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. 2010 Aug 31;49(34):7314-22.
doi: 10.1021/bi100359f.

Phospholipid Transfer Protein in Human Plasma Associates With Proteins Linked to Immunity and Inflammation

Free PMC article

Phospholipid Transfer Protein in Human Plasma Associates With Proteins Linked to Immunity and Inflammation

Marian C Cheung et al. Biochemistry. .
Free PMC article


Phospholipid transfer protein (PLTP), which associates with apolipoprotein A-I (the major HDL protein), plays a key role in lipoprotein remodeling. Because its level in plasma increases during acute inflammation, it may also play previously unsuspected roles in the innate immune system. To gain further insight into its potential physiological functions, we isolated complexes containing PLTP from plasma by immunoaffinity chromatography and determined their composition. Shotgun proteomics revealed that only 6 of the 24 proteins detected in the complexes were apolipoproteins. The most abundant proteins were clusterin (apoJ), PLTP itself, coagulation factors, complement factors, and apoA-I. Remarkably, 20 of the 24 proteins had known protein-protein interactions. Biochemical studies confirmed two previously established interactions and identified five new ones between PLTP and proteins. Moreover, clusterin, apoA-I, and apoE preserved the lipid-transfer activity of recombinant PLTP in the absence of lipid, indicating that these interactions may have functional significance. Unexpectedly, lipids accounted for only 3% of the mass of the PLTP complexes. Collectively, our observations indicate that PLTP in human plasma resides on lipid-poor complexes dominated by clusterin and proteins implicated in host defense and inflammation. They further suggest that protein-protein interactions drive the formation of PLTP complexes in plasma.


Figure 1
Figure 1. Protein composition of PLTP Complexes
(A) ApoA-I, clusterin, and PLTP in PLTP complexes were identified by SDS-PAGE and MALDI-TOF/TOF. PLTP complexes (2 μg) isolated from 4 plasma samples (lanes 1–4) were reduced, separated on 4%–12% SDS gel, and stained with Imperial Blue stain (Pierce). ApoA-I, clusterin, and PLTP were identified in prominent bands around the 30kDa, 40kDa, and 80 kDa positions by in-gel digestion and mass spectrometry. The apparent MWs of the proteins were deduced from the electrophoretic mobilities of a standard protein mixture. Fibronectin (FN) was shown by proteomics to be a non-specific contaminant. (* - various forms of immunoglobulin identified in the bands) (B) Immunoreactive protein S, vitronectin, and complement factor C1r were identified in PLTP particle. Immunoblot detection of PLTP, clusterin (CLU), protein S (PROS1), vitronectin (VTN), and complement C1r (C1R) in PLTP complexes subjected to SDS-PAGE. Lane C is either purified protein standard (PLTP, CLU, PROS1, VTN) or human plasma (C1R). Lanes 1-4 are PLTP complexes isolated from the plasma of 4 subjects.
Figure 2
Figure 2. PLTP and clusterin co-elute on size-exclusion chromatography (SEC)
Immunoaffinity isolated PLTP complexes were separated by FPLC, using a Superose 6 10/30 column. Column fractions were assayed for PLTP activity, PLTP mass, and clusterin. The panels, from top to bottom, show the distribution of PLTP activity, PLTP mass, and clusterin mass, respectively, of PLTP complexes isolated from 3 individuals. (Bars at the top indicate where LDL and HDL elute on this SEC column).
Figure 3
Figure 3. PLTP specifically binds to major proteins identified in PLTP complexes
Purified proteins immobilized on microtiter plates were reacted with rPLTP. After extensive washing, PLTP bound to the proteins was detected with anti-PLTP antibodies labeled with horseradish peroxidase; o-phenylenediamine dihydrochloride was used as the substrate. Data represent the mean and standard deviation of 3 experiments for apoA-I (APOA1), apoE (APOE), clusterin (CLU), fibrinogen (FG), plasminogen (PLG), transthyretin (TTR), and complement C1r (C1R) and 2 experiments for human albumin (ALB) and immunoglobulin G (IgG).
Figure 4
Figure 4. ApoA-I, clusterin, and apoE stabilize PLTP’s lipid-transfer activity
rPLTP was incubated alone or with apoA-I, clusterin, apoE, complement C1r (C1r), fibrinogen (FG), plasminogen (PLG), transthyretin (TTR), albumin, or immunoglobulin G (IgG) at a molar ratio of 1:2 (PLTP:protein) for 1 h at 37°C. The PLTP activity of each incubation mixture was immediately measured and compared with that of rPLTP diluted in phospholipid liposomes and incubated under the same conditions. The data represent the mean and standard deviation of 3 or 4 experiments, and are expressed as the % of the activity of the PLTP sample incubated in liposomes for 1 hr at 37°C. * denotes significantly different from PLTP incubated alone.
Figure 5
Figure 5. Most proteins in PLTP complexes have established protein–protein interactions
A protein interaction network for the proteins detected in PLTP complexes was constructed, using the MiMI plugin in Cytoscape and the Michigan Molecular Interactions database.
Figure 6
Figure 6. The proteome of PLTP complexes associates with components of the immune system
Using Gene Ontology annotation analysis and PubMed searches, we assigned major functional categories to the proteins that associated specifically with PLTP in plasma.

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