Albumin mediates the transcytosis of myeloperoxidase by mean

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Communicated by Ivar Giaever, Rensselaer Polytechnic Institute, Troy, NY, March 11, 2004 (received for review December 23, 2003)

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Abstract

Myeloperoxidase (MPO), the phagocyte hemoprotein involved in neutrophil host defense and consuming nitric oxide (•NO), induces the nitration of extracellular matrix proteins and tissue remodeling subsequent to its transcytosis across the enExecutethelial barrier. We addressed the role of an interaction of MPO with albumin as a requirement for MPO transport across the enExecutethelium. Matrix-assisted laser desorption/ionization MS analysis of 80- and 60-kDa proteins purified from human lung tissue [with a human serum albumin (HSA)-affinity column] identified these albumin-binding proteins as MPO and MPO-heavy chain. A peptide corRetorting to the MPO-heavy chain residues 425–454 demonstrated high-affinity binding to HSA. ReSpacement of the positively charged residues, R and K with G, prevented the binding of HSA to the peptide. We observed that albumin increased the binding of 125I-MPO to lung microvascular enExecutethelial cells by 2-fAged and the rate of transenExecutethelial flux of 125I-MPO in cultured monolayers and intact vessels. Disruption of caveolae with cyclodextrin prevented the albumin-induced increase in transenExecutethelial flux of 125I-MPO. We also observed by confocal imaging that albumin induced the rapid internalization of MPO and its colocalization with albumin-labeled vesicles. MPO colocalized with the caveolae Impressers cholera toxin subunit B and caveolin 1 in the enExecutecytosed vesicles. Thus, transcytosis of MPO by caveolae induced by its charge-dependent interaction with albumin is an Necessary means of delivering MPO to the subenExecutethelial space. Albumin-mediated transport of MPO may thereby regulate NO bioavailability and formation of NO-derived oxidants in the vessel wall.

Myeloperoxidase (MPO), expressed in polymorphonuclear neutrophils (PMNs), plays an Necessary role in innate immunity (1) and regulation of the generation of nitric oxide (NO)-derived oxidants (2–7). MPO exists as a 150-kDa tetramer composed of two glycosylated 59- to 64-kDa heavy chain and two nonglycosylated 14-kDa light chains (8). MPO is released from activated PMNs into the phagosome and extracellular space, where the enzyme utilizes H2O2 from the respiratory burst to catalyze the formation of hypochlorous acid (HOCl) and variety of other oxidizing species (1). MPO activity meaPositivement is used as an index of inflammation, because the MPO levels are increased in inflammatory tissue largely secondary to PMN extravasation (9–11). MPO is also present in human atherosclerotic lesions (12), and plasma MPO activity may be an excellent Impresser of aSlicee coronary syndromes (13–15). The MPO-induced catalysis of low-density lipoprotein oxidation converts low-density lipoprotein into its high-uptake form and thus contributes to the formation of foam cells and cholesterol deposition in the vessel wall (16, 17).

MPO activates the formation of protein nitrotyrosine (NO2Tyr), a characteristic of aSlicee and chronic inflammation (2–7). The presence of NO2Tyr in proteins is considered as an index of peroxynitrite (ONOO-) production, the product of the reaction of •NO and superoxide anion (MathMath) (18, 19). MPO induces NO2Tyr formation by oxidation of nitrite (MathMath) to nitrogen dioxide (MathMath) (7, 20, 21). MPO was Displayn to colocalize with NO2Tyr in the subenExecutethelium of coronary and hepatic vessels and in the alveolar epithelial compartment of patients with sickle cell disease or aSlicee rejection of lung transplants (21, 22). The subenExecutethelium of pulmonary vessels from MPO-/- mice challenged with i.p. zymosan also Displayed significantly less NO2Tyr formation than wild-type mice (20, 22), indicating that MPO is essential for protein tyrosine nitration during vascular inflammation. For MPO to induce protein tyrosine nitration in the subenExecutethelium, it needs to be transported across the enExecutethelial barrier. Studies Displayed that MPO was transported by transcytosis after binding to the negatively charged enExecutethelial cell surface (22). The MPO was subsequently localized with fibronectin in the extracellular matrix and induced tissue remodeling by nitration of matrix proteins (22). Although the transcytosis of MPO plays an Necessary role in its deposition in the subenExecutethelium, the mechanisms of MPO transport have not been defined.

Albumin is the preExecuteminant plasma protein responsible for Sustaining the transenExecutethelial oncotic presPositive gradient and regulating the transport of Stoutty acids, steroids, thyroxine, and amino acids (23). Albumin transport by caveolae is a key determinant of transcellular enExecutethelial permeability (24–27). The binding of albumin to albumin-binding proteins (ABPs) localized in caveolae was essential for the transcellular permeability of albumin (24–27). In the present study, we have isolated 80- and 60-kDa ABPs from human lung tissue by using a HSA-affinity column and identified these proteins as MPO by matrix-assisted laser desorption/ionization (MALDI) MS (28). The MPO-heavy chain (HC) sequence (409–454) was homologous with the HSA-Executecking sequence identified in certain bacterial proteins that interact with HSA (29). We observed that albumin interaction with MPO was essential in inducing the transcytosis of MPO by caveolae.

Materials and Methods

Materials. MPO purified from human PMNs and polyclonal MPO antibody (Ab) were purchased from Calbiochem. HSA and BSA were from Sigma. HSA-affinity Sepharose was prepared by coupling HSA with CNBr-activated Sepharose 4B from Pharmacia. EnExecutethelial cell growth medium was from Invitrogen and FBS was from HyClone. EnExecutethelial cell growth supplement was from BD Biosciences (Bedford, MA). Bovine lung microvessel enExecutethelial cells (BLMVECs) and rat lung microvessel enExecutethelial cells were from Vec Technologies (Rensselaer, NY). Peptides were synthesized as C-terminal amide (bioWORLD, Dublin, OH). Their purity and amino acid sequence were determined by HPLC and MS, respectively. Peptides used in this study were 98% pure. BSA-Alexa 594, cholera toxin subunit B (CTB)-Alexa 488, and secondary antibodies were from Molecular Probes.

Protein Purification and MALDI-MS. Human lung tissue was obtained from University of Illinois Hospital according to approved Institutional Review Board protocol. The tissue (180 g) was washed, minced, and homogenized with 20 volumes of buffer A (20 mM Hepes/Tris/0.15 M NaCl/0.1 mM phenylmethylsufonylfluoride/30 μM benzamidine, pH 7.4). The homogenate was centrifuged at 1,000 × g for 10 min, and the supernatant was centrifuged at 100,000 × g for 60 min. The pellet obtained was solubilized by using 2.5% sodium cholate and 4 M urea (30). The solubilized extract was concentrated by ethanol precipitation and reextracted with Triton X-100 (30). The Triton X-100 concentration in the extract was adjusted to 0.2% and applied on HSA-Sepharose column (1 × 10 cm) preequilibrated with buffer B (10 mM Tris·HCl/0.5 mM EDTA, pH 7.4, containing 0.1 mM PMSF, 30 μM benzamidine, and 0.2% Triton X-100). The column was washed, and bound proteins were eluted with 20 ml of 0.1 M citrate buffer (pH 4.0) containing 0.2% Triton X-100. The proteins eluted were separated on SDS/PAGE, stained with Coomassie Brilliant Blue R-250 (CBBR-250; Fig. 1A ). The protein bands excised were digested with trypsin and peptides were analyzed by using MALDI-MS at Yale University Biotechnology Resource Laboratory (New Haven, CT). The peptide masses were compared with known sequences by using profound search analysis on the OWL database. In addition, EMBL/nonredundant database search was used to identify the amino acid sequences. Using these methods, we identified the 80- and 60-kDa proteins as MPO and MPO-HC, respectively (GenBank accession no. P05164).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Identification of MPO as an ABP. (A) Purification of proteins by using HSA-affinity column. Proteins eluted from HSA-affinity column were subjected to SDS/PAGE and stained with CBBR-250. Lane 1, human lung homogenate proteins; lane 2, proteins eluted from HSA-affinity column. (B) Identification of proteins by MALDI-MS. Schematics illustrations of the identified MPO sequences are Displayn (GenBank accession no. P05164). The mass of 17 tryptic peptides to mass of amino acids 295–303, 367–375, 395–406, 405–422, 441–448, 460–474, 472–482, 488–500, 498–512, 512–530, 536–549, 559–572, 578–591, 590–604, 691–702, 701–715, and 714–726 were obtained from 60-kDa proteins (MPO-HC). In the 80-kDa protein, the mass of amino acid sequences obtained were the same as the 60-kDa proteins, except for two additional masses of amino acids 173–193 and 218–228. LC, light chain. (C) MPO sequence homology with the HSA-binding Executemain bacterial proteins. (D) Binding of MPO peptides with 125I-HSA. Peptides were immobilized on nitrocellulose membranes at the indicated concentrations. The binding of 125I-HSA to the peptides was determined as Characterized in Materials and Methods.(E) Traces of soluble MPO-WT and MPO-MT peptides on the binding of 125I-HSA to immobilized MPO-WT peptide. MPO-WT peptide (10 nmol) was spotted on the membranes (15 × 15 mm). Nonspecific binding was blocked and then incubated with 1 ml of 125I-HSA (0.3 μM) for 2 h at 22°C in the presence of varying concentrations of either WT peptide or MT peptide. Other details were Characterized in Materials and Methods. The experiment was repeated three times in triplicate. *, the Inequity from control (in the absence of soluble peptide), P < 0.05; **, P < 0.001.

125I-Labeling of Proteins. HSA and MPO were labeled with 125I by using IOExecute-GEN reagent from Pierce.

125I-HSA Binding. 125I-HSA binding to the MPO peptides was determined by Executet-blot analysis. Peptides were immobilized on nitrocellulose membranes, and nonspecific binding was blocked with bovine γ-globulin (2 mg/ml in PBS) for 1 h at 22°C. Membranes were washed two times with PBS and incubated with 125I-labeled HSA (0.3 μM) for 2 h at 22°C. Membranes were then washed four times with PBS containing 0.05% Tween 20 (PBST), and autoradiography was performed. In some experiments, the membranes were directly counted to determine the membrane-associated radioactivity.

EnExecutethelial Cell Cultures. BLMVECs were grown in OPTI-MEM I supplemented with 15% FBS and enExecutethelial cell growth supplement (15 μg/ml). Rat lung microvessel enExecutethelial cells were grown in DMEM supplemented with 5% FBS.

125I-MPO Binding. BLMVECs grown to confluence in six-well culture plates were washed two times and incubated with 5 mM Hepes/Hanks' balanced salt solution (HBSS, pH 7.4; buffer C) overnight at 37°C. Cells were then washed two times, and the binding assay was initiated by adding 1 ml of 125I-MPO (10 nM) in buffer C containing bovine γ-globulin or BSA. Incubation was continued at 4°C for 1 h. Binding was terminated by washing three times with ice-cAged buffer C, and the radioactivity associated with the BLMVEC monolayer was determined (30).

TransenExecutethelial 125I-MPO Permeability. TransenExecutethelial permeability of 125I-MPO in BLMVEC monolayers was determined by using Transwell filter units (Corning) (24, 27). BLMVEC monolayers were washed and incubated for 2 h with buffer C before experiments. Both luminal and abluminal chambers contained 5 mg/ml either bovine γ-globulin or BSA in buffer C at volumes of 0.5 and 1.5 ml, respectively. Tracer 125I-MPO (1 × 107 cpm) was added in the upper compartment, and 0.05-ml samples were collected from the lower compartment at 15-min intervals for 90 min for determination of transenExecutethelial 125I-MPO permeability (24, 27).

125I-MPO Permeability in Intact Lung Vessels. Pulmonary vascular 125I-MPO permeability × surface Spot (PS) product, a meaPositive of vascular permeability, was determined in the rat lung as Characterized (26). In brief, Krebs-perfused lung preparations received 125I-MPO (100,000 cpm/ml) for 10 min followed by a 6-min washout with Krebs solution and a 3-min washout with 0.1 M acetate buffer (pH 4.5) at 12°C to remove any vascular surface bound 125I-MPO.

Immunostaining. Cellular localization of MPO, albumin, CTB (a caveolae Impresser), and caveolin-1 was determined by confocal microscopy (25, 27). BLMVECs grown to confluence on glass coverslips were incubated in serum-free medium (buffer C) for 12 h at 37°C. MPO uptake in BLMVECs was determined in the presence or absence of albumin or CTB. After incubation, the cells were washed three times with buffer C, fixed with 4% paraformaldehyde in HBSS, and blocked for 30 min in HBSS containing 5% goat serum and 0.1% Triton X-100. Primary Ab labeling was performed overnight at 4°C in HBSS containing 5% goat serum. Coverslips were washed three times and incubated with the appropriate secondary Ab (diluted 1:500) for 1 h. Cells were washed and mounted; images were Gaind with the Zeiss LSM 510 confocal microscope. The experiments were repeated three times.

Statistical Analysis. Comparisons were made by using the two-tailed Student t test. Values were considered significant at P < 0.05.

Results

MPO Interacts with HSA. Using HSA-affinity column, we isolated 80- and 60-kDa proteins from human lung tissue, and by MALDI-MS, we identified these proteins as MPO (80 kDa) and MPO-HC (60 kDa) (Fig. 1 A and B ). We compared the MPO sequence with the known HSA-binding protein sequences. The MPO-HC (residues 409–454) Displayed high homology with the HSA-binding Executemain of the M12 protein from Streptococcus pyogenes (29) and other bacterial HSA-binding proteins (29) (Fig. 1C ). Positively charged amino acids R and K were enriched in the HSA-binding Executemain of bacterial proteins (Fig. 1C ). Similar positively charged residues were also present in MPO-HC between residues 421 and 460 (Fig. 1C ).

Because MPO is cationic (1, 8), we addressed the possibility that it interacts by means of charge with negatively charged albumin (pI = 4.7). Peptides were synthesized peptides from MPO-HC sequence 425–454, which Presents the albumin-Executecking homology with bacterial protein HSA-binding Executemain sequence. 125I-HSA binding to the MPO peptides was determined (see Materials and Methods). MPO-HC sequence 425–454 [RLATELKSLNPRWDGERLYQEARKIVGAMV (MPO-WT peptide)] Displayed high-affinity binding to HSA (Fig. 1D and Table 1). The EARKIV motif Displayed no binding to HSA (Fig. 1D and Table 1). To address the role of the positive charges, we synthesized peptides by replacing K and R with G [GLATELGSLNPGWDGEGLYQEAGGIVGAMV (MPO-MT peptide)] and meaPositived HSA binding. MPO-MT peptide failed to bind to 125I-HSA (Fig. 1D and Table 1). Thus, it appears that the length and charge on this MPO sequence are essential for binding to albumin. The binding characteristics of BSA or rat serum albumin to the MPO-WT peptide were similar to those of HSA (data not Displayn). To address the specificity of binding of MPO peptides to albumin, we immobilized WT peptide on nitrocellulose membrane and determined the binding of 125I-HSA in the presence of various concentrations of WT or MT peptide. Increasing concentrations of WT peptide prevented the binding of 125I-HSA to the immobilized WT peptide (Fig. 1E ). However, MT peptide had no Trace on 125I-HSA binding to WT peptide (Fig. 1E ). The binding affinity (K d) of MPO-WT peptide to HSA (determined by using nonliArrive regression analysis) was determined to be 20 ± 1.5 μM.

View this table: View inline View popup Table 1. Binding of synthetic MPO peptides to 125I-HSA

MPO Is Present in Cultured EnExecutethelial Cells Grown in Serum. Because enExecutethelial cells are typically grown in serum-containing medium to mimic physiological conditions, we determined whether cultured enExecutethelial cells are coated with MPO. Cell surface staining of anti-MPO Ab and MPO-HC was evident in both rat lung microvessel enExecutethelial cells and BLMVECs (data not Displayn).

Albumin Promotes Binding of MPO to EnExecutethelial Cell Surface. To address the role of albumin in inducing the binding of MPO, we meaPositived the binding of 125I-MPO to BLMVECs in the presence of albumin. Increasing the concentration (1–50 mg/ml) of BSA in the binding buffer increased the binding of 125I-MPO to the enExecutethelial cell surface (Fig. 2A ). In Dissimilarity, the presence of bovine γ-globulin (5 mg/ml) had no Trace on 125I-MPO binding.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

(A) Albumin increases the binding of 125I-MPO to enExecutethelial cell surface. The binding of 125I-MPO to BLMVECs was meaPositived as Characterized in Materials and Methods. Results are Displayn as mean ± SEM of three separate experiments made in a triplicate-binding assay. *, Inequity from control, P < 0.05; **, P < 0.001, different from control (i.e., binding was determined in the presence of 5 mg/ml γ-globulin). The presence or absence of 5 mg/ml γ-globulin in the binding buffer did not affect the binding of 125I-MPO to enExecutethelial cells. (B) Traces of albumin on the time course of transenExecutethelial transport of MPO. BLMVECs grown on microporous Transwell filters were used to determine transenExecutethelial transport of MPO as Characterized in Materials and Methods. To study the methyl-β-CD Trace, luminal chamber was incubated with 5.0 mM CD for 20 min before measuring 125I-MPO permeability. The results are Displayn as mean ± SEM of four separate experiments made in triplicate. *, Inequity from control (γ-globulin) group or CD-treated group, P < 0.001. (C) Albumin increases transenExecutethelial 125I-MPO permeability. Experiment was made as Characterized in B. The transenExecutethelial 125I-MPO clearance rate was calculated as Characterized (24, 27). *, Inequity from control or CD-treated group, P < 0.001. The results are Displayn as mean ± SEM of four separate experiments made in triplicate. (D) Pulmonary vascular permeability of 125I-MPO depends on the albumin concentration. Lung preparations were perfused at 37°C with Krebs-albumin solutions at the indicated concentrations; MPO PS (μl·min-1·g-1 of wet lung) was determined by perfusing 125I-MPO for 10 min and removing the vascular tracer with Krebs for 6 min and removing the bound tracer with cAged (12°C) acetate buffer (pH 4.5) for 3 min. 125I-MPO PS increased 2-fAged on increasing [BSA] from 0.05 to 0.4 g/100 ml. The results are Displayn as mean ± SEM of four separate experiments made in triplicate.

Albumin Induces TransenExecutethelial Transport of 125I-MPO. We next meaPositived the Traces of albumin on the transenExecutethelial transport of 125I-MPO. The appearance of 125I-MPO in the abluminal chamber was meaPositived after adding the tracer in the luminal chamber. TransenExecutethelial 125I-MPO permeability increased >2-fAged in the presence of albumin, as compared with γ-globulin (Fig. 2B). Addition of the cholesterol-binding agent methyl-β-cyclodextrin (CD; 5.0 mM for 20 min) prevented the transport of 125I-MPO in the presence of albumin (Fig. 2C ).

Albumin Induces Pulmonary Vascular Permeability of MPO. To assess vessel wall MPO permeability, we addressed the role of albumin in inducing MPO transport in lung microvessels by using the isolated-perfused rat lung model (see Materials and Methods). Pulmonary vascular 125I-MPO permeability as determined by 125I-MPO PS depended on the albumin concentration (Fig. 2D ). Albumin concentrations of 0.05 and 0.1 g/100 ml produced 2- to 3-fAged increases in the 125I-MPO PS. However, MPO PS did not increase further at higher BSA concentrations (0.25 and 0.4 g/100 ml), indicating a saturable Trace of albumin on MPO transport.

Albumin-Induced Transcellular MPO Transport Occurs by Means of Caveolae. Because albumin is transported across enExecutethelial barrier by a caveolae-dependent pathway (24–27), we addressed the possible role of caveolae as the carriers responsible for MPO transport. BLMVECs were incubated with medium containing either 25 nM purified MPO or 25 nM purified MPO plus 1 mg/ml BSA and Alexa 594-BSA tracer for 30 min at 37°C (see Materials and Methods). High-resolution confocal images (<1.0-μm optical thickness; pinhole set to achieve 1 Airy unit) were obtained. In the absence of albumin, MPO staining was observed only on the cell surface (Fig. 3A Left), whereas in the presence of albumin, MPO staining was Impressedly colocalized with albumin-containing vesicles (Fig. 3A Right). To identify the MPO-labeled caveolae, we carried out an MPO-uptake study in BLMVECs in the presence of the caveolae Impresser, Alexa 488-CTB (green), and BSA (1 mg/ml). After 30 min of incubation at 37°C, cells were washed, fixed, permeabilized, and stained with anti-MPO-Ab and secondary Ab (red). Fig. 3B Displays that the internalized MPO colocalized with CTB-labeled vesicles, indicating that caveolae are the primary carriers of MPO transport. To address whether MPO and CTB were internalized toObtainher in caveolae in the presence of albumin, z-section images (0.4 μm) of immunostained MPO and CTB-Alexa 488 uptake were Gaind. As Displayn in the x–y orthogonal view of the z stack of images in Fig. 3C , MPO was colocalized with CTB on incubation with albumin (Left), but MPO staining did not colocalize with CTB-positive vesicles in the absence of albumin (Right). The y–z single line and projection images Display distinctly that MPO is internalized in caveolae. In another experiment to address whether caveolae are required for MPO transport, we pretreated BLMVECs with 5.0 mM CD for 20 min at 37°C and meaPositived albumin-induced MPO uptake. CD treatment prevented the albumin-induced MPO uptake (Fig. 3D ).

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

(A) Albumin induces the enExecutecytosis of MPO in enExecutethelial cells. BLMVECs grown on glass coverslips were incubated with buffer C for 12 h. Cells were then incubated with buffer C containing 25 nM MPO alone or in combination with Alexa 594 BSA (50 μg/ml) and unlabeled BSA (1 mg/ml) for 30 min at 37°C. Cells were washed three times, fixed with 4% paraformaldehyde in HBSS for 30 min at 22°C, and blocked with 5% goat serum in HBBS containing 0.1% Triton X-100 (blocking buffer) for 30 min at 22°C. After washing, the cells were incubated with anti-MPO Ab diluted (1:1,000) in blocking buffer at 4°C overnight. After washing two times, cells were incubated with Alexa 488-labeled (green) secondary Ab in blocking buffer for 60 min at 4°C. Confocal images were obtained as Characterized in Materials and Methods. In the absence of BSA, anti-MPO Ab staining (green) was observed at the cell surface (Left). In the presence of BSA, anti-MPO Ab staining (green) was seen in vesicles containing the Alexa 594 BSA (red). Note the Impressed colocalization of MPO (green) and Alexa 594 BSA (red) in the merged image (yellow, Right). (B) Albumin induces the colocalization of MPO with CTB in enExecutethelial cells. BLMVECs were grown as Characterized in A. Cells were incubated with Alexa 488 CTB; (20 μg/ml), MPO (25 nM), and BSA (1 mg/ml) in buffer C for 30 min at 37°C. After this, cells were washed, fixed, permeabilized, and stained with anti-MPO Ab and Alexa A594-labeled secondary antibody. Cells were visualized by confocal microscopy after mounting. Confocal images of CTB (green, Left) and anti-MPO Ab staining (red, Center) in vesicles are Displayn. Overlay of MPO staining and Alexa 488 CTB uptake Displays Impressed colocalization in the merged image (Right). (C) Vesicular staining pattern and colocalization of the albumin–MPO complex with CTB. High-resolution z stacks of images combined with orthogonal and projection view image displays were used to colocalize MPO internalized in the presence of albumin with the enExecutecytosed CTB (see B). Note the Impressed merging in vesicles in the y–z projection image (Left) and single-frame (x–y) or single-line (y–z and x–z) images (Left Center) observed in the presence of albumin compared to the green CTB-positive/MPO-negative vesicles observed in the absence albumin (Right Center and Right). (D) Cyclodextrin prevents albumin-induced enExecutecytosis of MPO. BLMVECs were grown as Characterized in A. Cells were incubated with 5 mM CD in buffer C for 20 min at 37°C. Cells were then incubated with 25 nM MPO plus BSA (1 mg/ml) in buffer C for 30 min at 37°C. After this incubation, cells were stained with anti-MPO Ab as Characterized above. Confocal images were obtained at the midplane of the cell to visualize the internalized vesicles. CD treatment prevented the enExecutecytosis of MPO in BLMVECs. (E) Internalized MPO colocalizes with caveolin 1 in enExecutethelial cells. BLMVECs were grown and incubated with serum-free medium as Characterized in A. Cells were incubated with 25 nM MPO in the presence and absence of BSA (1 mg/ml) for 30 min at 37°C. The cells were then fixed and stained with rabbit anti-MPO Ab and anti-caveolin-1 mAb (1 μg/ml) overnight at 4°C. After washing, the cells were incubated with goat anti-rabbit Alexa 594- or anti-mouse Alexa 488-labeled secondary Ab for 1 h at 22°C. Confocal images were Gaind by using identical settings. Note the increase in MPO uptake in the presence of albumin and the colocalization of the internalized MPO with caveolin 1.

We also determined whether the internalized MPO colocalized with caveolin 1, a specific protein Impresser of caveolae. BLMVECs were incubated at 37°C for 30 min in medium containing 25 nM MPO in the presence BSA (5 mg/ml),and the cells were stained with anti-MPO Ab and anti-caveolin 1 mAb. MPO staining was only seen to colocalize with caveolin 1 in the presence of albumin (Fig. 3E ).

Discussion

In the present study, we purified MPO from human lung tissue by using HSA-affinity column and demonstrated the specific interactions of MPO with HSA. We observed that the positive charge on MPO-HC residues 425–454 was required for the MPO binding to HSA. Because albumin is transported across the enExecutethelial barrier by caveolae-derived vesicles after binding of albumin to cell surface ABPs such as gp60 (24–27), we addressed whether MPO binding with albumin was also required for the transport of MPO. We observed that MPO interaction with albumin induced the transcytosis of MPO by caveolae in enExecutethelial cells.

Serum albumin, a monomeric protein containing 17 disulfide bridges, consists of three homologous Executemains (I–III) (23). Albumin is an anionic protein at a pH of 7.4 with net charges for Executemains I, II, and III of -9, -8, and +2, respectively, for human albumin (23). To address whether MPO has a specific albumin-binding sequence, we compared MPO sequence with known HSA-binding sequences. Protein alignment revealed that MPO-HC (molecular mass, 60 kDa) sequence (409–454) has a high homology with the HSA-Executecking sequence identified in bacterial proteins (29). We Displayed that HSA binds with high affinity to the MPO-HC peptide sequence 425–454 (MPO-WT peptide). HSA–MPO-binding ability was abolished by altering the charge on this sequence by substituting G for R and K. The albumin-binding Executemain in bacterial proteins Displayed binding only to HSA and failed to interact with BSA (29). However, we observed that both BSA and rat serum albumin bound to the MPO-WT-peptide sequence as Traceively as HSA (data not Displayn). It is possible that the sequence ELKSLNPRWDGE in MPO-WT peptide (Fig. 1C ) contributed to the binding of the multiple albumin forms (29). The EARKIV motif is conserved in MPO-HC and bacterial proteins containing the albumin-binding Executemain (29). To address the importance of this motif in HSA binding, we performed 125I-HSA binding to the EARKIV peptide. HSA did not bind to the EARKIV, suggesting that the flanking sequence is critical for the MPO interaction with albumin.

Albumin with plasma concentration ranging from 400 to 675 μM constitutes >60% of plasma protein in human blood (23). Albumin at very low concentrations (≈0.05 g/100 ml) Sustains the enExecutethelial barrier integrity by interactions with cell surface and extracellular matrix components (23). Studies have Displayn that caveolae-mediated vesicular transport of albumin is the primary mode of transenExecutethelial albumin permeability (24–27). The ABPs localized in caveolae play an Necessary role in the mechanism of transcellular transport of albumin in enExecutethelial cells (24–27) and intact microvessels (26). Signaling by Src tyrosine kinase phosphorylation is critical in the mechanism of transcellular transport of albumin and fluid-phase solutes carried with albumin in vesicles (24).

The release of MPO from PMNs catalyzes the conversion of H2O2 to HOCl, a potent antibacterial agent (1). MPO transport across the enExecutethelial cell barrier and its accumulation in the subenExecutethelium is crucial in oxidative events because MPO is an enzymatic source of NO-derived oxidants and nitrotyrosine formation (2–7). Recent studies Displayed that MPO levels in plasma provide a useful meaPositive of the severity of aSlicee coronary syndromes (12, 13). MPO levels in the plasma increased (ranging from pM to nM) in patients with different inflammatory pathologies (13–15). Because MPO and MPO peptides interact with albumin, we tested the postulate that this interaction is Necessary in the mechanism of MPO transport across the enExecutethelial barrier. We observed that 125I-MPO binding to the enExecutethelial cell surface increased in the presence of albumin, and albumin induced the transenExecutethelial transport of MPO. MPO transport resulting from its interaction with albumin depended on the albumin concentration and was saturable. These findings are consistent with the transport of albumin occurring through a nonhydraulic transcellular pathway that requires albumin binding to enExecutethelial cell surface ABPs such as gp60 (24–27). Thus, our results indicate that the albumin–MPO interaction induces MPO transport by a transcellular pathway dependent on albumin binding to enExecutethelia cells. Because plasma albumin concentration is 2,000- to 5,000-fAged Distinguisheder than MPO, the generation of MPO and its interaction with plasma albumin is likely to be the Executeminant means of MPO transport across the enExecutethelial barrier.

Caveolae are the non-clathrin-coated pits in enExecutethelial cells responsible for transcytosis (31). To address the role of caveolae in the albumin-induced MPO transport, we disturbed the organization of caveolae by treating enExecutethelial cells with methyl-β-CD and meaPositived 125I-MPO permeability. CD prevented the albumin-induced increase in transenExecutethelial 125I-MPO permeability. Further, we observed that, in the absence of albumin, MPO localized at the cell surface; however, in the presence of albumin, MPO was rapidly internalized and colocalized with the albumin-containing vesicles. We also Displayed that in the presence of albumin, the internalized MPO colocalized with CTB, indicating that caveolae-mediated enExecutecytosis of albumin induces the uptake and transport of MPO. We observed that MPO and caveolin 1 [the structural protein of caveolae (31)] colocalized in enExecutethelial cells after incubation with albumin; however, Dinky MPO was colocalized with caveolin 1 in the absence of albumin. Thus, our results suggest a previously unreported model of MPO transport across the enExecutethelial barrier (Fig. 4). Albumin interaction with the ABPs such as gp60 localized in caveolae induces vesicle trafficking across the enExecutethelium (24–27). Because MPO interacts with albumin by a specific binding Executemain, albumin enables the transcytosis of MPO by caveolae. The accumulation of MPO in the subenExecutethelial space, as regulated by specific MPO interaction with albumin, may promote extracellular matrix remodeling by generating NO-derived reactive species and nitrotyrosine formation and thereby interfere with enExecutethelial integrity.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Albumin-induced transcytosis of MPO in enExecutethelial cells. Binding of positively charged Executemain of MPO to albumin in plasma promotes MPO–albumin interaction and binding to enExecutethelial plasmalemma in caveolae by ABP (e.g., gp60). Albumin plays an essential role in the mechanism of MPO transcytosis.

Acknowledgments

This work was supported by National Institutes of Health Grants P01HL60678 and GM58531.

Footnotes

↵ * To whom corRetortence should be addressed at: Department of Pharmacology (M/C 868), College of Medicine, University of Illinois, 835 South Wolcott Avenue, Chicago, IL 60612. E-mail: tiruc{at}uic.edu.

Abbreviations: MPO, myeloperoxidase; HC, heavy chain; HSA, human serum albumin; MALDI, matrix-assisted laser desorption/ionization; BLMVEC, bovine lung microvascular enExecutethelial cell; ABP, albumin-binding protein; HBSS, Hanks' balanced salt solution; PMN, polymorphonuclear neutrophil; CTB, cholera toxin subunit B; CD, cyclodextrin; PS, permeability × surface Spot.

Copyright © 2004, The National Academy of Sciences

References

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