HTRA1 cleaves A1AT to generate a 4.1 kD C-terminus cleavage fragment, A1ATM₃₈₃S-CF. (A) HTRA1 is known to cleave A1AT after the methionine 383 residue generating a 4132.226 Da carboxy-terminus fragment in amino acid sequence similar to the NIP, nNIF. (B) Cell-free assays were performed of A1AT incubated with increasing concentrations of recombinant HTRA1 (rHTRA1) protein (1-100 μg/mL). Western blotting with a primary antibody specific for the C-terminus of A1AT was used to detect A1ATM₃₈₃S-CF, an ∼4.1 kD cleavage fragment of A1AT. (C) Relative fluorescence intensity was used to quantitate A1ATM₃₈₃S-CF levels in the reaction products in panel B. The y-axis depicts the relative A1ATM₃₈₃S-CF levels after western blotting. (D) Western blotting was performed on cell-free reaction products using full-length A1AT alone, rHTRA1 alone, and A1AT incubated with rHTRA1. The western blot is representative of 3 separate experiments. (E) We incubated full-length A1AT with rHTRA1, HTRA1 protein extracted from umbilical cord blood, and HTRA1 protein extracted from placental homogenates and submitted them for analysis via mass spectroscopy. The sequence, resolved molecular weights (MWs), and expectation values from the analysis are shown. *P < .05 compared with baseline, arbitrarily set at 1 (dashed line).