Abstract
Neutrophils exhibit a dramatic enhancement of integrin-mediated cell adhesion in response to lipopolysaccharide (LPS). This response requires CD14 on the neutrophil and plasma proteins in solution. We have purified the factor from plasma that facilitates the adhesive response of neutrophil to LPS by using a combination of affinity and ion-exchange chromatography. Previous work has shown that the activity is associated with apolipoprotein A-I (apoA-I), and here we show that this activity is associated with an apoA-I-bearing complex of protein and phospholipid. Native polyacrylamide gel electrophoresis (PAGE) analysis showed a ladder of bands in the Mr 200,000 region, and electron microscopy revealed round, indented particles of 11.4 +/- 0.12 nm in diameter. Characterization of these particles revealed a density of 1.219-1.264 g/ml and approximately 10 molecules of lipid phosphate per Mr 200,000 complex. SDS-PAGE showed that each of the bands seen in native PAGE was composed of several polypeptides. These were identified as apoA-I, LPS binding protein (LBP), and factor H-related proteins (FHRPs). Physical association of apoA-I, LBP, and FHRP in these particles was further confirmed using double immunodiffusion, and association of LBP and FHRP in plasma was confirmed by coimmunoprecipitation. FHRPs are the numerically dominant protein components in these particles, and all plasma FHRP-1 appears to be associated with these particles. We suggest that FHRPs may be the defining constituent of this novel "lipoprotein" particle.
Highlights
§ To whom correspondence should be addressed: Laboratory of Cellular Physiology and Immunology, Box 303, The Rockefeller University, New York, NY 10021-6399
We found that this activity is borne on a very high density particle composed of phospholipids, apolipoprotein A-I (apoA-I), LPS binding protein (LBP), and FH-related proteins 1 and 2 (FHRP-1 and factor Hrelated proteins (FHRPs)-2)
Message has been linked to protein bands only for FHRP-1 and FHRP-2
Summary
Reagents—Fresh frozen normal human plasma (citrated) was obtained from the New York Blood Center (New York, NY). The column was washed with starting buffer until the absorbance (A280) came down to base line and eluted with a gradient of NaCl from 0 to 1 M at 4 ml/min with a fraction size of 5 ml. The sample was loaded, washed with starting buffer, and eluted with a gradient of potassium phosphate from 10 – 400 mM over 40 ml at a rate of 1 ml/min. Membranes were washed again and incubated with alkaline phosphatase-conjugated secondary antibodies diluted 1:1000 in Western buffer for 1 h at room temperature. Antibodycoupled beads (10 l) were incubated with freshly drawn plasma (500 l, diluted 1:1 with citrated PBS) at 4 °C for 1 h with gentle mixing by rotator and washed 5–7 times with 1 ml of PBS; adsorbed proteins were eluted in SDS sample buffer
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