Abstract
Antibacterial proteins are components of the innate immune system found in many organisms and produced by a variety of cell types. Human blood platelets contain a number of antibacterial proteins in their alpha-granules that are released upon thrombin activation. The present study was designed to purify these proteins obtained from human platelets and to characterize them chemically and biologically. Two antibacterial proteins were purified from platelet granules in a two-step protocol using cation exchange chromatography and continuous acid urea polyacrylamide gel electrophoresis and were designated thrombocidin (TC)-1 and TC-2. Characterization of these proteins using mass spectrometry and N-terminal sequencing revealed that TC-1 and TC-2 are variants of the CXC chemokines neutrophil-activating peptide-2 and connective tissue-activating peptide-III, respectively. TC-1 and TC-2 differ from these chemokines by a C-terminal truncation of 2 amino acids. Both TCs, but not neutrophil-activating peptide-2 and connective tissue-activating peptide-III, were bactericidal for Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Lactococcus lactis and fungicidal for Cryptococcus neoformans. Killing of B. subtilis by either TC appeared to be very rapid. Because TCs were unable to dissipate the membrane potential of L. lactis, the mechanism of TC-mediated killing most probably does not involve pore formation.
Highlights
During the last decade, antibacterial proteins have been recognized as effector molecules in the innate immune system of widely divergent animal species [1,2,3,4,5]
We show that the two major bactericidal proteins, TC-1 and TC-2, are truncated forms of NAP-2 and CTAP-III, respectively, differing from these CXC chemokines by the absence of the 2 C-terminal amino acids
TC-1 and TC-2 were bactericidal for the Grampositive B. subtilis and S. aureus as well as for the Gramnegative E. coli test strain, with MBCs ranging from 0.4 M (TC-1, B. subtilis) to 11 M (TC-2, S. aureus)
Summary
Isolation of Human Blood Platelets—Citrated human blood from healthy subjects was obtained from the Central Laboratory for Blood Transfusion (Amsterdam, The Netherlands). 25 ml of sonicate obtained from approximately 40 buffy coats and containing 3.5 mg protein/ml was applied to the column at 0.8 ml/min. Protein was dissolved in sample buffer (3 M urea in 5% acetic acid with methyl green as the tracking dye) and electrophorized at 40 mA with reversed polarity. Electrospray ionization mass spectrometry was performed on a hybrid quadrupole time-of-flight mass spectrometer (Micromass, Manchester, UK), equipped with an on-line nanoelectrospray interface (capillary tip, 20-m internal diameter ϫ 90-m outer diameter) with an approximate flow rate of 250 nl/min This flow was obtained by splitting of the 0.4 ml/min flow of a conventional high pressure gradient system 1 to 1000, using an Acurate flow splitter (LC Packings, Amsterdam, The Netherlands). AU-PAGE—CM-Sepharose and CAU-PAGE-purified proteins were analyzed using AU-PA slab gels (12.5% acrylamide, 5% acetic acid, 5 M urea). Suspensions of logarithmically growing test bacteria (Bacillus subtilis ATCC6633, E. coli ML35, or S. aureus 42D) were pre-
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