A1M/α1-microglobulin is proteolytically activated by myeloperoxidase, binds its heme group and inhibits low density lipoprotein oxidation

Abstract

α1-microglobulin (A1M) is a 26 kDa plasma and tissue protein with reductase activity and radical- and heme-binding anti-oxidative functions. In addition, exposure of A1M to hemoglobin has been shown to induce proteolytic elimination of a C-terminal tetrapeptide yielding a heme-degrading form, truncated A1M (t-A1M). Myeloperoxidase (MPO), a heme-containing enzyme that catalyzes the production of free radicals and hypochlorite, is released by neutrophils during the inflammatory response to bacterial infections. MPO-induced low density lipoprotein (LDL)-oxidation in blood has been suggested as a causative factor in atherosclerosis. In this study we have hypothesized that A1M interacts with MPO in a similar mode as with hemoglobin, and is a regulator of its activity. The results show that A1M is proteolytically cleaved, with formation of t-A1M, after exposure to MPO, and that t-A1M contains iron and heme-degradation products. The reaction is dependent of pH, time and concentration of substrates and a pH-value around 7 is shown to be optimal for cleavage. Furthermore, A1M inhibits MPO- and hydrogen peroxide-induced oxidation of LDL. The results suggest that A1M may have a role as an inhibitor of the damaging effects of the neutrophil respiratory burst on bystander tissue components.

Keywords: C-terminal proteolysis; heme binding; low density lipoprotein; myeloperoxidase; neutrophils; α1-microglobulin.

Figure 1

MPO-induced proteolytic cleavage of A1M. Lane 1 contains 3 μg A1M and lane 2 and 3 both contain 3 μg A1M mixed with 3 μg MPO in incubation buffer, incubated for 1 and 24 h respectively. (A) SDS-PAGE with mercaptoethanol. (B) Non-denaturing PAGE without mercaptoethanol.

Figure 2

Western blotting of A1M before and after MPO-induced cleavage. The samples, 0.1 μg of full-length A1M, MPO and a mixture of A1M/MPO, 0.1 μg of each, incubated for 4 h, were separated on a 12% SDS-PAGE prior to transfer to a PVDF-membrane and blotting. (A) Staining with Coomassie. (B) Immunoblot with anti-LIPR antibodies.

Figure 3

Time- and concentration dependence of MPO-induced cleavage of A1M. (A) A1M (0.195 mg/ml) was incubated with MPO at concentrations of 0, 0.3, or 1 mg/ml for 1, 4, or 24 h at room-temperature. Ten microliters of the samples were separated on a 12% SDS-PAGE gel in the presence of mercaptoethanol, and then stained with Coomassie. One representative experiment is shown. (B) A similar experiment with bovine catalase under the same conditions was performed.

Figure 4

pH-dependence of MPO-induced cleavage of A1M. A1M was mixed with MPO at a concentration of 0.195 mg/ml and 0.1 mg/ml respectively, in buffers with different pH. The samples were incubated for 1 h at 37°C. Ten microliters of the samples were then separated by SDS-PAGE on 12% gels in the presence of mercaptoethanol. The gels were stained with Coomassie and quantified by densitometry as described in Materials and Methods. One representative experiment is shown.

Figure 5

Ion exchange chromatography of an A1M/MPO mixture. (A) A1M and MPO (mol:mol, 6:1) were incubated at room temperature for 48 h and applied to a Mono-Q column. The column was eluted with a 0–0.5 M sodium chloride gradient in 20 mM Tris-HCl, pH 8. Fractions of 0.5 ml were collected and absorbance at 280 and 405 nm measured. (B) As a comparison, the same procedure was done on a sample with pure A1M. (C) SDS-PAGE analysis of the peak-fractions, marked 1–4 in (A,B), obtained after ion exchange chromatography of the A1M/MPO mix and the pure A1M sample. (D) Absorbance scanning between 250 and 750 nm of fractions obtained after ion exchange chromatography of the A1M/MPO mix shown to contain t-A1M by SDS-PAGE in (C) (lane 3). As a comparison, the same procedure was done on a sample with pure A1M and MPO. (E) Size-exclusion chromatography on Superose 12 of a mixture of A1M and MPO (molar ratio 6:1), incubated at room-temperature for 48 h, followed by analysis of A1M (absorbance and SDS-PAGE) and iron content measured by PIXE in selected fractions. The A1M-concentrations are given after dilution three times, for optimal illustration.

Figure 6

Cleavage of A1M in neutrophil culture medium. SDS-PAGE on 12% gels in the presence of mercaptoethanol followed by Western blotting with anti-A1M antibodies. The left lanes show A1M (0.1 mg/ml) incubated in fresh medium, the middle lanes show A1M incubated with neutrophil culture medium (0.1 mg/ml), and the right lanes show A1M (0.1 mg/ml) incubated with culture medium together with neutrophils activated by ionomycin. Culture conditions are described in Materials and Methods. Left panel, incubation for 3 h, and right panel shows the same procedure but with 24 h incubation.

Figure 7

A1M inhibits MPO-induced oxidation of LDL. (A) LDL, 0.2 mg/ml, was incubated with 8 mM H₂O₂ and a dilution series of MPO (25–100 μg/ml) in a total volume of 200 μl PBS at 24°C for 2 h followed by TBARS analysis. (B) LDL, 0.2 mg/ml, was incubated with 8 mM H₂O₂, 100 μg/ml MPO and albumin or A1M (8, 20 μM) in a total volume of 200 μl PBS at 24°C for 2 h followed by TBARS analysis. (C) LDL, 0.2 mg/ml, was incubated with 8 mM H₂O₂, 100 μg/ml MPO in PBS at 24°C. After 2 h, a dilution series of A1M (1–10 μM) was added in a total volume of 200 μl PBS, and the samples were incubated at 24°C for 1 h followed by TBARS analysis. Results are from triplicate experiments and presented as mean ± SD.

Figure 8

Tentative function of A1M in protection against bystander oxidative damage caused by neutrophils. The figure summarizes the findings of this paper in a physiological context. Recruitment and activation of neutrophils during inflammation involves MPO-mediated bacterial killing but may also yield LDL-oxidation as a side-effect. (1) Neutrophil culture medium or purified MPO induces heme-binding of A1M, and proteolytic cleavage of the protein which was previously shown to activate a heme-degradation activity of A1M (Allhorn et al., 2002). (2) The induced heme-degradation mechanism of A1M is tentatively “disarming” the peroxidase, preventing oxidative burst. (3) LDL-oxidation by MPO is inhibited by A1M, and pre-formed oxidation products on LDL are reduced.