Splice variants of the β-site APP-cleaving enzyme BACE1 in human brain and pancreas

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Abstract

BACE is the β-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 β-secretase activity. Using RNase protection we in addition show that the major pancreatic transcript is BACE1A. This suggests that the known absence of β-secretase activity in the pancreas is not due to a missing BACE1A transcript.

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Materials and methods

cDNA cloning of BACE1 and BACE2. Full-length BACE1A, BACE1B, and BACE2 were amplified by PCR from a human hippocampal QUICK-Clone cDNA library (Clontech; catalogue number 7169-1), and transferred by TA-cloning into the vector pGEMT (Promega). SalI and NotI restriction sites were present in the primers at the 5- and 3-ends, respectively. To allow the subsequent addition of a CFP-tag to the 3-end of BACE1 and BACE2, AflII and ClaI sites, respectively, were added upstream of the stop codon.

Results

While cloning BACE1 from a human hippocampal cDNA library and human exocrine pancreatic tissue we consistently observed two bands of similar size on agarose gels. Sequencing of a number of individually cloned PCR products revealed, in addition to the previously published BACE1 sequence [3], [4], [5], [6], [8], one and two shorter isoforms in hippocampus and pancreas, respectively (Fig. 1). We renamed BACE1 to BACE1A and called the two new isoforms BACE1B (GenBank AF338816) and BACE1C (GenBank

Discussion

In this paper we document the identification and further characterization of two splice variants of the β-secretase BACE1. During the preparation of this manuscript two other groups independently reported the identification of the same BACE1 isoforms [14], [15]. Although all sequences were identical we found a number of discrepancies between the recently published data and our results concerning tissue distribution of the transcripts and β-secretase activity of the respective proteins.

Do BACE1B

Acknowledgements

We thank Dr. Federico Bertuzzi for providing human exocrine pancreas, Bart De Strooper for antibodies B10/4 and B7/7, and Drs. Melissa Rolls and Jamie White for the gift of ER and Golgi markers, respectively. D.Z and D.D.P.T. gratefully acknowledge the continuous support of Dr. Fabio Grohovaz and his group. R.E. was supported by Grant EH196/1-1 from the Deutsche Forschungsgemeinschaft (DFG), P.K. by the Max Planck Gesellschaft, D.Z. by a Giovanni Armenise–Harvard Foundation collaborative basic

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    Abbreviations: APP, amyloid precursor protein; αAPPsec, α-cleaved ectodomain of APP; Aβ, β-amyloid fragment of APP; BACE, β-site APP-cleaving enzyme; βAPPsec, β-cleaved ectodomain of APP; CFP, cyan color variant of GFP; ER, endoplasmic reticulum; GFP, green fluorescent protein; YFP, yellow color variant of GFP.

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