Post-translational Processing of β-Secretase (β-Amyloid-converting Enzyme) and Its Ectodomain Shedding

Abstract

Processing of the ␤-amyloid precursor protein (␤APP) by ␤-and ␥-secretases generates the amyloidogenic peptide A␤, a major factor in the etiology of Alzheimer's disease. Following the recent identification of the ␤-secretase ␤-amyloid-converting enzyme (BACE), we herein investigate its zymogen processing, molecular properties, and cellular trafficking. Our data show that among the proprotein convertase family members, furin is the major converting enzyme of pro-BACE into BACE within the trans-Golgi network of HK293 cells. While we demonstrate that the 24-amino acid prosegment is required for the efficient exit of pro-BACE from the endoplasmic reticulum, it may not play a strong inhibitory role since we observe that pro-BACE can produce significant quantities of the Swedish mutant ␤APP sw ␤-secretase product C99. BACE is palmitoylated at three Cys residues within its transmembrane/cytosolic tail and is sulfated at mature N-glycosylated moieties. Data with three different antibodies show that a small fraction of membrane-bound BACE is shed into the medium and that the extent of ectodomain shedding is palmitoylationdependent. Overexpression of full-length BACE causes a significant increase in the production of C99 and a decrease in the ␣-secretase product APPs␣. Although there is little increase in the generation of A␤ by full-length BACE, overexpression of either a soluble form of BACE (equivalent to the shed form) or one lacking the prosegment leads to enhanced A␤ levels. These findings suggest that the shedding of BACE may play a role in the amyloidogenic processing of ␤APP.Alzheimer's disease is a progressive degenerative disorder of the brain characterized by mental deterioration, memory loss, confusion, and disorientation. Among the cellular mechanisms contributing to this pathology are two types of fibrous protein deposition in the brain, intracellular neurofibrillary tangles consisting of polymerized tau protein, and abundant extracellular fibrils largely composed of ␤-amyloid 1 (for reviews see Refs. 1-3). ␤-Amyloid, also known as A␤, arises from proteolytic processing of the ␤-amyloid precursor protein (␤APP) at the ␤-and ␥-secretase cleavage sites. The cellular toxicity and amyloid-forming capacity of the two major forms of A␤ (A␤ 40 and especially A␤ 42 ) have been well documented (1-3).An alternative, anti-amyloidogenic cleavage carried out by ␣-secretase(s) is located within the A␤ peptide sequence of ␤APP, thus precluding the formation of intact insoluble A␤. This cleavage by ␣-secretase within the (His-His-GlnLys2Leu-Val) sequence of ␤APP is the major physiological route of APP maturation. The products of this reaction are a soluble 100 -120-kDa N-terminal fragment (␤APPs␣) and a C-terminal membrane-bound ϳ9-kDa segment (C83). In several recent reports, metalloproteinases such as ADAM9, -10, and -17 were shown to be involved in the ␣-secretase cleavage

Regulation of the stepwise proteolytic cleavage and secretion of PDGF-B by the proprotein convertases

“…This finding is in agreement with the in vitro digestions that indicated PC5 to be the most efficient enzyme, followed by Furin, PC7 and then by PACE4 (Figure 3a and b). Using various PC-inhibitors, the processing of proPDGF-B was found to be blocked by a1-PDX, an engineered variant of a1-antitrypsin (Anderson et al, 1993;Benjannet et al, 1997) and by the inhibitory prosegments of PCs including pp-Furin, ppPC5 and ppPACE4 (Zhong et al, 1999;Benjannet et al, 2001 andNour et al, 2003).…”

“…(d) Cell lysates obtained from transiently cotransfected HK293 cells with vectors encoding proPDGF-B and each of the PC-inhibitors serpin a1-antitrypsin (a1-PDX) and a2-macroglobulin-Furin (a2-MG-F) and the prosegments preproFurin (ppFurin), ppPACE4, ppPC5 and ppPC7. After transfection, the cell lysates were analysed for ProPDGF-B processing using PDGF-B antibody (UpState) In order to assess the efficiency of PC-inhibitors on proPDGF-B processing by PC-like endogenous convertases, we co-transfected HK293 cells with vectors encoding proPDGF-B and each of the PC-inhibitors, including the PC-prosegments (Zhong et al, 1999), namely, the prosegments preproFurin (ppFurin), ppPACE4, ppPC5 and ppPC7 and the Furin-motif variants of a2-macroglobulin (a2-MG-F) and of the serpin a1-antitrypsin (a1-PDX) (Benjannet et al, 2001). After transfection, the processing of PDGF-B was analysed in cell lysates by immunoblotting using a PDGF-B antibody.…”

“…The Furin-deficient LoVo-C5 human colon adenocarcinoma cells were transiently cotransfected with the pcDNA3-zeo-flag empty vectors, pcDNA3-zeo-flag containing wild-type PDGF-B cDNA, or with the pcDNA3-zeo-flag vector containing PDGF-B and pIRES2-EGFP vectors that express full-length indicated convertases. Human embryonic kidney (HK293) cells were transiently cotransfected with the pIRES2-EGFP-V5 empty vector, wild-type or mutant pcDNA3-zeo-PDGF-B-flag constructs or with the pcDNA3-zeo-PDGF-B-flag and pIRES2-EGFP that expresses PCs inhibitors including preproFurin (ppFurin), ppPACE4, ppPC5, ppPC7, a2-macroglobulin (Benjannet et al, 2001;Khatib et al, 2002;Siegfried et al, 2003b). In several experiments HK293 cells were transiently transfected with pcDNA3-zeo-flag empty vector expressing either wildtype or mutated PDGF-B cDNAs or cotransfected with the PDGF-A and PDGF-B constructs.…”

“…Conditioned media were collected and cells lysed in buffer containing 150 mM NaCl, 50 mM Tris-HCl, pH 6.8, 0.5% Nonidet P-40, 0.5% and sodium deoxycholate (Roche Molecular Biochemicals). Cell lysates and conditioned media were subject to immunoprecipitation as previously described (Benjannet et al, 2001). …”

“…The basic aa-specific PCs are implicated in the processing of multiple protein precursors, including proteases, growth factors, and receptors at multibasic recognition sites exhibiting the general motif (K/R)-(X) n -(K/R)k (n ¼ 0, 2, 4 or 6) Benjannet et al, 2001;Khatib et al, 2002;Siegfried et al, 2003b). The convertase SKI-1 recognizes the motif (R/K)-X-(L,V)-Zk, where Z is any aa except Pro, Cys, Glu, and Val (Seidah and Prat, 2002), and NARC-1/PCSK9 recognizes the sequence VFAQk with Val at position P4 being critical (Benjannet et al, 2004).…”