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Originally published In Press as doi:10.1074/jbc.M402115200 on August 4, 2004

J. Biol. Chem., Vol. 279, Issue 43, 44924-44930, October 22, 2004
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Association of BAP31 with CD11b/CD18

POTENTIAL ROLE IN INTRACELLULAR TRAFFICKING OF CD11b/CD18 IN NEUTROPHILS*

Ke Zen{ddagger}§, Markus Utech¶||, Yuan Liu{ddagger}, Illena Soto{ddagger}, Asma Nusrat{ddagger}, and Charles A. Parkos{ddagger}

From the {ddagger}Epithelial Pathobiology Research Unit, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322 and Department of General Surgery, University of Münster, 48149 Münster, Germany

Received for publication, February 25, 2004 , and in revised form, July 26, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The {beta}2 integrin CD11b/CD18 is an integral membrane protein that is present in the plasma membrane and secondary granules of neutrophils and functions as a major adhesion molecule. Upon cellular activation, there is translocation of intracellular pools of CD11b/CD18 to the plasma membrane in concert with enhanced cellular adhesion. Although much is known about the function of CD11b/CD18, how this protein is transported within the cell is less well defined. Here we report that CD11b/CD18 specifically binds to BAP31, a member of a novel class of sorting proteins regulating cellular anterograde transport. Through experiments aimed at identifying CD11b/CD18-binding proteins, we produced a monoclonal antibody termed E1B2 that recognizes a 28-kDa membrane protein that co-precipitates with CD11b/CD18. Microsequence analysis of the E1B2 antigen revealed that it is BAP31. Co-association of CD11b/CD18 and BAP31 was confirmed in co-immunoprecipitation and protein binding assays. Additional experiments revealed that the binding of BAP31 to CD11b/CD18 was not dependent on divalent cations nor mediated by the I-domain of CD11b. Using glutathione S-transferase fusion chimeras, we determined that binding of CD11b/CD18 to BAP31 is mediated through interactions with the cytoplasmic tail of BAP31. Immunolocalization studies revealed colocalization of BAP31 and CD11b/CD18 within neutrophil secondary granules. Subcellular fractionation studies in polymorphonuclear leukocytes (PMN) revealed similar patterns of redistribution of BAP31 and CD11b/CD18 from fractions enriched in secondary granules to the plasma membrane following stimulation with formylmethionylleucylphenylalanine (fMLP). Given the known sorting properties of BAP31, these findings suggest that BAP31 may play a role in regulating intracellular trafficking of CD11b/CD18 in neutrophils.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The leukocyte {beta}2 integrin CD11b/CD18 (Mac-1, CR3) plays a central role in regulating PMN1 migration through tissues (1–3). Given the importance of this function, extensive investigation on the identification of ligands for CD11b/CD18 has been carried out. So far more than 30 protein/non-protein molecules have been reported to bind to CD11b/CD18. Known ligands with characterized functions include intercellular adhesion molecule 1 (ICAM-1) (4, 5), complement C3 fragment iC3b (6), junctional adhesive molecule C (JAM-C) (7, 53), fibrinogen (FBG) (8), heparin (9), neutrophil elastase (10), and neutrophil inhibitory factor (NIF) (11). CD11b/CD18 also interacts with complement factor H (12), glycoprotein Ib{alpha} (GPIb{alpha}) (13), uPAR (14), E-selectin (15), and many extracellular matrix proteins including laminin, collagens, vitronectin, Cyr61, and connective tissue growth factor (16–22). The diversity of ligands that bind to CD11b provides a molecular basis for its important role in many different biological events such as leukocyte adhesion/migration, phagocytosis, and pathogen invasion. The identification and characterization of CD11b/CD18-binding proteins have been central in understanding the role of CD11b/CD18 during different physiological events.

In neutrophils, CD11b/CD18 is mainly present in secondary granules under resting conditions. Upon cellular activation, CD11b/CD18 is translocated from intracellular pools to the plasma membrane. This increased surface expression in concert with activation-induced avidity changes serves as a basis for enhanced cellular adhesion. Although the localization of CD11b/CD18 within neutrophils and its translocation following cell activation are well documented (23–25), mechanisms that govern the intracellular trafficking of CD11b/CD18 are less well defined.

To identify new cellular proteins that bind to CD11b/CD18, we employed an antibody-based approach. We produced a mAb termed E1B2 that binds to a 28-kDa membrane protein that physically associates with CD11b/CD18. The antigen represented by E1B2 was identified as BAP31, a member of a family of proteins that associate with membrane IgD in B cells (26, 27) and regulate anterograde transport of certain membrane proteins including cystic fibrosis transmembrane conductance regulator (CFTR) (28) and cellubrevin (29). In this report, we characterize the interaction of BAP31 with CD11b/CD18 and its subcellular localization in PMN. The potential role of BAP31 in regulating intracellular trafficking of CD11b/CD18 in PMN is also discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells—Human colonic epithelial T84 cells were grown and passaged as described previously (30). The cells were grown on plastic culture dishes or permeable collagen-coated polycarbonate supports (31). HL-60 cells were obtained from the American Type Culture Collection (ATCC) and maintained at 37 °C in a humidified 5% CO2 atmosphere in RPMI 1640 medium supplemented with 20% (v/v) fetal calf serum. For the analysis of changes in antigen expression, HL-60 cells were cultured for 7 days in RPMI 1640 medium with or without 1.25% Me2SO (32). Human PMN were isolated from whole blood of normal human volunteers using Ficoll dextran sedimentation (33). Isolated PMN were resuspended in HBSS devoid of Ca2+ and Mg2+ (HBSS-) (4 °C) at a concentration of 5 x 107 cells/ml and used within 4 h of isolation.

CD11b/CD18 Purification—Functionally active CD11b/CD18 was isolated from human PMN (~1010 cells) by immunoaffinity chromatography using anti-CD11b mAb LM2/1 as described previously (9). SDS-PAGE of purified CD11b/CD18 revealed two prominent protein bands with molecular weight values of ~150,000 and 95,000, characteristic of CD11b and CD18, respectively. The functional activity of purified CD11b/CD18 was confirmed by binding assays with T84 cells (9, 34) and fibrinogen (FBG) (Sigma) (8).

Antibodies and Screening—Rat anti-BAP31 mAbs CC-1 and CC-4 (35) were generous gifts from Dr. Vanda A. Lennon (Mayo Clinic, Rochester, MN). Anti-CD11b mAbs LM2/1 and CBRM1/29 (36), anti-CD11a mAb TS1/22 (37), anti-CD11c mAb 4G1 (38), inhibitory rat anti-human {beta}1 integrin mAb 13 (39), and anti-CD47 mAb PF3.1 (33) were used as described previously. The polyclonal antibody against human CD11b (R7928A) and the antibody against human CD18 (R7928E) were prepared in rabbits using the peptide of the C terminus of CD11b and CD18, respectively, as described previously (40). Polyclonal antiactin antibody and the secondary antibodies conjugated with HRP, fluorescein isothiocyanate, rhodamine, and 6- and 12-nm gold particles were obtained from Jackson Immunoresearch Labs (West Grove, PA).

Monoclonal antibodies were generated by immunizing mice with T84 cell membrane fractions as described previously (31). Antibodies were screened for binding to CD11b/CD18-coated microtiter wells that had been preincubated with detergent lysates of T84 cell membranes. Specifically, CD11b/CD18 (~100 µg/ml in 150 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 50 mM triethylamine, 100 mM Tris, 1% N-octyl-{beta}-D-glucopyranoside, pH 7.4) was diluted 20-fold with HBSS and added to Linbro 96-well microtiter plates (ICN Biomedical, Aurora, OH) (50 µl/well) followed by incubation overnight at 4 °C. Nonspecific binding was then blocked by addition of 1% BSA in HBSS for 30 min. T84 cell membranes, prepared as described previously (41), were solubilized in HBSS containing 1% Triton X-100 (4 °C). The lysate was diluted with HBSS (final concentration of detergent, <0.25%) and added to CD11b/CD18-coated plates for incubation (37 °C, 1 h). Microtiter wells were then washed extensively with HBSS containing 0.1% Triton X-100 followed by incubation with individual hybridoma supernatant (50 µl/well) for 1 h at room temperature (R.T.). Wells were then washed and incubated with HRP-conjugated goat anti-mouse IgG followed by color development. BSA-coated plates were used as controls.

Immunofluorescence—For double staining human PMN and HL-60 cells with anti-BAP31 and anti-CD11b antibodies, cells were applied to glass slides and allowed to adhere and spread for 20 min at 37 °C. Cells were then fixed with 3.7% paraformaldehyde (15 min, R.T.) and permeabilized with 0.1% Triton X-100 (20 min, R.T.). After blocking non-specific protein with 5% normal goat serum in HBSS for 30 min, cells were then incubated with anti-BAP31 mAb CC-1 (rat IgG) and anti-CD11b mAb LM2/1 (mouse IgG) (1 h, R.T.) followed by incubation with fluorescein isothiocyanate- or rhodamine-conjugated secondary antibodies, accordingly. Epithelial cell monolayers were fixed and permeabilized in cold methanol (20 min, –20 °C). After blocking monolayers were incubated with primary antibodies including anti-E-cadherin and anti-BAP31 antibodies (CC-1 or CC-4) for 1 h at R.T. After washing, monolayers were incubated with fluorescein isothiocyanate- or rhodamine-conjugated secondary antibodies (30 min, R.T.). Washed cell monolayers were then mounted in ProLong antifade embedding solution (Molecular Probes, Eugene, OR). Monolayers or slides were visualized on a Zeiss LSM510 confocal microscope (Zeiss Microimaging, Thornwood, NY) (42). Images shown are representative of at least three experiments, with multiple images taken/slide. As a control for background labeling, control monolayers were incubated with comparable concentrations of normal mouse IgG and secondary antibody.

Immunoelectron Microscopy—800 µl of a suspension of PMN (5 x 107/ml) were layered on the top of 200 µl of 8% (w/v) paraformaldehyde fixative in a 1.5-ml Eppendorf tube and pelleted for 3 min at 3000 rpm. After removal of the supernatant, 200 µl of 4% (w/v) paraformaldehyde fixative were added and fixed for 4 h at R.T. For cryoprotection, the cell pellet was infiltrated in 2.3 M sucrose in phosphate-buffered saline containing 0.15 M glycine followed by mounting on an aluminum pin and freezing in liquid N2. Sectioning and labeling of ultrathin frozen sections were performed as described previously (43). Frozen sections (50 nm) were prepared with an ultracryomicrotome (Leica EM) at –110 °C and placed onto a formvar/carbon-coated copper grid. After blocking with 10% fetal calf serum, sections were incubated with respective primary antibody for 45 min, washed, and incubated for 45 min with the appropriate gold-conjugated secondary antibody. To prevent cross-reactivity, double labelings were performed with gold-conjugated antibodies from different species. The grids were contrasted and embedded in 2% methylcellulose solution (1 ml of methylcellulose contained 0.1 ml of 3% uranylacetate) and were examined with an electron microscope (Philips 201, Eindhoven, The Netherlands).

Co-immunoprecipitation—Cross-immunoprecipitation experiments were performed using mAb E1B2 and anti-CD11b antibody LM2/1. Lysates of detergent-solubilized cells were precleared with mouse IgG-Sepharose followed by incubation with mAb E1B2- and LM2/1-conjugated Sepharose 4B, respectively. After six washes, the beads were resuspended in SDS sample buffer and boiled for 5 min followed by centrifugation and collection of bead-free supernatants. Eluted samples of E1B2-Sepharose and LM2/1-Sepharose were then analyzed with SDS-PAGE and Western blots probed with a polyclonal anti-CD11b antibody R7928A (for E1B2 antibody immunoprecipitation) and anti-BAP31 antibody (for CD11b antibody immunoprecipitation), respectively. As a control, immunoprecipitated products were probed with anti-CD47 mAb PF3.1 (44). Co-immunoprecipitation experiments in PMN were also performed by using mAb E1B2 with mAb TS1/22, mAb 4G1, and mAb 13, respectively. The products immunoprecipitated by mAb TS1/22 or 4G1 were probed with anti-CD18 antibody R7928E.

Protein Purification and Microsequencing—The antigen defined by E1B2 IgG was immunopurified from large scale cultures of T84 cells (~1010 cells). Cells were harvested and resuspended in lysis buffer containing a mixture of protease inhibitors. The lysate was sequentially subjected to low speed (2,000 x g, 10 min, 4 °C) and high speed (180,000 x g, 45 min, 4 °C) centrifugation followed by passage through a 0.2-µm filter. The extract was then pumped at a flow rate of 20 ml/h sequentially through a column of mouse IgG-Sepharose (4 ml, 2 mg of IgG/ml of beads) followed in tandem by a column of E1B2-Sepharose (6 ml, 2 mg of IgG/ml of beads). The E1B2 column was then washed extensively with phosphate-buffered saline consisting of 0.1% Triton X-100 and eluted with high pH buffer (150 mM NaCl, 2 mM MgCl2,2mM CaCl2,50mM triethylamine, and 0.1% Triton X-100, pH 10.5). Fractions of 2 ml were collected and neutralized with 0.2 ml of 2.0 M Tris (pH 7.0) and were analyzed by SDS-PAGE and Western blot.

For protein microsequencing, the peak fraction of immunopurified protein was concentrated (YM10, Amicon Inc.) and subsequently was denatured, reduced, and subjected to SDS-PAGE on a 4–20% gradient polyacrylamide gel followed by electrophoretic transfer to a polyvinylidene difluoride membrane (Millipore Inc., Billerica, MA). The transferred protein was visualized by stain with GelCode Blue stain reagent (Pierce, catalog no. 24590) followed by excision of the band and submission to the Emory Microchemical Facility (Atlanta, GA) for N terminus amino acid sequencing.

Glutathione S-Transferase (GST) Fusion Proteins of the Extracellular Domain of bap31—GST fusion proteins of the BAP31 intravesicular domain (bapN, residues 68–102) and the BAP31 cytoplasmic domain (bapC, residues 124–246) were generated, respectively. For bapN, the forward primer was 5'-GGATCCCGCGAAATTCGGAAGTATGAT-3', and the reverse primer was 5'-CTCGAGATTCCTCTGGGCACGGAAAAG-3'. For bapC, the forward primer was 5'-GGATCCTCGCAGCAGGCCACGCTG-3', and the reverse primer was 5'-CTCGAGCTCTTCCTTCTTGTCCATGGG-3'. The primers included BamHI and XhoI restriction sites, respectively (underlined). Amplified products were obtained using the human carcinoma library (Clontech, BD Biosciences) as template and by cycling 30 times: 94 °C for 1 min, 52 °C for 50 s, and 72 °C for 1 min. After digestion, cDNA products of bapN and bapC were inserted downstream of the GST moiety in the pGEX-4T1 vector (Amersham Biosciences). The expressed GST-BAP31 extracellular domain was purified on glutathione-agarose (Sigma) and eluted with 10 mM reduced glutathione, and the purity was verified by SDS-PAGE and Western blot analysis.

Binding Assays—Purified E1B2 antigen (10 µg/ml), GST-bapN (10 µg/ml), GST-bapC (10 µg/ml), and FBG (10 µg/ml) were first immobilized onto 96-well microtiter plates as described before. After blocking with 2% BSA in HBSS, purified CD11b/CD18 (5 µg/ml in HBSS containing 0.1% Triton X-100) was added to plates and incubated for 1 h at 37 °C. After washing with HBSS containing 0.1% Triton X-100, bound CD11b/CD18 was verified using anti-CD11b mAb LM2/1 followed by HRP-conjugated secondary antibody and color development. For inhibition experiments, CD11b/CD18 was first immobilized in microtiter wells. After blocking, purified BAP31 was added to wells in the absence or presence of inhibitors. Bound BAP31 was detected by anti-BAP31 mAb CC-1 followed by HRP-conjugated secondary antibody and optical density measurement. In experiments with EDTA treatment, HBSS- was used instead of HBSS.

Subcellular Fractionation of PMN—Freshly isolated, unstimulated PMN or 10-7 M fMLP-stimulated (37 °C, 30 min) PMN (2 x 108/condition) were resuspended in 5 ml of nitrogen cavitation buffer followed by nitrogen cavitation (15 min, 400 psi, 4 °C). The lysate was centrifuged (1000 x g), and the supernatant was subjected to isopycnic sucrose density gradient fractionation on linear 20–55% sucrose gradients in a Beckman SW-28 swinging bucket rotor (100,000 x g; 3 h, 4 °C) as described previously (45). Sucrose gradients were then collected in 1.5-ml fractions and analyzed for sucrose density, protein, plasma membrane (alkaline phosphatase), primary granules (myeloperoxidase (MPO)), and specific (secondary) granules (lactoferrin) (45). To better define the distribution of BAP31 and CD11b/CD18 in the different granule populations within PMN, we employed three-layer Percoll gradient subcellular fractionation (Percoll densities of 1.050, 1.090, and 1.120 g/ml) to separate specific (secondary) granules from gelatinase (tertiary) granules exactly as described previously (46, 47). Lactoferrin and gelatinase were used as marker proteins for secondary granules and tertiary granules, respectively (47).

Statistics—Data are presented as the mean ± S.E. and were compared by Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
E1B2 mAb Recognizes a Cell Membrane Protein That Specifically Binds to CD11b/CD18 —By screening mAbs against the binding complex of CD11b/CD18 and all T84 cell membrane proteins, we identified a mAb termed E1B2. The hybridoma supernatant from E1B2 clone strongly bound to microtiter wells that were preincubated with CD11b/CD18 and lysates of T84 cell membrane fractions. In contrast, there was no binding when E1B2 was incubated with microtiter wells that were preincubated with BSA and T84 cell membrane lysate or CD11b/CD18 alone. As shown in Fig. 1, immunofluorescence labeling indicates that the E1B2 antigen is strongly expressed in both T84 cells (A) and human PMN (B). In T84 cells, the E1B2 antigen mainly localized to basolateral aspects of cell membranes and in submembranous vesicular organelles. In labeled suspensions of PMN, mAb E1B2 is visualized in granular cytoplasmic structures. Without cell permeabilization, there was no apparent staining with mAb E1B2 in both T84 cells and PMN (data not shown), suggesting that the epitope of mAb E1B2 is not accessible to extracellular labeling. In Fig. 1C, Western blot analysis indicated that E1B2 antigen represents a ~28-kDa protein in both human PMN and T84 cells.



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  		FIG. 1.
mAb E1B2 labeling of PMN and epithelial cells. mAb E1B2 was identified as labeling T84 cell membrane extracts that bound to immobilized CD11b/CD18 as detailed under "Materials and Methods." A and B show immunofluorescence labeling and confocal microscopy of T84 monolayers and freshly isolated human PMN cells adherent to glass slides, respectively. Note that E1B2 strongly labels the basolateral aspect of T84 monolayers and stains PMN cytoplasm in a granular pattern. Bars,20 µm. In C, Western blot of T84 cells and PMN with E1B2 mAb shows a single protein band of ~28 kDa.

 
The binding of CD11b/CD18 to the E1B2 antigen was investigated by co-immunoprecipitation experiments. Cross-immunoprecipitations of E1B2 antigen and CD11b/CD18 from detergent lysates of T84 cells mixed with purified CD11b/CD18 were then probed with mAb E1B2 or anti-CD11b polyclonal antibody (R7928A), respectively (Fig. 2A). In parallel, because the antigen is expressed in PMN, we performed similar cross-immunoprecipitation experiments using Triton X-100 lysates of human PMN (Fig. 2B). Western blot results from T84 cell lysates mixed with purified CD11b/CD18 and human PMN lysates alone both indicated that the E1B2 antigen could be pulled down by anti-CD11b antibody and that integrin CD11b/CD18 could be pulled down by E1B2 IgG, respectively. As a control, CD47, an Ig superfamily membrane protein expressed in both epithelial cells and neutrophils, was not detected in either immunoprecipitated product by anti-CD47 mAb PF3.1 (33), suggesting the association of E1B2 antigen with CD11b/CD18 is specific. Experiments were also performed to examine whether the E1B2 antigen associates with other integrins in PMN. In these experiments, a PMN lysate was first immunoprecipitated with mAb TS1/22 (for CD11a), mAb 4G1 (for CD11c), or mAb 13 (for {beta}1). The immunoprecipitates were then probed with mAb E1B2. As shown in Fig. 2C, no E1B2 antigen was detectable in immunoprecipitates of CD11a/CD18, CD11c/CD18, or {beta}1 integrin.



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  		FIG. 2.
The E1B2 antigen co-precipitates with CD11b/CD18. In A, T84 cell detergent lysates mixed with purified CD11b/CD18 were immunoprecipitated (IP) with anti-CD11b mAb and E1B2, respectively. In B, detergent lysates of whole PMN were immunoprecipitated with anti-CD11b and E1B2 mAb, respectively. In C, Western blots of immunoprecipitates of CD11a/CD18 (TS1/22), CD11c/CD18 (4G1), and {beta}1 integrin (mAb 13) from PMN lysates were probed with anti-CD18, anti-{beta}1 integrin, and mAb E1B2 as labeled.

 
The Antigen for mAb E1B2 Is BAP31—The antigen defined by mAb E1B2 was immunopurified from large scale cultures of T84 cells using an affinity E1B2-Sepharose column. The purified protein was subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane for N terminus amino acid sequencing. We obtained the N-terminal sequence, SLQWTAVATFLY, which is identical to the terminal amino acid sequence of BAP31, an integral membrane protein originally identified to be associated with membrane IgD in B-cells (26, 27) (Fig. 3A). Two known BAP31 mAbs (CC-1 and CC-4) (35) also recognized the purified E1B2 antigen, confirming that E1B2 antigen is BAP31 (data not shown). To determine the region on BAP31 that binds to CD11b/CD18, we prepared two GST fusion proteins consisting of the putative intravesicular portion of BAP31 (bapN, amino acid residues 60–108) and the cytoplasmic domain of BAP31 (bapC, residues 124–246) (Fig. 3B), respectively. In a binding assay, isolated BAP31, BAP31-GST chimeras, and fibrinogen were immobilized on microtiter plates. After blocking, purified CD11b/CD18 was added followed by incubation at 37 °C for 1 h. After washing, the bound CD11b/CD18 was detected by HRP-conjugated anti-CD11b antibody. As shown in Fig. 4A, full-length BAP31 isolated from epithelial T84 cells bound to CD11b/CD18. GST-bapC also bound to CD11b/CD18, although to a slightly lesser extent than the full-length protein. Interestingly, no significant binding between GST-bapN and CD11b/CD18 was detected. Furthermore, such binding of CD11b to BAP31 could be blocked by GST-bapC but not by GST-bapN (Fig. 4B). Taken together, these results suggest that the cytoplasmic domain of BAP31 mediates binding to CD11b/CD18. Further experiments were performed to characterize the binding properties of CD11b binding to BAP31. As shown in Fig. 4C, anti-I-domain-specific mAb (CBRM1/29) (9, 34, 48), fibrinogen (8), and treatment with EDTA each did not disrupt binding of CD11b/CD18 to BAP31. These results suggest that BAP31 does not bind to the I-domain of CD11b and that the binding of BAP31 with CD11b/CD18 is not divalent cation-dependent.



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  		FIG. 3.
Identification of E1B2 antigen as BAP31. The N-terminal amino acid sequence of purified E1B2 antigen is identical to the N-terminal sequence of BAP31 (A). Based on the putative structure of BAP31 (B), GST fusion chimeras were constructed that consisted of the putative intravesicular domain of BAP31 (GST-bapN, amino acid residues 68–102) and the cytoplasmic tail of BAP31 (GST-bapC, amino acid residues 124–246).

 



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  		FIG. 4.
The binding of CD11b/CD18 to BAP31 and GST-BAP31 chimeras. In A, BAP31, GST-bapC, GST-bapN, GST only, and FBG were immobilized in microtiter plates as detailed under "Materials and Methods." After blocking, wells were incubated with CD11b/CD18 (5 µg/ml in HBSS with 0.1% Triton X-100) for 1 h at 37 °C. Bound CD11b was detected with anti-CD11b mAb. In B, BAP31-coated wells were incubated with CD11b/CD18 in the presence of GST-bapC and GST-bapN at various concentrations. After 1 h incubation (37 °C), bound CD11b/CD18 was detected with anti-CD11b mAb. In C, CD11b/CD18 was first immobilized in microtiter wells. After blocking, the wells were incubated with BAP31 (10 µg/ml) in the presence of CBRM1/29 (5 µg/ml), FBG (10 µg/ml), or 2.5 mM EDTA, respectively. For CBRM1/29 and FBG, the binding buffers were HBSS with 0.1% Triton X-100. For EDTA treatment, the binding buffer was HBSS- with 0.1% Triton X-100. After1hof incubation (37 °C), bound BAP31 was detected with mAb CC-1. Data represent the mean ± S.E. of three independent experiments performed in duplicate.

 
Colocalization of BAP31 and CD11b/CD18 in Neutrophils—The association of BAP31 and CD11b/CD18 in PMN was further investigated by immunofluorescence and immunoelectron microscopy. For immunofluorescence staining, PMN were double labeled with anti-CD11b mAb (LM2/1, mouse IgG) and anti-BAP31 mAb (CC-1, rat IgG), respectively. As shown in Fig. 1B, freshly isolated human PMN in suspension were stained brightly by BAP31 antibody. However, the intracellular localization of BAP31 and colocalization with CD11b/CD18 in PMN were difficult to assess because of intense labeling within the cytoplasm. To better localize BAP31 and CD11b/CD18 in PMN, adherent and spread cells were used. In these experiments, PMN were allowed to adhere and spread on glass slides. As shown in Fig. 5, BAP31 and CD11b/CD18 were largely colocalized to cellular granular structures (arrowheads). The colocalization of CD11b/CD18 and BAP31 to the membranes of PMN granules was further confirmed by double immunogold labeling and electron microscopy (Fig. 6). Interestingly, CD11b (Fig. 6, large arrowheads, gold particle size of 12 nm) and BAP31 (small arrowheads, gold particle size of 6 nm) were often observed very close to each other in the same membrane segment.



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  		FIG. 5.
Colocalization of BAP31 and CD11b/CD18 in adherent and spread PMN. PMN adherent to glass slides were fixed in 3.7% paraformaldehyde and permeabilized with 0.1% Triton X-100 followed by labeling with mAbs E1B2 (green) and LM2/1 (red). Note that BAP31 and CD11b colocalize in PMN granular structures (arrowheads). Bar, 10 µm.

 



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  		FIG. 6.
Double immunogold labeling and electron microscopy of CD11b (6-nm gold particles) and BAP31 (12-nm gold particles) in PMN. Note that CD11b and BAP31 are colocalized in granular structures of PMN. The inset shows a higher magnification image of an intracellular granule demonstrating colocalization of CD11b (small arrowheads) and BAP31 (large arrowheads). PM, plasma membrane. Bars, 0.1 µm.

 
It is well known that in resting PMN, the majority of CD11b/CD18 is localized within secondary granules and secretory vesicles. Upon PMN activation, secondary granule contents are released after fusion with the plasma membrane. As a membrane receptor within secondary granules, CD11b/CD18 is incorporated into the plasma membrane following PMN activation (23, 49). We investigated whether the cell surface expression of BAP31 was increased in a pattern similar to that observed for CD11b/CD18 following stimulation by fMLP. As shown in the sucrose gradient profile in Fig. 7, the distribution of BAP31 and CD11b/CD18 in resting PMN is quite similar. Both proteins mainly localized to secondary granule-enriched fractions (17–20) with only a minor fraction of protein colocalizing with plasma membrane-enriched fractions (11–15). After stimulation with fMLP (10-7 M, 30 min), there was significant redistribution of BAP31 from cellular granule-enriched fractions (particularly secondary granules) to plasma membrane-enriched fractions similar to that observed for CD11b/CD18. After fMLP stimulation, more than half of total BAP31 colocalized with plasma membrane-enriched fractions, and the remaining protein colocalized with granule-enriched fractions. In subcellular fractionation experiments of resting/stimulated PMN, total CD11b/CD18 and BAP31 recovery in fractions (%) was 91.6/85.7 and 90.5/86.2, respectively. The similar redistribution profiles of BAP31 and CD11b/CD18 from secondary granule-enriched fractions to plasma membrane-enriched fractions upon PMN activation suggested that BAP31 association with CD11b/CD18 might mediate intracellular trafficking of the integrin following cell activation.



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  		FIG. 7.
Co-distribution of BAP31 and CD11b/CD18 in secondary granule-enriched fractions with redistribution to the plasma membrane fractions after fMLP stimulation. In A, PMN (2 x 108/condition), either unstimulated or stimulated with 10-7 M fMLP (37 °C, 30 min), were disrupted by nitrogen cavitation, subjected to isopycnic sucrose gradient centrifugation, and fractionated as detailed under "Materials and Methods." The panels represent immunoblots of BAP31 and CD11b/CD18 from subcellular organelle-containing sucrose fractions (30-µl sample/lane) using mAb E1B2 and polyclonal anti-CD11b (R7928A) antibody, respectively. In B, nitrogen-cavitated unstimulated PMN (2 x 108) were subjected to three-layer Percoll gradient centrifugation and fractionated as detailed under "Materials and Methods." The panels represent immunoblots of CD11b/CD18 and BAP31 from subcellular organelle-containing fractions (25-µl sample/lane).

 
Because CD11b/CD18 has been localized to both secondary and tertiary granules and because the above sucrose fractionation protocol does not adequately resolve these two granule fractions, experiments were performed to separate secondary granules from tertiary granules using three-layer Percoll density gradients (47). We then determined the localization of CD11b/CD18 and BAP31 in Percoll fractions enriched in the tertiary granule marker gelatinase and secondary granule marker lactoferrin. Western blotting results confirmed localization of CD11b/CD18 in both tertiary and secondary granule-enriched fractions; however, BAP31 cosedimented with secondary granule-containing fractions only (Fig. 7C). These findings explain why not all of the CD11b/CD18 colocalizes with BAP31 in the immunofluorescence images in Fig. 5.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
BAP31 and its related protein (BAP29) were originally identified as membrane proteins co-purifying with membrane-bound immunoglobulin from lysates of B leukocytes (27). The DNA sequence of BAP31 (26) predicts a 28-kDa (246-residue) protein (Fig. 3A) that, in agreement with other members of this family of proteins, has three putative transmembrane domains. It has a cytoplasmic C terminus containing {alpha}-helical domains and a terminal dilysine ER-homing motif (26). Although cloning of human and murine cDNA showed that BAP31 is ubiquitously expressed in all tissues (26), BAP31 protein expression seems restricted to a minority of cells in tissues (35). In the present study, we have demonstrated the presence of BAP31 in human intestinal epithelial cells and PMN. By immunofluorescence, we also found expression of BAP31 in HL-60 or Me2SO-induced HL-60 cells (data not shown). In epithelial cells, a significant amount of BAP31 was observed along the basolateral cell membrane (Fig. 1A). Previous studies on BAP31 have suggested that BAP31 is a component of the rough ER and ER-derived trafficking vesicles. It mainly localizes within ER-like structures and has been proposed to participate in regulating the export of selected membrane proteins from the ER to the Golgi apparatus (28, 29, 35, 50). The localization pattern of BAP31 observed in human colonic epithelial cells would argue that BAP31 might also be involved in protein sorting or vesicle transport to cell basolateral surfaces. The idea that BAP31 plays a role in protein/vesicle transport to the cell surface is also supported by our observation of up-regulation of surface expression of BAP31 on activated PMN (Fig. 7).

The mechanism of BAP31 in protein/vesicle sorting is not clear, although it has been suggested that BAP31 may be part of the common machinery involved in vesicle trafficking and membrane fusion. Annaert et al. (29) demonstrated that BAP31 selectively binds not only to cellubrevin but also to synaptobrevin I. The alteration of BAP31 structure by site-directed mutagenesis of BAP31 significantly affected the BAP31-cellubrevin binding and perturbed the sorting of cellubrevin. It has also been shown that BAP31 specifically associates with nonmuscle myosin heavy chain {beta} and {gamma}-actin, two components of the cytoskeleton actomyosin complex (51), suggesting that BAP31 may play a fundamental role in the structural organization of the cytoplasm. Interestingly, besides being a common component of protein/vesicle-sorting machinery, BAP31 also selectively binds to individual membrane proteins and regulates their intracellular transport and cell surface expression. Lambert et al. (28) showed that BAP31 directly binds to cystic fibrosis transmembrane conductance regulator (CFTR) and controls the CFTR surface expression in epithelial cells. Recently Paguet et al. (52) reported that BAP31 binds to two allotypes of class I molecules and plays an important role in recruiting class I molecules to transport vesicles. In the present study, we have observed that BAP31 specifically associates with leukocyte integrin CD11b/CD18. The binding assay using different GST-BAP31 chimeras suggested that binding of BAP31 to CD11b/CD18 requires the intracellular portion of BAP31 (Fig. 4).

The BAP31-binding site on CD11b is not clear. As expected, the I-domain of CD11b seems not to be involved, and the binding is not divalent cation-dependent (Fig. 4C). We speculate that binding of BAP31 to CD11b/CD18 may be similar to the binding of BAP31 to the membrane-associated IgD, in which the binding requires the involvement of the transmembrane domain of IgD. In other words, the binding of BAP31 to CD11b/CD18 may depend on both the transmembrane segment and intracellular domain of the integrin. Further studies are necessary to confirm this hypothesis.

Although there is no apparent physiological basis for BAP31-CD11b/CD18 interaction in epithelial cells because these cells do not express CD11b/CD18, this interaction is likely to be physiologically relevant in PMN and other cells that express both BAP31 and CD11b/CD18 proteins. In particular, we believe that the association of BAP31 with CD11b/CD18 may be important in regulation of trafficking of CD11b/CD18 in cells that co-express these proteins. Although the localization of CD11b/CD18 within PMN secondary granules and its increased expression on the cell surface following PMN activation are well documented (23–25), the mechanisms that govern the intracellular trafficking of CD11b/CD18 are not clear. By immunofluorescence staining, we observed that BAP31 and CD11b/CD18 were largely colocalized in granule-like structures in PMN (Fig. 5, arrowheads). We did observe a few punctate structures adjacent to neutrophil cell boundaries that were not co-labeled with anti-CD11b and anti-BAP31 antibodies (Fig. 5). This may represent the protein-sorting nature of BAP31. If BAP31 is responsible for protein transport between certain organelles (28, 29, 35), it would be expected to associate and dissociate with the proteins along the sorting pathway. Indeed, our subcellular fractionation studies suggest that BAP31 and CD11b/CD18 colocalize in secondary granules but not in other CD11b/CD18-containing granules such as tertiary granules. Given what is known about BAP31 in other systems, this colocalization pattern suggests that BAP31 may be involved in CD11b/CD18 trafficking to/from secondary granules. Thus, we propose that BAP31 may be a regulator of intracellular transport and cell surface expression of CD11b/CD18 in neutrophils. In support of this, we have preliminary observations on the effects of BAP31 knockdown on expression and transport of CD11b/CD18 in Me2SO-induced HL-60 cells that suggest decreased expression of CD11b/CD18 within granules and at the cell surface after BAP31 knockdown (data not shown). However, further detailed studies on the effects of BAP31 on CD11b/CD18 expression and translocation are required to better define the role of BAP31 in regulating CD11b/CD18 trafficking in human neutrophils.


   FOOTNOTES
 
* This work was supported by Grants HL54229 and HL72124 from the National Institutes of Health (to C. A. P.). Tissue culture and monoclonal antibody production were supported by Digestive Diseases Mini-center Grant DK277640 from the NIDDK, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| Supported by Grant UT 42/1-1 from the German Research Foundation (Deutsche Forschungsgemeinschaft). Back

§ To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, Emory University, Whitehead Biomedical Bldg., Rm. 115, 615 Michael St., Atlanta, GA 30322. Tel.: 404-727-8541; Fax: 404-727-3321; E-mail: kzen{at}emory.edu.

1 The abbreviations used are: PMN, polymorphonuclear leukocyte(s); mAb, monoclonal antibody; BAP31, B cell antigen-associated protein 31; HBSS, Hanks' balanced salt buffer; HBSS-, HBSS devoid of Ca2+ and Mg2+; FBG, fibrinogen; HRP, horseradish peroxidase; BSA, bovine serum albumin; R.T., room temperature; GST, glutathione S-transferase; bapN, BAP31 intravesicular domain (residues 68–102); bapC, BAP31 cytoplasmic domain (residues 124–246); fMLP, formylmethionylleucylphenylalanine; ER, endoplasmic reticulum. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Vanda A. Lennon for providing anti-BAP31 antibodies (CC-1 and CC-4). We also thank Dr. Jan Pohl (Emory Microbiochemical Facility) for N-terminal sequencing of BAP31 and Susan Voss and Ingrid McCall for excellent cell culture assistance.



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