produced larger amounts of mature ADAM10 therefore secreted approximately twice as many APPs as HEK ADAM10 cells, in which ADAM10 maturation is unaffected. This indicates that enhanced maturation of ADAM10 leads to enhanced APPs release. That ADAM10-PC7 cells secreted threefold more APPs than HEK-PC7 cells demonstrates that PC7 exerts its -secretase-enhancing effect by processing of ADAM10. Moreover, in ADAM10RKKR cells, which express mutated ADAM10 lacking the proprotein convertase consensus sequence, -secretase activity was significantly lower compared with wild-type ADAM10-overexpressing cells and was not significantly enhanced by overexpression of PC7.

4. Dual function of the ADAM10 prodomain
To prove that the prodomain maintains ADAM10 in an inactive proform, we constructed a mutant form of ADAM10 containing the signal sequence but lacking the prodomain (ADAM10Pro). Expecting to obtain a constitutive active proteinase, we transfected HEK cells either with this mutant or wild-type ADAM10 as control. The expression of ADAM10 in HEK cells led to an increase of APPs release (Fig. 2 ). In contrast, ADAM10Pro is proteolytically inactive. HEK cells expressing ADAM10Pro secreted the same amount of APPs as untransfected HEK cells. Cell surface biotinylation revealed that ADAM10Pro is translocated from the endoplasmic reticulum to the cell surface.

View larger version (24K): [in this window] [in a new window]   Figure 2. Proteolytic activity of ADAM10Pro and ADAM10 in the presence of the ADAM10 prodomain in trans. HEK cells stably expressing either ADAM10 or ADAM10Pro and HEK cells as control were stably transfected with the vector pIRES1hyg alone or with the cDNA encoding the prodomain of ADAM10 in pIRES1hyg. For each cell line, the same number of cells were incubated with culture medium for 4.5 h and the proteins of the medium were subsequently precipitated. APPs was immunologically detected by Western blot with antibody 6E10 and quantified. Statistical significance between control HEK and HEK + prodomain, HEK ADAM10, and HEK ADAM10 + prodomain as well as between HEK ADAM10Pro and HEK ADAM10Pro + prodomain was determined by Student’s unpaired t test (*P<0,05; **P<0,001).

To investigate the involvement of the ADAM10 prodomain in maturation, we constructed an expression vector containing the signal sequence of ADAM10 followed by the coding region of the prodomain. After expression of the prodomain in HEK cells and in HEK cells already expressing either ADAM10Pro or ADAM10, the proteolytic activities of ADAM10Pro and ADAM10 were determined by measuring APPs secretion. As shown in Fig. 2 , the additional expression of the prodomain in cells expressing ADAM10Pro doubled the APPs secretion. This observation indicates that when expressed in trans as an independent polypeptide, the prodomain is able to rescue the -secretase activity of ADAM10Pro.

Furthermore, expression of the ADAM10 prodomain in trans together with wild-type ADAM10 led to a significant decrease of APPs release, indicating an inhibitory function of the prodomain with regard to the proteolytic activity of ADAM10 (Fig. 2) . The overexpressed ADAM10 prodomain also impaired the release of APPs by endogenous ADAM10.

CONCLUSIONS

Overexpression of the proprotein convertase PC7 has been reported to enhance APPs secretion and simultaneously decrease formation of Aß40 and Aß42. However, as no studies of the maturation of ADAM10 or TACE had been performed, the relation between PC7 and APP processing remained obscure.

Our results demonstrate that PC7 as well as furin affects ADAM10 maturation by proteolytic processing at the predicted proprotein convertase recognition site. We were able to establish that increased maturation of ADAM10 induced by overexpression of either PC7 or furin is accompanied by an increased -secretase activity. In contrast, maturation of ADAM10 with a mutated PC7/furin cleavage site (ADAM10RKKR) is totally blocked. This confirms first that this site is essential for prodomain removal; second, that proprotein convertases are involved in the maturation of ADAM10; and, finally, that blocked maturation of ADAM10 is accompanied by a loss of -secretase activity. The finding that APPs secretion in wild-type ADAM10-PC7 cells is further enhanced compared to HEK-PC7 cells, combined with the finding that after overexpression of PC7 in ADAM10RKKR cells APPs secretion is not significantly enhanced, demonstrates that PC7 acts upstream from APP as a pro--secretase-activating enzyme. A direct cleavage of APP by PC7 is improbable since APP does not contain a PC7/furin recognition motif at or close to the -secretase cleavage site.

By Western blot analysis of LoVo cells, we demonstrate that endogenous ADAM10 is proteolytically processed, indicating that furin is not required and can be compensated for by PC7 or PACE4. Compared with the proteolytic processing of endogenous ADAM10 in untransfected HEK cells, maturation is lowered significantly, which might be due to the missing furin.

Because activation of the ß-secretase BACE is performed by furin, but not by PC7, and activation of ADAM10 can be induced by both PC7 and furin, the competition between BACE and ADAM10 with regard to APP cleavage might be shifted to the nonamyloidogenic -secretase pathway by an inhibition of furin and/or a simultaneous stimulation of PC7 (Fig. 3 ). Considering the resemblance between PC7 and furin, this might be difficult to achieve. However, pathways that lead to enhanced gene expression of PC7 might be beneficial.

View larger version (28K): [in this window] [in a new window]   Figure 3. Model for regulation of APP processing by proprotein convertases via proteolytical activation of the -secretase ADAM10 and the ß-secretase BACE. The prodomain of ADAM10 is necessary for the correct folding and maintains the proteinase in a latent state. Prodomain removal results in the activation of the zymogen ADAM10. In contrast, the prodomain of BACE is cleaved by furin but not by PC7. Arrows indicate consecutive steps in proteinase maturation and function whereby subcellular compartments are not considered.

Our finding that ADAM10 lacking the prodomain is proteolytically inactive, although it is expressed in high amounts and is present on the plasma membrane, prompted us to examine the possible involvement of the ADAM10 prodomain in protein folding. The results of our study reveal that the ADAM10 prodomain, when expressed in trans as an independent polypeptide, restores the proteolytic activity of prodomain-deleted ADAM10. In accordance with our experiments, it was reported earlier that the prodomain of membrane type 1 matrix metalloproteinase (MT1-MMP) acted as intramolecular chaperone when expressed in trans with the mature protein.

Furthermore, we observed that the separately expressed prodomain inhibited the proteolytic activity of endogenous and overexpressed ADAM10. As the prodomain of ADAM10 reaches the cell surface and can even be secreted, we assume that the overexpressed prodomain is able to interact directly with mature ADAM10, thereby inhibiting the catalytic activity. Our results suggest that ADAM10, especially its prodomain, is a potential pharmaceutical target. Inhibitors with the ability to prevent the association of the prodomain should be able to increase the -secretase activity of ADAM10. In addition, targeted overexpression of PC7 together with ADAM10 should favor the nonamyloidogenic -secretase pathway.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0007fje ; to cite this article, use FASEB J. (June 27, 2001) 10.1096/fj.01-0007fje

This Article Full Text (PDF) All Versions of this Article: 15/10/1837 01-0007fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by ANDERS, A. Articles by FAHRENHOLZ, F. Search for Related Content PubMed PubMed Citation Articles by ANDERS, A. Articles by FAHRENHOLZ, F.