PURPOSE: In human plasma the normal M-type A1PI occurs in several isoforms that are molecules with different numbers of sialic acids (isoforms M1, M2, M4, M6) or missing 5 N-terminal amino acids (M7, M8) In addition alpha1-proteinase inhibitor (A1PI) concentrates lack the C terminal amino acid lysine. The latter was shown to derive from the action of basic carboxypeptidases during the ethanol fractionation process. Having previously shown that carboxypeptidase M can cleave off the C-terminal lysine and lung tissue contains high levels of this enzyme, we investigated human BAL solutions for the occurrence of the C-terminal truncated A1PI isoform.
METHODS: BAL samples from two non-A1PI deficient patients (COPD, cancer) were obtained from Vilnius University Allergy Centre, Lithuania. Isoelectric focusing was done on Immobiline IPG 4.2-4.9 using 1% Pharmalyte 4.2-4.9 and 20% glycerol for re-hydrating the gels and 0.2 M orthophosphoric acid and 0.2 M NaOH as anolyte and catholyte, respectively. Samples were concentrated/desalted by precipitation with 50% PEG 5000 in the presence of rabbit serum, applied cathodically, focused for 6 hours and transferred to nitrocelluose. For the immunodetection we used rabbit anti-human a1-antitrypsin, goat anti-rabbit IgG-peroxidase and the Opti 4 CN kit. For the mass spectrometric (MS) analysis, the proteins contained in BAL were concentrated on a C4 column, reduced, carboxymethylated and subjected to trypsin digestion in the presence of urea.
RESULTS: Isoelectric focusing showed an A1PI isoform pattern for both BAL solutions similar to a pattern indicative for C-terminal truncated A1PI. Targeted MS and MS-MS analysis unambiguously demonstrated the presence of C-terminal truncated A1PI in the BAL solution of the cancer patient.
CONCLUSION: We show that the C-terminal truncated isoform of A1PI that until now was associated only with the manufacturing processes of A1PI concentrates also occurs naturally in a human biological fluid.
CLINICAL IMPLICATIONS: Our finding suggests that exposure to des-Lys form in A1PI products is unlikely to cause an immune response in patients on chronic augmentation therapy.
DISCLOSURE: Alfred Weber, Grant monies (from industry related sources) Daniel Kolarich and Friedrich Altmann received grant monies from Baxter BioScience; Employee Alfred Weber, Andrea Engelmaier, Peter Turecek and Hans-Peter Schwarz are employees of Baxter BioScience.