Human aspartic protease memapsin 2 cleaves the β-secretase site of β-amyloid precursor protein

Abstract

The cDNAs of two new human membrane-associated aspartic proteases, memapsin 1 and memapsin 2, have been cloned and sequenced. The deduced amino acid sequences show that each contains the typical pre, pro, and aspartic protease regions, but each also has a C-terminal extension of over 80 residues, which includes a single transmembrane domain and a C-terminal cytosolic domain. Memapsin 2 mRNA is abundant in human brain. The protease domain of memapsin 2 cDNA was expressed in Escherichia coli and was purified. Recombinant memapsin 2 specifically hydrolyzed peptides derived from the ␤-secretase site of both the wild-type and Swedish mutant ␤-amyloid precursor protein (APP) with over 60-fold increase of catalytic efficiency for the latter. Expression of APP and memapsin 2 in HeLa cells showed that memapsin 2 cleaved the ␤-secretase site of APP intracellularly. These and other results suggest that memapsin 2 fits all of the criteria of ␤-secretase, which catalyzes the rate-limiting step of the in vivo production of the ␤-amyloid (A␤) peptide leading to the progression of Alzheimer's disease. Recombinant memapsin 2 also cleaved a peptide derived from the processing site of presenilin 1, albeit with poor kinetic efficiency. Alignment of cleavage site sequences of peptides indicates that the specificity of memapsin 2 resides mainly at the S 1 subsite, which prefers small side chains such as Ala, Ser, and Asp. membrane aspartic proteases ͉ Alzheimer's disease M any proteases are intimately involved in the regulation of cellular and physiological functions. There are five well studied human aspartic proteases. Pepsin and gastricsin participate in digestion in the stomach whereas cathepsins D and E function in intracellular protein degradation in lysosomes and endosomes. Human cathepsin D has been implicated in the metastasis of breast cancer and in Alzheimer's disease. Renin catalyzes the conversion of angiotensinogen to angiotensin 1, a clinically important step in hemostasis. The intimate involvement of human aspartic proteases in physiology and disease illustrates their central role in biology and medicine.The emergence of human gene sequences in the expressed sequence tag (EST) database represents an important new resource for identifying novel human enzymes. We report here the cloning of two new human aspartic proteases, memapsin 1 (M1) and memapsin 2 (M2), initially identified from the human EST database. These proteases are unique among aspartic proteases in that they are membrane-anchored. The presence of M2 in the brain led us to test its capacity to hydrolyze the ␤-amyloid precursor protein (APP). Detailed enzymic and cellular studies suggest that M2 fits all of the criteria of ␤-secretase. Like M2, APP is a type I integral transmembrane protein and is known to be processed in vivo at three sites. The evidence suggests that cleavage at the ␣-secretase site by a membraneassociated metalloprotease is a physiological event. This site is located in APP 12 residues away from the luminal surface of the plasma membrane....

Receptor tyrosine kinases positively regulate BACE activity and Amyloid-β production through enhancing BACE internalization

“…Up to now, studies of BACE have concentrated on its structure [4,45], trafficking [11,16,[46][47][48][49][50], and specific inhibition [10], but how its activity is regulated remains largely unclear. In this study, we demonstrated that BACE activity both in vitro and in vivo could be efficiently regulated by RTKs, and the regulation of BACE activity led to alterations of subsequent Aβ production.…”

“…Nowadays, β-secretase is considered the initial and rate-limiting enzyme during this process. β-site APP cleavage enzyme (BACE), a type I integral membrane aspartic protease, was identified as the main β-secretase [3][4][5][6][7]. As formation of senile plaques could not be detected in BACE-deficient mice [8,9], BACE has attracted major attentions in AD research during recent years.…”

Ion channel regulation by β-secretase BACE1 – enzymatic and non-enzymatic effects beyond Alzheimer's disease

“…In 1999, BACE1 (also: b-secretase 1 or memapsin 2) was identified as a b-site APP-cleaving enzyme, [7][8][9][10][11] and, 2 years later, confirmed as the major b-secretase in vivo. 12 BACE1 initiates the amyloidogenic pathway of APP processing.…”

Trafficking and Proteolytic Processing of APP

“…Expression cloning or biochemical purification led to the identification of a unique b-secretase enzyme (Sinha et al 1999;Vassar et al 1999;Yan et al 1999;Hussain et al 2000;Lin et al 2000). Although many different names were originally used to describe this activity, such as memapsin, aspartyl protease 2, or BACE1 (b-site APP cleaving enzyme-1), BACE1 is now the generally accepted term for the enzyme harboring b-secretase activity.…”