CD147 is a regulatory subunit of the gamma-secretase complex in Alzheimer's disease amyloid beta-peptide production

Abstract

gamma-Secretase is a membrane protein complex that cleaves the beta-amyloid precursor protein (APP) within the transmembrane region, after prior processing by beta-secretase, producing amyloid beta-peptides Abeta(40) and Abeta(42). Errant production of Abeta-peptides that substantially increases Abeta(42) production has been associated with the formation of amyloid plaques in Alzheimer's disease patients. Biophysical and genetic studies indicate that presenilin-1, which contains the proteolytic active site, and three other membrane proteins [nicastrin, anterior pharynx defective-1 (APH-1), and presenilin enhancer-2 (PEN-2)] are required to form the core of the active gamma-secretase complex. Here, we report the purification of the native gamma-secretase complexes from HeLa cell membranes and the identification of an additional gamma-secretase complex subunit, CD147, a transmembrane glycoprotein with two Ig-like domains. The presence of this subunit as an integral part of the complex itself was confirmed through coimmunoprecipitation studies of the purified protein from HeLa cells and of solubilized complexes from other cell lines such as neural cell HCN-1A and HEK293. Depletion of CD147 by RNA interference was found to increase the production of Abeta peptides without changing the expression level of the other gamma-secretase components or APP substrates whereas CD147 overexpression had no statistically significant effect on Abeta-peptide production, other gamma-secretase components or APP substrates, indicating that the presence of the CD147 subunit within the gamma-secretase complex down-modulates the production of Abeta-peptides.

Fig. 1.

CD147 is found in the purified γ-secretase complex. (A) Purified sample was analyzed by SDS/PAGE (4–20% Tris·HCl gel) where the gel was stained with Coomassie blue. (B) The presence of the previously identified components of the γ-secretase complex was confirmed by Western blot. (C) The 50-kDa band, initially identified as CD147 through amino acid sequencing, was confirmed by Western blot by using two different anti-CD147 antibodies.

Fig. 2.

CD147 coelutes with γ-secretase components during chromatographic purification. (A) In the Q-Sepharose HP chromatographic step, CD147, together with Psn-1 CTF and Nct, was found only on fractions eluting at a salt concentration of 200 mM. (Elution steps: 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, and 1 M.) (B) In molecular sieve chromatography, CD147, Psn-1 CTF, and Nct coeluted at a molecular mass of 250–300 kDa.

Fig. 3.

Coimmunoprecipitation experiments using purified proteins indicate that CD147 is an integral member of the γ-secretase complex. (A) In purified γ-secretase complex, antibody to human CD147 coimmunoprecipitates Psn-1 CTF and Nct. (B) Anti-Psn-1 CTF antibody coimmunoprecipitates CD147 and Nct. (C) Anti-Nct antibody coimmunoprecipitates CD147 and Psn-1 CTF. The negative controls are shown in lane 3. Asterisks correspond to immunoglobulins.

Fig. 4.

CD147 as a member of the γ-secretase complex regulates the γ-secretase proteolytic activity in the production of Aβ₄₀ and Aβ₄₂ peptides. (A) CD147 RNAi specifically reduces CD147 expression in CHO-APP₆₉₅ cells without changing the expression of other γ-secretase components. The expression levels were measured by quantitative Western blots. CD147 RNAi does not change the amount of sAPP_α, sAPP_β, and sAPP in culture media. (B) Graded concentrations of Stealth CD147 siRNA duplexes show a dose-dependent reduction in CD147 expression. The levels of Aβ₄₀ (C) and Aβ₄₂ (D) peptides in culture media were measured by ELISA, showing that the depletion of CD147 increased the production of Aβ-peptides. Error bars represent standard deviation. Asterisks indicate a significant difference from control (in absence of siRNA), as determined by one-way ANOVA (P < 0.01). The results in B–D were from six repeated experiments.

Fig. 5.

Overexpression of CD147 on CHO-APP₆₉₅ cells does not lead to significant changes in the production of Aβ₄₀ and Aβ₄₂ or in the expression of other γ-secretase components and APP. (A) The expression levels of CD147, APP, Nct, and Psn-1 CTF were measured by Western blots. The expression level of CD147 was substantially increased in CD147 overexpressed CHO-APP₆₉₅ (lane 2) compared with CHO-APP₆₉₅ (lane 1) cell line; the expression levels of APP, Nct, and Psn-1 CTF in the CHO-APP₆₉₅ (lane 1) and CD147 overexpressed CHO-APP₆₉₅ cell lines (lane 2) were comparable. The levels of Aβ₄₀ and Aβ₄₂ are shown in B and C, respectively. Error bars represent standard deviation. There are no statistically significant differences in the production levels of Aβ₄₀ and Aβ₄₂ by the CHO-APP₆₉₅ and CD147 overexpressed CHO-APP₆₉₅ cell lines, based on Student t test analysis (P > 0.05).