Metalloprotease Meprin β Generates Nontoxic N-terminal Amyloid Precursor Protein Fragments in Vivo

Abstract

Identification of physiologically relevant substrates is still the most challenging part in protease research for understanding the biological activity of these enzymes. The zinc-dependent metalloprotease meprin ␤ is known to be expressed in many tissues with functions in health and disease. Here, we demonstrate unique interactions between meprin ␤ and the amyloid precursor protein (APP). Although APP is intensively studied as a ubiquitously expressed cell surface protein, which is involved in Alzheimer disease, its precise physiological role and relevance remain elusive. Based on a novel proteomics technique termed terminal amine isotopic labeling of substrates (TAILS), APP was identified as a substrate for meprin ␤. Processing of APP by meprin ␤ was subsequently validated using in vitro and in vivo approaches. N-terminal APP fragments of about 11 and 20 kDa were found in human and mouse brain lysates but not in meprin ␤ ؊/؊ mouse brain lysates. Although these APP fragments were in the range of those responsible for caspase-induced neurodegeneration, we did not detect cytotoxicity to primary neurons treated by these fragments. Our data demonstrate that meprin ␤ is a physiologically relevant enzyme in APP processing.

The Metalloprotease Meprin β Generates Amino Terminal-truncated Amyloid β Peptide Species

“…Recently, we were able to demonstrate that the metalloprotease meprin ␤ can cleave the amyloid precursor protein in its N-terminal region (15). The work presented here extends this investigation by showing that meprin ␤, although in a smaller extend than BACE1, can also generate different A␤ species with several cleavage sites identical or proximate to the known ␤-secretase cleavage site.…”

“…After direct loading of tissue culture supernatant, we were able to detect the previously described N-APP20 fragment when cells were incubated with the active, soluble meprin ␤ enzyme (Fig. 8, lower panel) (15). As a control, we applied exogenously a soluble inactive E90A mutant of meprin ␤ revealing no detectable increase in N-APP20.…”

“…For human meprin ␤, a striking preference for aspartate and glutamate residues around the cleavage site in native substrates has been revealed, demonstrating an exceptionally high specificity for a metalloprotease (22). Recently, APP was found to be a substrate of human meprin ␤, using a cell culture-based degradomic approach (15). Together, these observations identify meprin ␤ as a protease candidate for the generation of A␤ peptides, exhibiting acid amino acid residues in p1, p2, and p3 position.…”

“…Moreover, we observed that the level of full length APP decreases after meprin ␤ overexpression, which might indicate further meprin ␤ cleavage sites within the APP sequence (15). To further determine specific meprin ␤ cleavage sites within the APP amino acid sequence, three peptide substrates derived from APP representing ␤-secretase cleavage sites were analyzed by MALDI-TOF after meprin ␤ incubation (Fig.…”

Functional and structural insights into astacin metallopeptidases

“…Of special interest is the fact that meprins cleave components of the extracellular matrix, in particular the basal lamina but also adhesion proteins at the cell-cell interface (Sterchi et al , 2008 ;Ambort et al , 2010 ;Vazeille et al , 2011 ). Recent proteomics approaches have identifi ed previously known and new physiologically relevant in vivo substrates such as vascular endothelial growth factor (Sch ü tte et al, 2010 ), amyloid precursor protein (Jefferson et al , 2011 ), procollagens I and III (Kronenberg et al , 2010 ), interleukin-1 β (Herzog et al , 2005 ), interleukin 18 (Banerjee and Bond , 2008 ), prokallikrein 7 (Ohler et al , 2010 ), and fi broblast growth factor 19 (Becker -Pauly et al, 2011 ).…”