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Abstract: Poster Presentations |

DETECTION OF A NEW ALPHA1-PROTEINASE INHIBITOR ISOFORM MISSING THE C TERMINAL LYSINE IN A HUMAN BRONCHEOALVEOLAR LAVAGE (BAL) SOLUTION FREE TO VIEW

Alfred Weber, PhD*; Andrea Engelmaier; Daniel Kolarich, PhD; Friedrich Altmann, PhD; Raimundas Sakalauskas, MD; Peter L. Turecek, PhD; Hans P. Schwarz, MD
Author and Funding Information

Baxter BioScience, Wien, Austria



Chest. 2006;130(4_MeetingAbstracts):183S. doi:10.1378/chest.130.4_MeetingAbstracts.183S-a
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Abstract

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.

Wednesday, October 25, 2006

12:30 PM - 2:00 PM


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