Increased generation of alternatively cleaved beta-amyloid peptides in cells expressing mutants of the amyloid precursor protein defective in endocytosis.

The subcellular location of the secretases processing the beta-amyloid precursor protein (APP) is not established yet. We analyzed the generation of the beta-amyloid peptide (Abeta) in human embryonic kidney 293 cell lines stably expressing wild-type and noninternalizing mutants of human APP. APP lacking the entire cytoplasmic domain or with both tyrosine residues of the motif GYENPTY mutated to alanine showed at least fivefold reduced endocytosis. In these cell lines, the production of Abeta1-40 was substantially reduced, but accompanied by the appearance of two prominent alternative Abeta peptides differing at the amino-termini. Based on antibody reactivity and mobility in high-resolution gels in comparison with defined Abeta fragments, these peptides were identified as Abeta3-40 and Abeta5-40. Notably, these alternative Abeta peptides were not generated when the APP mutants were retained in the early secretory pathway by treatment with brefeldin A. These results indicate that the alternative processing is the result of APP accumulation at the plasma membrane and provide evidence of distinct beta-secretase activities. Cleavage amino-terminal to position 1 of Abeta occurs predominantly in endosomes, whereas the processing at positions 3 or 5 takes place at the plasma membrane.

Analysis of constitutive and constitutive-like secretion in semi-intact pituitary cells.

To study biosynthetic transport through the constitutive and regulated secretory pathways, we have designed a semi-intact mammalian cell system that restores the transport of secretory proteins from the trans-Golgi/trans-Golgi network (TGN) to the cell surface. The mouse pituitary AtT-20 cell line is a suitable model to biochemically analyze molecular sorting in the secretory pathway. The prohormone proopiomelanocortin is sulfated on N-linked carbohydrate chains in the trans-Golgi prior to proteolytic processing in the secretory granule. Radiolabeling with [35S]sulfate therefore provides a convenient tool to selectively follow molecular events in the regulated secretory pathway without interference from earlier steps. Likewise, transport through the constitutive secretory pathway may be monitored using sulfate-labeled glycosaminoglycan chains. We show that export from the TGN is efficiently reconstituted in cells made semi-intact with streptolysin O, and is dependent on temperature, ATP and GTP hydrolysis, and cytosol. Packaging of proopiomelanocortin into immature secretory granules also activates the proteolytic processing machinery which eventually converts the prohormone to its bioactive mature product, adrenocorticotropic hormone. In addition, a large fraction of incompletely processed proopiomelanocortin is secreted as the processing intermediates from immature secretory granules. This process of constitutive-like secretion can be clearly distinguished from direct constitutive secretion from the trans-Golgi network by kinetic and compositional criteria. Furthermore, we have found that specific inhibitors of different protein phosphatases and kinases are potent blockers of constitutive and constitutive-like secretion. This experimental model should provide a valuable system to elucidate the molecular mechanism regulating post-Golgi traffic during secretory granule biogenesis.

Cloning of the PABA peptide hydrolase alpha subunit (PPH alpha) from human small intestine and its expression in COS-1 cells.

PABA peptide hydrolase (PPH) from human enterocytes is comprised of two subunits, alpha and beta. PPH alpha is over 70% identical to meprin, a protease isolated from mouse and rat kidney. The enzyme shows a modular organization in that it contains an astacin protease domain, an adhesive domain, an EGF-like domain, an a putative C-terminal membrane spanning domain. Expression of a chimeric meprin-PPH alpha cDNA in COS-1 cells led to the synthesis of immature, transport-incompetent homodimers. In addition, complex glycosylated forms were detected in the culture medium, suggesting that the enzyme is secreted after proteolytic removal of the membrane anchor.

Expression of the alpha subunit of PABA peptide hydrolase (EC 3.4.24.18) in MDCK cells. Synthesis and secretion of an enzymatically inactive homodimer.

In this paper, we report the expression of PPH alpha in the polarized cell line MDCK (Madin Darby canine kidney). In these cells, the enzyme was synthesized in an inactive proform, which upon treatment with trypsin was activated. The enzyme isolated from cell extracts was core-glycosylated and appeared to be retained in the ER as a homodimer. No PPH alpha was detectable on the surface of intact cells by immunofluorescence. However, a complex glycosylated soluble but inactive form was present in the culture medium, suggesting that proteolytic removal of the C-terminal membrane anchoring peptide leads to the secretion of PPH alpha.

The astacin family of metalloendopeptidases.

Molecular cloning of a human intestinal brush border metalloendopeptidase (N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase, PPH) and a mouse kidney brush border metalloendopeptidase (meprin A) has revealed 82% identity in the NH2-terminal amino acid sequences (198 residues) of the mature enzymes. Furthermore, searching of protein sequence data bases with the inferred peptide sequences as probes revealed strong similarities to astacin, a crayfish digestive protease, and an NH2-terminal domain of a human bone morphogenetic protein (BMP-1). Meprin A and PPH both have approximately 30% identity with astacin and BMP-1. Multiple alignment analysis indicated that 37 residues, including 3 cysteine residues, are strictly conserved for the four proteins in a sequence frame equivalent to the complete 200-amino acid astacin sequence. The four proteins contain a zinc-binding motif (HEXXH), found at the active site of most metalloendopeptidases, within an extended sequence of HEXXHXXGFXHE which is unique to this subgroup of metalloendopeptidases. In addition, the four proteins have 54% identity in a 24-amino acid sequence that includes the putative active site. A fifth protein, Xenopus laevis developmentally regulated protein UVS.2, also shares sequence identity with the metalloendopeptidases. These data provide strong evidence for an evolutionary relationship of these proteins. It is suggested that this new family of metalloendopeptidases be called the "astacin family."

Effects of seven different mutations in the pho1 gene on enzymatic activity, glycosylation and secretion of acid phosphatase in Schizosaccharomyces pombe.

Structural gene mutants of the cell-surface glycoprotein acid phosphatase of Schizosaccharomyces pombe were analysed to define structural determinants that are responsible for enzymatic activity, N-glycosylation and secretion. All seven defined mutations cause a single amino acid substitution in the mature acid phosphatase protein and destroy the enzymatic activity. The mutational lesions are distributed throughout the pho1 gene. A ser to phe substitution at position 349 abolishes enzymatic activity only and does not affect glycosylation and secretion. Two mutations create a new N-glycosylation site by substitution of pro at position 56 by phe and ser, respectively. This new site is apparently used in the mutants. Their core-glycosylated acid phosphatase is slightly larger than that of the wild type. Overglycosylation seems not to affect secretion. Four different mutations (a gly to asp substitution at position 281 and ser to phe substitutions at positions 150, 271 and 277) cause intracellular accumulation of enzymatically inactive core-glycosylated acid phosphatase precursor. These mutational lesions apparently block transport of acid phosphatase from the endoplasmic reticulum to the Golgi apparatus.

Identification and characterization of thiamin repressible acid phosphatase in yeast.

We have identified a genetic locus, pho4, in Schizosaccharomyces pombe which encodes a minor expressed cell surface acid phosphatase that is repressed by low concentrations (0.5 microM) of thiamin. The enzyme was purified from a strain that overproduces the enzyme. It is an Asn-linked glycoprotein. Removal of the carbohydrates by endoglycosidase H does not abolish enzymatic activity. The molecular mass of deglycosylated and unglycosylated enzyme that accumulates in membranes when cells are grown in the presence of tunicamycin is 56 kDa as determined by sodium dodecyl sulfate-gel electrophoresis. Thiamin regulation, at least in part, operates by reducing the level of pho4-mRNA. Pho4 is not genetically linked to the phosphate repressible acid phosphatase gene pho1. Phosphate and thiamin repressible acid phosphatase differ in their substrate specificity. Their protein moieties are immunologically related. Pho4 and pho1 are the only genes in S. pombe that express cell surface acid phosphatases being enzymatically active with nitrophenyl phosphate as substrate. S. pombe is not unique in having a thiamin repressible acid phosphatase. In Saccharomyces cerevisiae this enzyme is encoded by PHO3.