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. 2009 Aug 21;284(34):22590-600.
doi: 10.1074/jbc.M109.032599. Epub 2009 Jun 29.

Role of ADAMs in the ectodomain shedding and conformational conversion of the prion protein

David R Taylor  1 , Edward T Parkin, Sarah L Cocklin, James R Ault, Alison E Ashcroft, Anthony J Turner, Nigel M Hooper
Affiliations

Role of ADAMs in the ectodomain shedding and conformational conversion of the prion protein

David R Taylor et al. J Biol Chem. .

Abstract

The cellular prion protein (PrP(C)) is essential for the pathogenesis and transmission of prion diseases. PrP(C) is bound to the plasma membrane via a glycosylphosphatidylinositol anchor, although a secreted, soluble form has also been identified. Previously we reported that PrP(C) is subject to ectodomain shedding from the membrane by zinc metalloproteinases with a similar inhibition profile to those involved in shedding the amyloid precursor protein. Here we have used gain-of-function (overexpression) and loss-of-function (small interfering RNA knockdown) experiments in cells to identify the ADAMs (a disintegrin and metalloproteinases) involved in the ectodomain shedding of PrP(C). These experiments revealed that ADAM9 and ADAM10, but not ADAM17, are involved in the shedding of PrP(C) and that ADAM9 exerts its effect on PrP(C) shedding via ADAM10. Using dominant negative, catalytically inactive mutants, we show that the catalytic activity of ADAM9 is required for its effect on ADAM10. Mass spectrometric analysis revealed that ADAM10, but not ADAM9, cleaved PrP between Gly(228) and Arg(229), three residues away from the site of glycosylphosphatidylinositol anchor attachment. The shedding of another membrane protein, the amyloid precursor protein beta-secretase BACE1, by ADAM9 is also mediated via ADAM10. Furthermore, we show that pharmacological inhibition of PrP(C) shedding or activation of both PrP(C) and PrP(Sc) shedding by ADAM10 overexpression in scrapie-infected neuroblastoma N2a cells does not alter the formation of proteinase K-resistant PrP(Sc). Collectively, these data indicate that although PrP(C) can be shed through the action of ADAM family members, modulation of PrP(C) or PrP(Sc) ectodomain shedding does not regulate prion conversion.

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Figures

FIGURE 1.
FIGURE 1.
Shedding of PrPC is enhanced by ADAM9 and ADAM10. HEK cells stably expressing PrPC were transiently transfected with either empty vector (Mock) or the cDNAs encoding ADAM9 (AD9), ADAM10 (AD10), or ADAM17 (AD17). Transfected cells were incubated for 5 h in serum-free medium before harvesting the conditioned medium and preparing cell lysates. A, immunodetection of ADAMs in cell lysates. B, immunodetection of PrPC (antibody 3F4), APP (antibody Ab54), and actin in cell lysates. C, immunodetection of sPrP in conditioned medium (antibody 3F4). D, immunodetection of sAPPα in conditioned media (antibody 6E10). C and D, multiple immunoblots were quantified by densitometric analysis. Results are the mean ± S.D. (n = 3). *, significant at p ≤ 0.05.
FIGURE 2.
FIGURE 2.
Knockdown of ADAM9 and ADAM10 by siRNA reduces the shedding of PrPC. HEK cells stably expressing PrPC were transiently transfected with one of three different siRNA duplexes to either ADAM9 (AD9) (A–C) or ADAM10 (AD10) (D–F) as detailed under “Experimental Procedures.” Transfected cells were incubated for 5 h in serum-free medium before harvesting the conditioned medium and preparing cell lysates. A and D, immunodetection of ADAM10, PrPC, APP, and actin in cell lysates. ADAM9 was detected by semi-quantitative RT-PCR. Immunodetection of sPrP (B and E) and sAPPα (C and F) in conditioned medium using antibodies 3F4 and 6E10, respectively, is shown. Multiple immunoblots were quantified by densitometric analysis. Results are the mean ± S.D. (n = 3). *, significant at p ≤ 0.05. Ctrl, control.
FIGURE 3.
FIGURE 3.
Knockdown of ADAM10 by siRNA inhibits the increased shedding of PrPC by ADAM9. HEK cells stably expressing PrPC were transiently transfected with or without ADAM10 (AD10) siRNA duplexes as detailed under “Experimental Procedures.” After 18 h cells were transfected with or without the cDNA encoding ADAM9 (AD9). After a further 25 h, cells were incubated for 5 h in serum-free medium before harvesting the conditioned medium and preparing cell lysates. A, immunodetection of ADAM9, ADAM10, PrPC, APP, and actin in cell lysates. Immunodetection of sPrP (B) and sAPPα (C) in conditioned medium using antibodies 3F4 and 6E10, respectively, is shown. Multiple immunoblots were quantified by densitometric analysis. Results are the mean ± S.D. (n = 3). *, significant at p ≤ 0.05. O/E, overexpressing.
FIGURE 4.
FIGURE 4.
Proteolytic activities of both ADAM9 and ADAM10 are required for the shedding of PrPC. HEK cells stably expressing PrPC were transiently transfected with either empty vector (Mock) or the cDNAs encoding ADAM9 (AD9), dnADAM9 (dnAD9), ADAM10, or dnADAM10 (dnAD10). Transfected cells were incubated for 5 h in serum-free medium after which the conditioned medium was harvested and cell lysates prepared. A, immunodetection of ADAMs, PrPC, APP, and actin in cell lysates. B, immunodetection of sPrP in conditioned medium. C, immunodetection of sAPPα in conditioned medium. B and C, multiple immunoblots were quantified by densitometric analysis. Results are the mean ± S.D. (n = 3). *, significant at p ≤ 0.05. O/E, overexpressing.
FIGURE 5.
FIGURE 5.
ADAM10, but not ADAM9, cleaves recombinant PrP. A, recombinant PrP (final concentration 10 μm) was incubated with 200 ng of recombinant ADAM9 or ADAM10 for 3 h at 37 °C. Samples were then prepared for analysis by nanoelectrospray mass spectrometry as described under “Experimental Procedures.” Nanoelectrospray mass spectra of rPrP only, rPrP with ADAM9(AD9), and rPrP with ADAM10 (AD10) showing the 9+ and 8+ charge state envelope are shown. PrP (●) is present in all samples, and the proteolytically cleaved product (■) is present in the ADAM10-treated sample only. The m/z ratios for the [M + nH]n+ signals are labeled, where n is the charge state; adjacent signals correspond to sodium substitution adducts of the protonated signal. B, C-terminal sequence of PrPC showing the site of GPI anchor addition and the determined cleavage site by ADAM10. C, activity of recombinant ADAM9 and ADAM10 was assessed by measuring the cleavage of a fluorogenic peptide substrate Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Ser-Arg-NH2 for 3 h at 37 °C in the presence or absence of the inhibitor GW4023. Values are mean ± S.D. (n = 3). Mca, 7,4-methoxycoumarin-4-acetyl; Dpa, Nβ-(2,4-dinitrophenyl)-l-2,3-diaminopropionic acid.
FIGURE 6.
FIGURE 6.
ADAM9 and -10 are involved in the shedding of BACE1. HEK cells stably expressing BACE1 were transiently transfected with either empty vector (Mock) or the cDNAs encoding either ADAM9 (AD9) or ADAM10 (AD10). Transfected cells were incubated for 5 h in serum-free medium before harvesting the conditioned medium and preparing cell lysates. Immunodetection of overexpressed ADAM9 (A) or ADAM10 (B) along with BACE1 and actin in cell lysates and sBACE1 in conditioned medium is shown. HEK cells stably expressing BACE1 were transiently transfected with or without ADAM10 siRNA duplexes as detailed under “Experimental Procedures.” After 18 h cells were transfected with or without the cDNA encoding ADAM9. After a further 25 h, cells were incubated for 5 h in serum-free medium before harvesting the conditioned medium and preparing cell lysates. C, immunodetection of ADAM9, ADAM10, and actin in cell lysates. D, immunodetection of BACE1 in lysates and conditioned medium. Multiple immunoblots were quantified by densitometric analysis. Results are the mean ± S.D. (n = 3). *, significant at p ≤ 0.05. O/E, overexpressing.
FIGURE 7.
FIGURE 7.
ADAM9, -10, and -17 are not involved in the endoproteolytic α-cleavage of PrPC. HEK cells stably expressing PrPC were transfected with either empty vector (Mock) or the cDNAs encoding ADAM9 (AD9), ADAM10 (AD10), or ADAM17 (AD17) (A) or siRNA targeted to either ADAM9 or ADAM10 (B). Cell lysates were prepared and deglycosylated as described under “Experimental Procedures.” Full-length (FL) PrPC and its C1 fragment in cell lysates were immunodetected using antibody 6H4. Multiple immunoblots were quantified by densitometric analysis. Results are the mean ± S.D. (n = 3).
FIGURE 8.
FIGURE 8.
Inhibition of PrPC shedding does not alter PrPSc formation. A, ScN2a cells were incubated for 12 h in the absence or presence of GW0264 (10 μm). sPrP in conditioned medium was detected using antibody 6D11. B, ScN2a cells were incubated in the presence or absence of GW0264 (10 μm) for 96 h (inhibitor was replaced every 12 h). As a positive control for anti-PrPSc activity, cells were treated with 1 μg/ml Congo red for 96 h (with media replaced every 12 h). Cells were harvested, lysed, and digested with PK as described under “Experimental Procedures.” Immunoblotting for PrP was performed using antibody 6D11. C, densitometric analysis of PK-resistant PrPSc levels for each treatment, relative to those of mock-treated cells, from multiple blots from three independent experiments is shown. D, to confirm that shedding had been inhibited in ScN2a cells treated with GW0264 for 4 days, medium samples from each day of incubation were pooled and then concentrated. Equal volumes of concentrated medium were resolved by SDS-PAGE and immunoblotted using 6D11. *, significant at p ≤ 0.05.
FIGURE 9.
FIGURE 9.
ADAM10 overexpression increases the shedding of PrPC and PrPSc but does not alter prion conversion. ScN2a cells were transiently transfected with empty vector (Mock) or the cDNA encoding ADAM10 (AD10) or treated with 1 μg/ml Congo red. The cells were then left for 96 h. A, 12 h prior to the end of the experiment, the medium was replaced with serum-free medium before harvesting the conditioned medium and preparing cell lysates. Immunodetection of PrP, ADAM10, and actin in cell lysates is shown. Medium samples were concentrated and then treated with PK, where indicated. Equal volumes of concentrated medium were resolved by SDS-PAGE and immunoblotted using 6D11 for sPrP. B, cells were harvested, lysed, and digested with PK as described under “Experimental Procedures.” PK-resistant PrPSc was detected using antibody 6D11. C, densitometric analysis of PK-resistant PrPSc levels of ADAM10-transfected and Congo red-treated cells, relative to those of mock-treated cells. Data from multiple blots from three independent experiments are shown. *, significant at p ≤ 0.05.
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