Aspergillus fumigatus complement inhibitor: production, characterization, and purification by hydrophobic interaction and thin-layer chromatography.
Aspergillus fumigatus has previously been shown to produce a soluble extracellular inhibitor of the alternative complement pathway, called Aspergillus complement inhibitor, or CI. We now report an efficient method for production of CI which relies on the fact that poorly conidiating cultures yielded CI activity with approximately sevenfold-higher potency than CI produced by conidiating cultures. CI from poorly conidiating cultures provided 50% inhibition of alternative pathway-mediated binding of 125I-labeled complement component C3 to cryptococcal blastoconidia at a mean concentration of 60 micrograms/ml. The ability of crude CI to inhibit the alternative complement pathway seemed to be independent of intact protein or polysaccharide structure, as evidenced by resistance of inhibitory activity to digestion by proteases, including subtilisin, alpha-chymotrypsin, papain, and pepsin as well as endoglycosidases F and H. Separation of the active inhibitory component of CI from contaminating materials contained in crude CI preparations was achieved by using Phenylsuperose hydrophobic interaction chromatography in a fast protein liquid chromatography system. The active material proved to be extremely hydrophobic, desorbing from the column only during elution with ethanol; it contained only 15% protein and 5% polysaccharide. Furthermore, results from preparative thin-layer chromatography indicated that lipids which comigrated with phosphatidylserine/phosphatidylinositol and phosphatidylethanolamine possessed significant complement-inhibitory activity. Taken together, these data suggested that phospholipids from A. fumigatus contributed to the functional activity of CI.
Washburn
R G
RG
Department of Medicine, Wake Forest University Medical Center, Winston-Salem, North Carolina 27103.
DeHart
D J
DJ
Agwu
D E
DE
Bryant-Varela
B J
BJ
Julian
N C
NC
D001232:Aspergillus fumigatus / Q000187:drug effects / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D003169:Complement Inactivator Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D003170:Complement Pathway, Alternative / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006358:Hot Temperature; D007456:Iodine Isotopes; D010714:Phosphatidylethanolamines / Q000302:isolation & purification; D010716:Phosphatidylinositols / Q000302:isolation & purification; D010718:Phosphatidylserines / Q000302:isolation & purification; D010743:Phospholipids / Q000096:biosynthesis* / Q000494:pharmacology; D011134:Polysaccharides / Q000096:biosynthesis* / Q000494:pharmacology; D012995:Solubility
D003169:Complement Inactivator Proteins; D007456:Iodine Isotopes; D010714:Phosphatidylethanolamines; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D010743:Phospholipids; D011134:Polysaccharides; D006026:Glycoside Hydrolases