Splice variants of the b-site APP-cleaving enzyme BACE1 in human brain and pancreas q Robert Ehehalt, a Beate Michel, a Davide De Pietri Tonelli, b Daniele Zacchetti, b Kai Simons, a and Patrick Keller a, * a Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany b Cellular Neurophysiology Unit, Department of Neurosciences, Dibit, San Raffaele Scientific Institute, via Olgettina 58, I-20132 Milano, Italy Received 15 March 2002 Abstract BACE is the b-secretase responsible for the first step in amyloidogenic processing of the amyloid precursor protein APP. We have identified two BACE isoforms, BACE1B and BACE1C, lacking 25 and 44 amino acids, respectively. Whereas the BACE1B transcript is present in human pancreas and brain, the BACE1C transcript is found in pancreas only. In transfected cells both BACE1A, which encodes the originally described full-length BACE1 protein and the close homolog BACE2 localized mainly to post-Golgi membranes. In contrast, the two shorter isoforms were found in the endoplasmic reticulum only, and they did not display b-secretase activity. Using RNase protection we in addition show that the major pancreatic transcript is BACE1A. This suggests that the known absence of b-secretase activity in the pancreas is not due to a missing BACE1A transcript. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Amyloid precursor protein; APP; Alzheimer; BACE isoforms; b-Secretase A hallmark of Alzheimer’s disease is the progressive formation in the brain of insoluble amyloid plaques containing b-amyloid (Ab). Ab is produced by proteo- lytic cleavage from the amyloid precursor protein (APP), a transmembrane protein with a large N-terminal ect- odomain and a short C-terminal cytoplasmic tail [1]. During its intracellular transport APP can also be cleaved by an alternative pathway (a-cleavage) to release the nonamyloidogenic secreted ectodomain of APP (aAPPsec). Ab generation occurs in two steps [2]. First, APPiscleavedinitsluminaldomain(b-cleavage) to leave behind a C-terminal 10 kDa fragment. This C-terminal stub subsequently is the substrate for c-secretase, which cleaves within the transmembrane domain to release Ab. Two enzymes capable of b-cleavage, the so-called b-site APP-cleaving enzymes (BACE1, originally referred to as BACE; and BACE2), have been identified [3–9]. BACE1 mRNA is found at very high levels in pancreas, at moderate levels in brain, and at low levels in most peripheral tissues [5,6]. Interestingly, b-secretase activity is high in brain only, but almost undetectable in pancreas [4]. The homologous protein BACE2 has a more broad mRNA distribution. It is widely expressed in peripheral tissues, but only at low levels in brain [7,9,10]. While both enzymes are capable of promoting b-cleavage in vitro and in cell culture [3–6,8,11], data from BACE1 knockout mice indicate that BACE1 at least in the brain of mice is the enzyme responsible for b-cleavage of APP in vivo [12,13]. Recently, a shorter transcript of BACE1 (BACE457) has been found in pancreas, but not in brain [14]. However, the same transcript (BACE-I-457), together with two more (BACE-I-476 and BACE-I-432), now has also been cloned from human brain [15]. While the BACE457 product was shown to be localized to the endoplasmic reticulum (ER) and to be devoid of b-sec- Biochemical and Biophysical Research Communications 293 (2002) 30–37 www.academicpress.com q Abbreviations: APP, amyloid precursor protein; aAPPsec, a- cleaved ectodomain of APP; Ab, b-amyloid fragment of APP; BACE, b-site APP-cleaving enzyme; bAPPsec, b-cleaved ectodomain of APP; CFP, cyan color variant of GFP; ER, endoplasmic reticulum; GFP, green fluorescent protein; YFP, yellow color variant of GFP. * Corresponding author. Fax: +49-351-210-29-00. E-mail address: keller@mpi-cbg.de (P. Keller). 0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S0006-291X(02)00169-9