A 28,000-dalton protein of normal mouse serum binds specifically to the inner core region of bacterial lipopolysaccharide

Abstract

Normal mouse serum was found to contain a protein, referred to here as factor, which binds to the inner core region of lipopolysaccharides (LPSs) of various bacterial families. Since factor-LPS interactions resulted in activation of guinea pig complement, factor activity could be assayed by a passive hemolysis test with sheep erythrocytes coated with LPS or lipid A from Acinetobacter calcoaceticus (which was found earlier to bind particularly well to factor). Factor was purified by G-50 and hydroxyapatite chromatography whereby the specific hemolytic activity was enriched 1,675-fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed the presence of a 28,000-dalton protein as the main band. The identity of this band was determined by absorption experiments with LPS-coated sheep erythrocytes or latex beads, whereby the 28,000-dalton band disappeared after specific absorption and could be recovered from the absorbent. The binding specificity of factor was determined in a passive hemolysis inhibition assay with defined oligosaccharides representative for the inner core region of LPS. Thus, the di- and trisaccharides alpha-D-mannoheptopyranosyl-(1----5)-2-keto-3-deoxy-D-mannoocto nic acid and alpha-D-mannoheptopyranosyl-(1----3)-alpha-D-mannoheptopyranosy l-(1----5)-2- keto-3-deoxy-D-mannooctonic acid, respectively, were able to inhibit binding of factor to LPS. The results are in accordance with our earlier observation that the heptose-2-keto-3-deoxy-D-mannooctonic acid region represents a common antigen of bacterial LPS. Rabbit hyperimmune serum directed against this common antigen and purified factor was found to exhibit the same specificity for LPS. Factor activity was followed in mice in vivo after injection of LPS; it disappeared completely 15 min after the injection of LPS and reappeared within 1 h.