PURPOSE: Recently the characteristic pattern of A1PI seen on high resolution isoelectric focusing (IEF) gels was elucidated by the description of a new A1PI isoform missing the C-terminal amino acid, lysine. As this modification of A1PI could be induced in vitro by the action of basic carboxypeptidases we were interested in whether it can also be found in vivo. After intravenous application of intact human A1PI in rats, we determined the IEF pattern of A1PI in broncheoalveolar lavage (BAL) and checked for the presence of the C-terminal truncated peptide using mass spectrometry (MS).
METHODS: Male CD rats were intravenously infused with 600 mg/kg human plasma derived A1PI, and BAL solution was obtained. A1PI levels were measured with an ELISA. The A1PI isoform pattern was investigated by IEF on IPG 4.2-4.9 followed by an immunoblotting procedure. The BAL samples were concentrated/desalted by precipitation with 50% PEG in the presence of rabbit serum. For the immunodetection we used rabbit anti-human a1-antitrypsin and goat anti-rabbit IgG-peroxidase. Peroxidase was detected using the Opti 4 CN kit. Targeted MS and MS-MS analysis of tryptic peptides served to investigate the possible Lys truncation of A1PI.
RESULTS: Human A1PI was measurable in all rat BAL samples. IEF analysis showed an altered isoform pattern similar to that of a C-terminal truncated A1PI preparation, although the band intensities suggested a different level of truncation. MS and MS-MS analysis of the tryptic A1PI peptide unambiguously proved the presence of C-terminal truncated peptide.
CONCLUSION: Partially lysine-truncated A1PI was found in vivo after diffusion through the vasculature into BAL. This is probably due to the presence of lipid-anchored carboxypeptidase M in lung tissue, which appears to cleave human A1PI.
CLINICAL IMPLICATIONS: Lys-truncation in A1PI seen in A1PI concentrates is not only associated with manufacturing processes but is also a physiological process.
DISCLOSURE: Eva Muchitsch, Grant monies (from industry related sources) D. Kolarich and F. Altmann recieved grant monies from Baxter; Employee E. Muchitsch, A. Weber, A. Englmaier, W. Auer, P.L. Turecek and H.P. Schwarz are employees of Baxter BioScience.