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....

The generation of the 40/42-residue amyloid β (Aβ) peptide in human brain by proteolysis of the membrane anchored β-amyloid precursor protein (APP) is a key event in the progression of Alzheimer's disease. 1 Proteases involved in the production of Aβ peptide are known as γand β-secretases. β-Secretase, which catalyzes the rate-limiting step in Aβ Supporting Information Available: Experimental procedures for the synthesis of Leu-Ala dipeptide isostere and solid-phase synthesis of OM99-1 and OM99-2 (PDF). This material is available free of charge via the Internet at http://pubs.acs.org.