Exact Q9VAS1 is 671 residues. Independent MAFFT L-INS-i and G-INS-i alignments agree at all five inspected catalytic/zinc-binding sites, including the surrounding 17-column windows. Human neprilysin P08473 is the active reference; mouse neprilysin Q61391 is the positive control.
| Human site | Human role | Nepl19 mapped site | Mouse control |
|---|---|---|---|
| H584 | zinc ligand | Q511 | H584 |
| E585 | catalytic glutamate | Q512 | E585 |
| H588 | zinc ligand | H515 | H588 |
| E647 | zinc ligand | E560 | E647 |
| D651 | catalytic proton donor | D564 | D651 |
The reference HEITH motif at 584–588 maps to QQLAH at Nepl19 511–515. Nepl19 retains the M13 fold and downstream ENIAD region but replaces the first zinc-binding histidine and catalytic glutamate with glutamines. Both alignment strategies recover every active-reference site unchanged in the mouse control. This specific catalytic-site disruption strongly supports loss of conventional neprilysin proteolysis; it does not identify the protein’s noncatalytic physiological role or exclude every possible unrelated chemistry.
The selected UniProt sequence has a SignalP-derived signal peptide at 1–24 and a mature chain at 25–671, with no annotated transmembrane segment. The mature C-terminal sequence ends in TSPQKCQLFAVNLD, lacking a hydrophobic membrane anchor. These observations support the secreted Nepl-family interpretation in PMID:34189422 rather than a default membrane-tethered neprilysin location.