Involvement of Cell Surface Glycosyl-phosphatidylinositol-linked Aspartyl Proteases in α-Secretase-type Cleavage and Ectodomain Solubilization of Human Alzheimer β-Amyloid Precursor Protein in Yeast

Abstract

Human ␤-amyloid precursor protein (APP) introduced into yeast undergoes ␣-secretase-type cleavage, suggesting that yeast have ␣-secretase-like protease(s). Here we report that two structurally and functionally related glycosyl-phosphatidylinositol-linked yeast aspartyl proteases, Mkc7p and Yap3p (collectively termed yapsin), are responsible for ␣-secretase-type cleavage of APP expressed in yeast, resulting in release of soluble APP into the extracellular space. Disruption of MKC7 and YAP3 in a vacuolar protease-deficient strain abolished this APP cleavage/release, and APP cleavage/release could be restored by introduction of MKC7 or YAP3 on a single copy plasmid. Purified Mkc7p cleaved an internally quenched fluorogenic APP peptide substrate at the ␣-secretase cleavage site. Measurement of proteolytic activity either in yeast homogenates or on the yeast cell surface revealed that most Mkc7p and Yap3p activities were localized at the cell surface. These results establish a molecular basis for ␣-secretase-type cleavage in yeast and support the generally held concept that ␣-secretase cleavage of APP occurs at the cell surface.The ␤-amyloid precursor protein (APP), 1 a type-1 transmembrane protein, can undergo at least two alternative pathways of proteolytic processing. ␤-Amyloid peptide (A␤), the component of cerebral plaques associated with Alzheimer's disease, is produced by proteolytic processing of APP and includes 28 residues of the N-terminal lumenal domain and the first 12-14 residues of the transmembrane domain (1). Alternatively, cleavage of a Lys-Leu bond the lumenal portion of the A␤ region of APP by a membrane protein-solubilizing proteolytic activity, termed "␣-secretase," releases the lumenal portion of APP as a soluble protein and precludes the formation of A␤ (1).There has been intensive interest in identifying the enzymes that cleave APP to determine their relationship to the etiology of Alzheimer's disease. However, the responsible molecules remain unknown. In particular, molecular identification of ␣-secretase could provide insight into the general mechanism of integral membrane protein cleavage (2) and clarify the role of this enzyme in the pathogenesis of Alzheimer's disease. We have reported that the yeast Saccharomyces cerevisiae has ␣-secretase-like activities: human APP expressed in S. cerevisiae undergoes proteolytic cleavage at the ␣-secretase site (3, 4). Existence of an ␣-secretase-like activity was also observed in insect cells (5), suggesting that the enzymes are widely conserved among metazoans. Therefore, identification of yeast secretases could lead to the discovery of counterparts among other phyla. Here we demonstrate that homologous glycosyl-phosphatidylinositol (GPI)-linked aspartyl proteases, Mkc7p (6) and Yap3p (7, 10 -12), termed "yapsin" (33), are responsible for ␣-secretase cleavage of human APP in yeast. EXPERIMENTAL PROCEDURESYeast Media and Strains-Liquid synthetic media were as described (13). All strains were derived from the W303 background as described (14). The str...

Enhanced peptide secretion by gene disruption of CYM1, a novel protease in Saccharomyces cerevisiae

“…Another metalloprotease, ADAM10, has been shown to contain a-secretase activity that cleaves APP, where it participates in the constitutive as well as the regulated secretion of the N-terminal fragment of APP [32]. However, after introduction into yeast, APP undergoes a-secretase-type cleavage, but Yps1p and Yps2p were identified as the active enzymes [5]. Our data indicates that yeast contain a metalloprotease, which could be a novel a-secretase, in addition to the two aspartic proteases, Yps1 and Yps2.…”

“…To enrich the secretion of full length peptides, the host strains used are often made protease deficient by inactivation of the vacuolar protease Pep4p and the glycosyl-phophatidylinositol (GPI)-anchored aspartyl protease, Yps1p [2]. Yps1p has been shown to be responsible for both Arg and Lys specific cleavage in the maturation of heterologous peptides such as prosomatostatin, human b-amyloid precursor protein and the elastasespecific inhibitor, pre-elafin [3][4][5]. Protease activity within the secretory pathway is dominated by the serine proteases, Kex2 and Kex1 and the aspartic protease, Yps1.…”

Glycosylphosphatidylinositol-anchored Proteins Play an Important Role in the Biogenesis of the Alzheimer's Amyloid β-Protein

“…It was recently demonstrated, however, that APP is proteolytically processed by an ␣-secretase-like pathway in the yeast Saccharomyces cerevisiae (23,24). This processing pathway was shown to involve the glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases YAP3 and MKC7 (25,26). In mammals, GPI-anchored proteins are a relatively small class of membrane proteins that are anchored to the outer leaflet of the cell membrane by a glycolipid anchor consisting of ethanolamine, mannose, glucosamine, and phosphatidylinositol (27).…”

The Functions of the Amyloid Precursor Protein Gene and Its Derivative Peptides: II Experimental Evidence and Clinical Studies

“…After injection of Aβ into the cerebral cortex of rats neuronal loss and degenerating neurites were observed; this was prevented by substance P administered systemically [280]. Retroviral expression of a C-terminal fragment containing the Aβ region in PC12 cells, when injected into the brains of newborn mice, caused atrophy of the cerebral cortex [281].…”