Identification of human plasma proteins as major clients for the extracellular chaperone clusterin

Abstract

Clusterin (CLU) is an extracellular chaperone that is likely to play an important role in protein folding quality control. This study identified three deposition disease-associated proteins as major plasma clients for clusterin by studying CLU-client complexes formed in response to physiologically relevant stress (shear stress, approximately 36 dynes/cm(2) at 37 degrees C). Analysis of plasma samples by size exclusion chromatography indicated that (i) relative to control plasma, stressed plasma contained proportionally more soluble protein species of high molecular weight, and (ii) high molecular weight species were far more abundant when proteins purified by anti-CLU immunoaffinity chromatography from stressed plasma were compared with those purified from control plasma. SDS-PAGE and Western blot analyses indicated that a variety of proteins co-purified with CLU from both stressed and control plasma; however, several proteins were uniquely present or much more abundant when plasma was stressed. These proteins were identified by mass spectrometry as ceruloplasmin, fibrinogen, and albumin. Immunodot blot analysis of size exclusion chromatography fractionated plasma suggested that CLU-client complexes generated in situ are very large and may reach >or=4 x 10(7) Da. Lastly, sandwich enzyme-linked immunosorbent assay detected complexes containing CLU and ceruloplasmin, fibrinogen, or albumin in stressed but not control plasma. We have previously proposed that CLU-client complexes serve as vehicles to dispose of damaged misfolded extracellular proteins in vivo via receptor-mediated endocytosis. A better understanding of these mechanisms is likely to ultimately lead to the identification of new therapies for extracellular protein deposition disorders.

FIGURE 1.

Turbidity of control and stressed plasma at day 0 and day 10. The plasma samples were diluted 1:2 in PBS on days 0 and 10, and the A_{360 nm} was measured. The figure shows the average A_{360 nm} (n = 3 ± standard error) for each sample. *, significantly increased turbidity relative to the three other sample types (Tukey HSD, p < 0.0001 in all cases). These results are representative of three independent experiments.

FIGURE 2.

SEC of stressed or control plasma (A) and anti-CLU immunoaffinity-purified proteins (B) from stressed or control plasma. The samples analyzed were: whole plasma exposed to shear stress for 10 days or freshly isolated control plasma (A) and proteins purified by anti-CLU immunoaffinity chromatography from the plasma samples described in A (B). The positions of molecular mass markers are indicated by labeled arrows; the exclusion limit (V_o) ≥ 4 × 10⁷ Da. The results shown are representative of two independent experiments.

FIGURE 3.

SDS-PAGE of proteins purified from stressed or control human plasma by anti-CLU immunoaffinity chromatography. The proteins were separated using 12% SDS-PAGE under reducing conditions. The figure shows molecular mass markers (left lane, masses indicated in kDa) and proteins immunoaffinity-purified from freshly isolated control plasma (labeled C) or stressed plasma (labeled S). The open arrows indicate bands that were selected for mass spectrometry analysis. The solid black arrow indicates the position of CLU. The results shown are representative of many independent experiments.

FIGURE 4.

Western blot analysis of proteins purified by anti-CLU immunoaffinity chromatography from control or stressed human plasma. The proteins separated by SDS-PAGE (10 μg of total protein loaded per lane) were transferred to nitrocellulose membrane and probed with anti-CERU (A), anti-FGN (B), or anti-HSA antisera (C). Each panel shows the position of molecular mass markers (left lanes, masses in kDa indicated), and proteins immunoaffinity-purified from freshly isolated control plasma (labeled C) or stressed plasma (labeled S). In B, the position of the α, β, and γ subunits of FGN are indicated. The results shown are representative of three independent experiments.

FIGURE 5.

Immunodot blot analyses of SEC-fractionated control or stressed human plasma. Freshly isolated control plasma (C) or stressed plasma (S) were fractionated using a Superose^TM 6 column (V₀ ≥ 4 × 10⁷ Da). Aliquots from each fraction were spotted onto a nitrocellulose membrane that was then incubated with antibodies against CLU (A), CERU (B), FGN (C), or HSA (D) followed by the appropriate HRP-conjugated secondary antibody prior to development by ECL. The approximate molecular mass of the SEC fractions is indicated at the top of the figure. The results shown are representative of three independent experiments.

FIGURE 6.

Detection of CLU-client protein complexes in stressed plasma by sandwich ELISA. The results of sandwich ELISA detecting CLU-client protein complexes containing CERU (A), FGN (B), or HSA (C) in human plasma. The results for “control antisera” were obtained using species-matched control antisera as the primary detection antibody in each case. The results shown are the average A_{490 nm} (n = 3, ± standard error) relative to the nonspecific binding generated in wells coated with mouse IgG₁ control antibody. *, increased A_{490 nm} relative to wells incubated with control plasma (stored static at 4 °C; Student's t test, p ≤ 0.02). The results shown are representative of three independent experiments.