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Am J Physiol Lung Cell Mol Physiol 293: L354-L363, 2007. First published May 18, 2007; doi:10.1152/ajplung.00412.2006
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Glycosylation and annexin II cell surface translocation mediate airway epithelial wound repair

Benjamin J. Patchell,1 Kimberly R. Wojcik,2 Ting-Lin Yang,1 Steven R. White,2 and Delbert R. Dorscheid1

1James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, University of British Columbia, Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada; and 2Section of Pulmonary and Critical Care Medicine, Department of Medicine, Division of Biological Sciences, University of Chicago, Chicago, Illinois

Submitted 16 October 2006 ; accepted in final form 11 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Glycosylation of cell surface proteins can regulate multiple cellular functions. We hypothesized that glycosylation and expression of glycoproteins after epithelial injury is important in mediating repair. We report the use of an in vitro culture model of human airway epithelial cells (1HAEo–) to identify mediators of epithelial repair. We characterized carbohydrate moieties associated with repair by their interaction with the lectin from Cicer arietinum, chickpea agglutinin (CPA). Using CPA, we identified changes in cell surface glycosylation during wound repair. Following mechanical wounding of confluent monolayers of 1HAEo– cells, CPA staining increases on the cell surface of groups of cells in proximity to the wound edge. Blocking the CPA carbohydrate ligand inhibited wound repair highlighting the role of the CPA carbohydrate ligand in epithelial repair. Annexin II (AII), a calcium-dependent, membrane-associated protein, was identified as a protein associated with the CPA ligand. By membrane protein biotinylation and immunodetection, we have shown that following mechanical wounding, the presentation of AII on the cell surface increases coordinate with repair. Cell surface AII accumulates in proximity to the wound. Furthermore, translocation of AII to the cell surface is N-glycosylation dependent. We are the first to demonstrate that following injury, N-glycosylation events and AII presentation on the cell surface of airway epithelial cells are important mediators in repair.

airway epithelial repair; Cicer arietinum agglutinin

THE AIRWAY EPITHELIUM ACTS as a protective barrier, preventing the exposure of the underlying tissue to noxious particles. The epithelium is routinely challenged by allergens, pollutants, and virus particles, resulting in damage that requires repair to restore barrier integrity. Damage is commonly seen in diseases such as asthma, with the ciliated columnar cells as the most damaged cell type (32). Other studies have shown an increase in the presence of sloughed epithelial cells in the bronchoalveolar lavage fluid isolated from patients with mild asthma (39). Epithelial loss can result in sensory nerve exposure with the release of neuropeptides that may worsen bronchoconstriction (reviewed in Ref. 5). Furthermore, the epithelium is not only a passive barrier, but also a source of inflammatory cytokines (reviewed in Refs. 36 and 17). The ability of the epithelium to act as a physical barrier and its involvement in the regulation of airway diameter and inflammatory events highlight the role of an intact epithelium and the importance of epithelial repair.

The mechanisms and regulators of epithelial repair are poorly understood. One important modifier of cell function is the glycosylation of cell surface proteins. Carbohydrate structures attached to and presented on the cell surface play a role in several cellular functions. Glycosylated proteins have been shown to participate in cell adhesion (43), proliferation (37), and growth potential (42), and alterations in glycosylation can have profound effects on these functions (11, 22, 26). Work by Gipson et al. (19) showed that the inhibition of N-glycosylation by treating the cells with tunicamycin, an N-glycosylation inhibitor, inhibits corneal epithelial wound repair. The authors suggested that asparagine-linked glycoproteins required for migration include cell surface glycoproteins. Our laboratory (14) has demonstrated similar results in airway epithelial cells. In both studies, the same global inhibitor of N-glycosylation was used, but the specific cell surface glycoproteins that were altered by this inhibition were not identified. Further work by Adam et al. (1) has shown that in culture, functional carbohydrate structures can be identified on the surface of cells by lectin histochemistry and wound closure inhibition (40).

Lectins are a valuable tool in glycobiology. Like the antibody to protein structure, lectins are carbohydrate binding proteins highly specific to carbohydrate structures. Chickpea agglutinin (CPA) is a lectin with unknown carbohydrate specificity. Previously, we (13) have shown CPA binds airway epithelial cells in human lung tissue. Its complex carbohydrate specificity and differential binding to the epithelial cell subsets in intact airway epithelial tissue made CPA a lectin of interest for further study of epithelial repair. Furthermore, the ability of CPA to inhibit wound closure suggests it binds a functional ligand involved in repair. The purpose of this study was to use CPA as a marker of basal cell glycosylation during repair and to identify novel carbohydrate-associated proteins that participate in the normal processes of repair. Using that same lectin, we purified the protein(s) associated with the carbohydrate structure. We have identified annexin II (AII) as a protein precipitated by the lectin CPA. AII is presented on the cell surface, and repair is coordinate with this cell surface translocation. We have also demonstrated that the recovery of AII with CPA and its cell surface presentation during repair are N-glycosylation dependent.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Normal human airway epithelial cells (1HAEo–) were a gift from Dr. D. Gruenert, University of Burlington, Vermont (9). 1HAEo– cells are SV40-transformed normal human airway epithelial cells that have been characterized previously (10, 21) and express multiple surface carbohydrate markers of primary basal airway epithelial cells (13). They were grown in culture for lectin histochemistry, wound repair kinetics, and protein extraction as previously described (13).

Monolayer wound creation and lectin cytochemistry. Monolayers were grown to confluence, and small wounds (~1.0–2.0 mm2) were created using a small rubber dental GUM Stimulator (Sunstar Butler, Guelph, ON, Canada). Lectin cytochemistry was carried out as described previously (14). At 0, 6, 12, and 24 h after the creation of the wound, cells were fixed with Clark's solution (95% ethanol-5% acetic acid). After quenching endogenous peroxidases with 0.03% H2O2, slides were blocked with Dako universal block before incubation with biotinylated CPA lectin and Vicia villosa agglutinin (VVA; 5 µg/ml and 10 µg/ml, respectively; EY Laboratories) in HEPES buffer without BSA for 60 min. Following incubation in ABC horseradish peroxidase (HRP) complex followed by 3,3-diaminobenzidine acid (DAB; Dako), monolayers were imaged on a Nikon 50i series upright microscope equipped with a digital camera.

Monolayer wound repair assay. After wounding as described above, cells were washed with DMEM without fetal bovine serum (FBS; Invitrogen, Burlington, ON, Canada). Treatment of the cells included an unstimulated control incubated in DMEM without FBS. The remaining five wells were incubated in DMEM with FBS, each with 15 ng/ml human epidermal growth factor (EGF; Invitrogen) and unconjugated CPA or VVA (EY Laboratories) at 0, 10, 25, 50, and 100 µg/ml. Monolayers were treated with lectin added immediately after wound creation (0 h), 6 h after wounding, and added 6 h and replaced at 12 h after wounding. Wounds were imaged at t = 0, 6, 12, 18, and 24 h using a Nikon Eclipse TE200 inverted microscope equipped with a Nikon Coolpix E995. Wound area was calculated by manual tracing and area calculation software (Image-Pro Plus; Media Cybernetics, Silver Spring, MD).

Lectin precipitation assay. The 1HAEo– cells were grown to confluence and mechanically wounded using a cell scraper (Sarstedt, Montreal, PQ, Canada). The wound size was large enough such that following 18 h of repair, wounds would not have fully closed. Monolayers were washed with phosphate-buffered saline (PBS) followed by the addition of DMEM + 10% FBS. After 18 h in culture, protein collection and lectin precipitation was carried out as previously described (40). Biotinylated lectin (20 µg of CPA or VVA, EY Laboratories) was added to the supernatant and incubated overnight at 4°C. Protein recovery was determined by densitometry. Confirmation of CPA precipitation of the identified protein was carried out by SDS-PAGE separation of the lectin precipitated proteins. Gel contents were transferred to a nitrocellulose membrane for Western blot analysis. Precipitations following tunicamycin treatment were carried out as above; however, following mechanical injury, monolayers were treated with the indicated concentration of tunicamycin (Sigma) added to the culture media. DMSO-treated monolayers were included as a vehicle control.

Protein purification and sequencing. Following SDS-PAGE of the precipitated proteins, the gel was stained with Coomassie brilliant blue R-250 and destained overnight in 30% methanol-10% acetic acid. The purified 36-kDa band was excised from the gel and sequenced by one-dimensional reverse-phase chromatography with on-line mass spectrometry [Applied Biosystems-MDS Sciex QSTAR hybrid liquid chromatography/mass spectrometer/mass spectrometer (LC/MS/MS) quadruple time-of-flight system, University of Victoria-Genome British Columbia Proteomics Centre, http://www.proteincentre.com/]. Complete details can be found in the data supplement online at the AJP-Lung Cellular and Molecular Physiology web site. Fragment data was submitted to ProID (proprietary Applied Biosystems software) for bioinformatics analysis of public protein databases (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/) and identification.

AII biotinylation and detection. Before protein collection, 1HAEo– cell surface proteins were biotinylated as previously described on endothelial cells (41). Following transfer, the membrane was reacted with mouse anti-AII (BD Pharmingen, San Diego, CA) or mouse anti-beta-actin (AC-74, Sigma-Aldrich) followed by HRP-conjugated rabbit anti-mouse (Santa Cruz Biotech), visualized using ECL (Amersham Biosciences, Piscataway, NJ), and quantified using ImageJ version 1.27z software.

AII elution with EGTA. EGTA elution of cell surface AII was carried out as described before (23). Briefly, 1HAEo– monolayers were grown to confluence. Monolayers were mechanically wounded, treated with either tunicamycin (10–6 or 10–7 M) or DMSO, and allowed to repair for 18 h. Repairing monolayers were equilibrated on ice for 5 min and washed once with ice-cold PBS to remove cell debris or nonadherent cells. Monolayers were incubated in equivalent volumes of ice-cold HEPES-buffered saline (HBS) containing EGTA for 10 min at 4°C. The buffer was collected and spun to remove any cells that may have lifted during the incubation. Following centrifugation, equivalent volumes of collected EGTA containing HBS were subjected to standard immunoblot procedures and probed for AII.

AII immunostaining. The 1HAEo– cells were grown to confluence on collagen IV (Sigma)-coated chamber slides and mechanically wounded. Following 18 h of repair, monolayers were fixed for 10 min in 4% paraformaldehyde diluted in HBS. Nonspecific sites were blocked with a universal serum block (Dako). Monolayers were incubated overnight at 4°C in the dark with FITC-conjugated primary antibody diluted in HBS. Following several HBS washes, the cells were incubated for 10 min in Hoechst 33342 (diluted 1:1,000 in distilled H2O; Molecular Probes, Eugene, OR). Monolayers were visualized and imaged using a Nikon TE300 inverted microscope equipped with a spot camera. For double labeling experiments, 1HAEo– monolayers were fixed as above and incubated with FITC-conjugated CPA followed by anti-AII (BD Pharmingen). Alexa 546-conjugated secondary antibody was used, and nuclei were counterstained with Hoechst 33342 (Molecular Probes) in preparation for confocal image analysis. Images were obtained using a Leica AOBS SP2 confocal microscope. The z-plane reconstructions were generated using Volocity (Improvision, Boston, MA).

Similarly, monolayers of primary airway epithelial cells were grown to confluence on collagen IV (Sigma)-coated chamber slides and mechanically wounded. Following repair, cells were fixed as above. Slides were incubated with the primary antibodies, anti-AII followed by rabbit anti-mouse immunoglobulins (Dako) and alkaline phosphatase-anti-alkaline phosphatase (APAAP) mouse (Dako). New fuchsin was used as the chromogenic substrate for color development. Slides were counterstained and imaged using a Sony spot camera mounted on a Nikon E600 microscope. Sample handling and processing were carried out simultaneously to maintain experimental conditions for all samples. During image acquisition, image capture settings were preserved throughout each session.

Statistics. Values are presented as means ± SE. The significance of differences between means was assessed by analysis of variance; when significant differences were found, a Student's t-test was used to compare the means with the level of significance set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Lectin staining of wounded airway epithelial cell monolayers. Small circular wounds were generated in 1HAEo– monolayers reliably without cell lifting. These wounds were ~1–2 mm2 in all experiments (Fig. 1). The inset images in each panel of Fig. 1 represent the intensity of lectin or control staining in the confluent areas for each wounded monolayer. Note the intensity differences between those areas and the areas in proximity to the wound. Staining for CPA was throughout the confluent areas of monolayers, but intensity of CPA staining was markedly greater in areas in proximity to the wound (Fig. 1A). This area of increased staining was either at the perimeter of the wound or in groupings of cells proximal to the wound. Monolayers fixed after 0 h (Fig. 1A, panel A), 6 h (Fig. 1A, panel B), 12 h (Fig. 1A, panel C), and 24 h (Fig. 1A, panel D) of repair each show an increase in CPA staining intensity of cells proximal to the edge of the repairing wound. VVA staining was virtually absent from intact areas of the monolayers (Fig. 1B, insets). Similar to CPA, VVA staining increases at the wound edge after mechanical wounding. Monolayers fixed after 0 h (Fig. 1B, panel A), 6 h (Fig. 1B, panel B), 12 h (Fig. 1B, panel C), and 24 h (Fig. 1B, panel D) of repair demonstrate VVA staining of cells is localized to the edge of the repairing wound with no change at more distal areas. As was noted for both CPA and VVA at time 0, in the absence of lectin (Fig. 1C), there is nonspecific staining proximal to the wounds (Fig. 1C, panel A). This artifact staining may be a result of the physical disturbance, cellular debris, and compression of cells during the wounding process and may explain the increased lectin binding at 0 h. Following 6–24 h of repair, artifact staining is lost (Fig. 1C, panel B; 24-h repair representative of 6- to 24-h time points).


Figure 1
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  		Fig. 1. Cicer arietinum, chickpea agglutinin (CPA) and Vicia villosa agglutinin (VVA) bind to the surface of human airway epithelial cell (1HAEo–) monolayers. Biotinylated lectin binding was detected using streptavidin-horseradish peroxidase conjugate followed by 3,3-diaminobenzidine acid (DAB) to generate a brown precipitate. The inset images in each panel of Fig. 1 represent the intensity of lectin or control staining in the confluent areas for each wounded monolayer. A: CPA positive staining increases following mechanical wounding over time and accumulates at and near the site of injury. Baseline CPA staining was initially present at 0 h in confluent areas immediately following wound creation (panel A). Following 6-h repair (panel B), 12-h repair (panel C), and 24-h repair (panel D), CPA staining as described is markedly increased. B: VVA positive staining was absent at baseline in confluent monolayers (insets) and increases following mechanical wounding over time and accumulates at and near the site of injury. VVA staining is present immediately following wound creation (panel A), 6-h repair (panel B), 12-h repair (panel C), and 24-h repair (panel D). C: negative control staining was absent in intact areas of 1HAEo– monolayers (insets). Immediately following wound creation, there is nonspecific staining proximal to the wound (panel A). After 6–24 h of repair, there is no nonspecific staining remaining on the repairing 1HAEo– monolayers (panel B; 24-h repair is representative of 6- to 24-h images). Original magnification, x200.

 
The effect of CPA on wounded airway epithelial monolayer repair. Mean wound starting areas for each group were not statistically different. Furthermore, in experiments where the addition of CPA lectin was delayed 6 h, wound areas were not statistically different at the initial time of lectin incubation. The addition of CPA immediately following wound creation had no effect on wound closure (Fig. 2A). We have demonstrated that CPA staining is initially weak on the surface of 1HAEo– cells. As a result, the addition of CPA immediately following mechanical injury would result in little to no lectin binding on the cells participating in repair. A delay of 6 h before CPA addition to the culture medium at the higher doses (50 and 100 µg/ml) disrupted wound closure temporarily for 12–18 h relative to EGF-stimulated wound repair, however, these results were not statistically significant. By 24 h, the wound repair of all the CPA-treated monolayers was complete and comparable to EGF-stimulated wounds (Fig. 2B). Treatment of 1HAEo– monolayers with CPA at 6 h and replaced 12 h after wound creation inhibited repair in a dose-dependent manner (Fig. 2C). Following 24 h of repair, wounds in the presence of 100 µg/ml CPA demonstrated an average of 32 ± 10% of initial wound area remaining and was significantly different relative to the EGF-stimulated closure at 24 h (9 ± 4%; P < 0.05). At the highest dose of CPA (100 µg/ml), wound repair rates closely resembled those of the negative unstimulated control. Wounds incubated in the absence of lectin, serum, and EGF demonstrated an average of 42 ± 12% of initial wound area remaining at 24 h. There was no statistical difference observed between the serum-free control and the 100 µg/ml CPA at each of the measured time points. The use of the lectin VVA in wound repair experiments similar to those with CPA did not generate significant inhibition of wound closure (data not shown). VVA and CPA generate similar binding patterns with respect to airway epithelial cell wounds but bind to distinct sugar moieties.


Figure 2
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  		Fig. 2. Wound closure of 1HAEo– monolayers in the presence of CPA. Cells were grown to confluence, mechanically wounded, and treated with CPA added at 0 h (A), 6 h (B), or 6 h and replaced at 12 h (C) in increasing doses (10, 25, 50, and 100 µg/ml for CPA 10, CPA 25, CPA 50, and CPA 100, respectively, grown in serum + EGF). Controls included negative serum-free unstimulated (Ctl) and serum + EGF (EGF)-stimulated monolayers without lectin treatment. Wound closure was followed using time lapse videomicroscopy. Remaining wound area was determined using Image-Pro Plus and presented as the percentage of the initial wound remaining. Arrows indicate time points of CPA exposure. Error bars were omitted for clarity; *P < 0.05 relative to EGF-stimulated repair. n ≥ 3 experiments for all groups.

 
The purification of CPA-specific glycoprotein ligands. Cell protein lysates were precipitated with CPA lectin (Fig. 3A) or VVA lectin (Fig. 3B) to identify glycoproteins or glycoprotein-associated proteins with increased recovery during repair. Selection of the protein band for sequencing was based on the relative increase in protein band intensity in precipitations from wounded monolayers relative to confluent monolayers. Similarities were observed between CPA and VVA precipitations of cell protein lysates after wounding. A protein band of 36 kDa was present in both the CPA (Fig. 3A) and VVA (Fig. 3B) lectin precipitations and demonstrated the largest increase as visualized with Coomassie staining. By densitometry, this 36-kDa protein band was almost twofold greater in precipitations of wounded monolayers relative to confluent monolayer preparations for both lectins. Following protein sequencing of both bands, the protein candidate was identified as AII.


Figure 3
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  		Fig. 3. Lectin precipitation profiles of 1HAEo– monolayer protein extracts. Protein extracts from confluent and wounded monolayers were incubated with biotinylated lectins CPA (A) or VVA (B) at a concentration of 15 µg/ml and avidin-agarose beads as described in MATERIALS AND METHODS. Precipitated proteins were separated by 10% SDS-PAGE, and the gel was stained with Coomassie brilliant blue R-250. The 36-kDa band (arrow) had a 2-fold increase in recovery when precipitated from wounded monolayers relative to nonwounded controls and was subsequently identified as annexin II (AII).

 
AII expression and recovery with CPA. Following mechanical wounding, the expression of AII is unchanged following normalization to beta-actin; results were not statistically significant (Fig. 4). However, CPA lectin-based precipitation of the 36-kDa band changed over the course of wound repair, suggesting changes in the glycosylation of AII or its associated proteins result in the greater recovery during repair (Figs. 3 and 5A). CPA precipitation of the 36-kDa band increased by 2 h relative to time 0 and was greatest at 12–18 h after mechanical wounding (Fig. 5A). The small wounds created during this experiment typically close in ~24–30 h. As wound closure neared completion (24 h), the recovery of the 36-kDa band returned to baseline levels (0 h).


Figure 4
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  		Fig. 4. AII expression in mechanically wounded monolayers of 1HAEo– cells. Total AII expression remains unchanged following mechanical injury. This representative immunoblot demonstrates that when AII levels are normalized to beta-actin to account for differences in protein loading, AII expression does not significantly change following mechanical injury. The results of 3 independent experiments are shown.

 

Figure 5
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  		Fig. 5. CPA precipitation profiles of wounded 1HAEo– monolayers protein extracts collected serially. Protein extracts from wounded monolayers collected over a 24-h period were incubated with biotinylated CPA (15 µg/ml) and avidin-agarose beads as described in MATERIALS AND METHODS. Precipitation of the 36-kDa protein(s) with CPA increases following mechanical injury to 1HAEo– monolayers, reaching a maximum at 12–18 h after wounding (A). Precipitations were split such that a duplicate gel could be transferred to a membrane and probed for AII by Western blot analysis (B).

 
Sequencing results that identified this 36-kDa protein as AII are based on peptide profiles and database searches and therefore were confirmed by Western blot analysis (Fig. 5B). Our results confirmed that AII is precipitated by CPA and demonstrated that its recovery approximated the recovery pattern of the 36-kDa protein, reaching a maximum recovery at 12 h after mechanical injury (Fig. 5, A and B).

The 36-kDa band in Fig. 3 is the result of a carbohydrate-dependent recovery with CPA. We have shown that the twofold increase in AII recovery with CPA is not a result of an increase in AII expression (Fig. 4). Changes in the recovery of AII as demonstrated in Fig. 3 and 5 must therefore be a result of protein modification such as the glycosylation of AII with the CPA-specific carbohydrate.

Cell surface presentation of AII. Quantification of cell surface AII was achieved through nonspecific membrane protein biotinylation. The presentation of AII on the surface of 1HAEo– cells occurred by 2 h and was maximal 18 h after mechanical injury to confluent monolayers (Fig. 6A). Similarly to the CPA precipitation of AII, the amount of AII on the cell surface began to decrease as wound closure neared completion after 18 h, suggesting a link between glycosylation and AII translocation. Western blots were probed for an intracellular negative control protein, beta-actin. This protein was undetectable in our avidin-agarose precipitations and present only in the whole protein lysate, confirming only extracellular proteins were recovered (Fig. 6A, panel B). The amount of AII presented on the cell surface increased ~2-fold 6 h after wound creation, reaching a maximum at 18 h, where there is a 2.5-fold change in AII on the surface (Fig. 6A, panel C).


Figure 6
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  		Fig. 6. A: membrane protein biotinylation of AII on the surface of wounded 1HAEo– monolayers. Biotinylated membrane proteins were collected, separated by SDS-PAGE, and transferred to membranes for immunodetection to detect AII on the cell surface of 1HAEo– cells ("a"). As an intracellular control, membranes were probed for beta-actin ("b"). Quantification of the cell surface AII was done by densitometry and is shown ("c"). *P < 0.05 relative to 0 h; {dagger}P < 0.1 relative to 0 h. B: immunofluorescent detection of cell surface AII on 1HAEo– cells. Following wounding, 1HAEo– cells were fixed in the absence of detergent and stained for AII (green) and nuclei (blue). Confluent monolayer (panel A) and wounded monolayer 18 h after injury (panel B) are shown. Original magnification, x200. Images were taken with the same exposure settings. C: immunohistochemical detection of cell surface AII on primary airway epithelial cells. Following wounding, cells were fixed as before and stained for cell surface AII using standard immunohistochemistry as described in MATERIALS AND METHODS. Staining was absent in both the isotype control (panel A) and monolayers fixed immediately after injury (panel B). Cell surface AII staining accumulated at the wound edge at 12 h (panel C) and 24 h after mechanical wounding (panel D).

 
By immunocytochemistry on nonpermeabilized monolayers, we confirmed cell surface translocation of AII during repair (Fig. 6B). Immunofluorescence in regions distal to the wound demonstrated minimal to no AII staining (Fig. 6B, panel A). After mechanical wounding and 18 h of repair, the levels of cell surface AII dramatically increased (Fig. 6B, panel B). AII staining was visible on cells that had migrated into the wounded area as well as those proximal to the wound edge. The findings from the 1HAEo– cells were confirmed in primary airway epithelial cells grown in culture (Fig. 6C). No background staining was observed with the isotype control antibody (Fig. 6C, panel A) and monolayers fixed immediately after mechanical wounding (Fig. 6C, panel B). Cell surface AII accumulates proximal to the wound edge following 12-h repair (Fig. 6C, panel C) and 24-h repair (Fig. 6C, panel D). Furthermore, the staining pattern closely resembles that of the original increase in CPA lectin staining (Fig. 1A) and the VVA lectin staining (Fig. 1B).

The inhibition of glycosylation. The effect of inhibiting N-glycosylation was observed both quantitatively and qualitatively with respect to AII. Following addition of tunicamycin, a global N-glycosylation inhibitor, the amount of AII on the cell surface was dramatically reduced (Fig. 7A). Previously, we (14) have shown that wound closure of airway epithelial cells is inhibited by tunicamycin. Calcium chelation removes an essential element for the association of AII with the plasma membrane and therefore can be used to elute and quantify AII on the cell surface. The addition of tunicamycin at the time of injury resulted in the inhibition of AII translocation to the cell surface as determined by EGTA elution. Tunicamycin treatment also decreased the amount of AII that can be recovered by CPA precipitation to 33% relative to the DMSO-treated control (Fig. 7B).


Figure 7
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  		Fig. 7. AII recovery following tunicamycin treatments. Following mechanical injury, 1HAEo– monolayers were treated with increasing doses of tunicamycin. Following 12-h repair, monolayers were incubated with EGTA to elute any calcium-dependent membrane-associated proteins such as AII. In the presence of 10–6 M tunicamycin, AII recovery dropped below 20% of that of the DMSO control (A). Similarly, protein was isolated from monolayers 12 h after mechanical wounding and subjected to CPA lectin precipitation as before. CPA-recovered AII was detected by standard immunoblotting techniques. Following tunicamycin treatment, AII recovery levels dropped to levels approaching the no lectin control (B).

 
The effect of tunicamycin on AII translocation was visualized by confocal microscopy (Fig. 8). In the presence of DMSO alone, AII (red) can be easily visualized on the surface of 1HAEo– cells (Fig. 8A). AII staining is in fact colocalized with the CPA staining (green) as shown in the z-x (top) and z-y (left) plane images. Following tunicamycin treatment, the presence of AII on the cell surface is virtually eliminated (Fig. 8B), further confirming our quantitative results (Fig. 7).


Figure 8
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  		Fig. 8. AII and CPA double staining of mechanically wounded 1HAEo– monolayers. Following injury, wounded monolayers were treated with DMSO (A) or 10–6 M tunicamycin (B). Monolayers were fixed without permeabilization and stained for CPA (green), AII (red), and nuclei (blue) as described in MATERIALS AND METHODS. In the absence of tunicamycin, cell surface AII is abundantly present; furthermore, colocalization of the CPA and AII is indicated by the presence of yellow staining on cells in proximity to the wound (A). Tunicamycin treatment disrupts the translocation of AII to the cell surface such that there are low levels of cell surface AII and no colocalization of the CPA and AII staining (B). The z-x and z-y plane images are shown; white crosshairs indicate location of z-plane image acquisition. Scale bar, 10 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Research has suggested that the asthmatic epithelium is defective in mechanisms of repair (24, 29, 30). However, this pathway in healthy individuals is not fully understood. We have shown that after mechanical injury to airway epithelial cell monolayers, there is an increase in CPA carbohydrate ligand presentation on the surface of epithelial cells proximal to the wound (Fig. 1A). Following lectin precipitation, AII was identified. Subsequently we have shown a significant increase of AII on the cell surface after mechanical injury (Fig. 5A). Cells at and near the site of injury demonstrate the increased cell surface presentation of AII relative to areas of intact epithelium. The increase in AII recovery by lectin precipitation is carbohydrate dependent and occurs temporally before AII translocation to the cell surface. This suggests that glycosylation occurs first and may act as a regulatory signal in the AII translocation. Wound repair disruption with CPA and the presentation of AII on the cell surface along with CPA/AII double staining provides some insight into the timing and mechanics of these events. In the absence of AII on the cell surface, CPA would be free to bind other carbohydrate targets as demonstrated by the baseline staining by CPA. Following 6 h of repair, AII is presented on the surface of a subset of cells permitting the binding of CPA at this time. Combined with the affect of CPA replacement at 12 h, wound repair was significantly inhibited. CPA staining was abundant proximal to the edge of the wound (Fig. 1). The leading edge cells are replaced during repair; the high turnover of cells at the wound edge may explain the requirement for repeated CPA treatments to affect wound closure inhibition. This data supports and extends our previous findings (14, 40) that carbohydrates play an essential role in the processes of wound repair.

Our results confirm the association of AII with the CPA carbohydrate ligand. As we have demonstrated, our recovery of AII by CPA mediated precipitation increases after mechanical injury (Fig. 4A). Studies have suggested that AII is a glycosylated protein (20) based on its retention in lectin affinity columns. AII was also purified from human placental cell membrane extracts using concanavalin A, a lectin that binds D-glucose and D-mannose structures (49). AII is a glycoprotein but may also function as a lectin. The ability of AII to bind glycoproteins such as heparin (25, 27) and fucose-containing saccharides (16) has been reported. The increased recovery of AII by CPA would be a direct result of a change in glycosylation. This would result from repair-associated glycosylation of AII. Alternatively, the subsequent increase in the CPA recovery of AII may be indirect. CPA binding of a neoglycosylated protein that forms a complex with AII during repair through the lectin function of AII would also permit this increased AII recovery. The loss of AII recovery with CPA and the inhibition of cell surface translocation following tunicamycin treatment highlights the role N-glycosylation plays in this regulation of AII function.

Annexins are a family of Ca2+-dependent lipid binding proteins that bind acidic phospholipids. AII is an abundant member of the annexin family and has been shown to exist as a 36-kDa monomer, a homodimer, or a heterotetramer (AIIt) consisting of two AII heavy chain subunits and two 11-kDa AII light chain subunits (p11). The monomer is largely cytosolic, whereas the formation of the AIIt results in association of the protein complex with the plasma membrane. Initially, AII was thought to participate in endocytosis (15), exocytosis (44), and cell-cell adhesion (46). It is unclear whether the interaction of AII with biological membranes is solely due to a phospholipid association or whether a receptor is required. AII on the extracellular face of the plasma membrane has been demonstrated in endothelium (6), skin keratinocytes (33), and many tumor cells (48). Matsuda et al. (38) have shown that following mechanical injury to rat cornea, AII translocates to the cell surface of epithelial cells and interacts with an extracellular AII ligand, tenascin (7, 8, 45), to participate in cell-cell and cell-matrix interactions (45). However, this work did not investigate the role of glycosylation as a form of regulation. Recent reports have shown that AII plays a role in the regulation of cell migration in other model cell systems (3, 4). Our study is the first demonstration of AII on the surface of airway epithelial cells during repair and the essential involvement of glycosylation to regulate this process.

Previous studies have highlighted the potential importance of carbohydrates in epithelial repair after injury (1, 2, 13, 40); however, very little is known about the identity or how these glycosylated structures regulate these events. With no known carbohydrate ligand for CPA, it is difficult to infer any structural information of the carbohydrate ligand responsible for the CPA staining of epithelial wounds. The ability of VVA to produce a similar cell surface staining pattern and also to purify AII from mechanically wounded 1HAEo– monolayers suggest the presence of N-acetylgalactosamine (GalNAc) residues on the glycosylated protein as an important carbohydrate modification during repair. During rat corneal epithelial repair, the addition of GalNAc slowed epithelial migration, highlighting the potential importance of GalNAc-containing structures in epithelial wound repair (18). However, the inability of VVA to significantly disrupt wound repair suggests that GalNAc is not critical. The complex carbohydrate recognized by CPA, not GalNAc, is the regulatory structure. Our laboratory has characterized the importance of several carbohydrate structures involved in airway epithelial repair such as galactose containing serum glycoproteins (40) and sialyl LewisX structures (2).

As a result of their association with the plasma membrane, annexins are thought to be involved in the regulation of membrane events. The ability of AII to bind a variety of proteins in a regulated manner may help us identify its specific function during airway epithelial repair. AII may act as a scaffold protein that facilitates protein complex assembly that binds the glycoprotein on the cell surface. The carbohydrate binding of AII has been shown to be tightly regulated by phosphorylation (25). Furthermore, changes in glycosylation, as detected by the binding of CPA and VVA during repair would confer specificity of specific cell surface glycoproteins to associate with AII and effect wound repair. Kauffman et al. (28) have demonstrated that the cell surface glycosylation of asthmatics is altered. This altered glycosylation could affect protein interactions that would result in the accumulation of epithelial damage as repair would be impaired. During wound repair, there is an increase in tenascin expression with the accumulation between the migrating cells and the underlying matrix (34). Our findings may be applied to disease considering that there is a marked increase in tenascin expression in the basement membrane of asthmatic airways (31) and that the binding of tenascin-C with AII on the surface of endothelial cells results in cellular responses including cell migration and the loss of focal adhesions (8). As a receptor for tenascin, the translocation of AII to the cell surface may facilitate epithelial wound repair through the interaction with the extracellular matrix. A defect in this pathway may result in cellular compensation and the increased deposition of tenascin in the basement membrane of the remodeled asthmatic airway. However, whether tenascin deposition is an effect of such a defect is not known.

Many unknowns remain with regard to AII and the airway epithelium. Our experiments suggest that AII is a mediator of epithelial repair; however, the regulation of this mechanism is unknown. As a lipid-associated protein that is translocated to the cell surface, AII lacks a transmembrane and cytoplasmic domain and has no known enzymatic function (47). It is likely that AII facilitates the interaction of the cell with extracellular ligands. This may result from either direct binding of tenascin or as a scaffold protein that facilitates complex assembly. By presenting functional proteins on the epithelial cell surface such as its association with tissue plasminogen activator (t-PA) and plasminogen facilitating the generation of plasmin on the cell surface of endothelial cells (12, 35), cell surface AII may be a key regulatory protein in airway epithelial repair.

In summary, we demonstrate that the identification of AII as a glycoprotein-associated mediator of airway epithelial repair and that glycosylation is critical in its regulation and function. Identification of how AII interacts with the extracellular environment and other proteins during repair remains to be determined. The carbohydrate modification of AII or AII-associated glycoproteins to direct expression at the cell surface may lead to new insights regarding epithelial cell motility and repair in asthma. The implications of AII and glycosylation as they relate to airway epithelial repair suggest that the chronic damage of the epithelium characteristic of asthma and the treatment of airway remodeling may require a more complete understanding of normal repair mechanisms.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
B. J. Patchell is supported by graduate student fellowships from the Michael Smith Foundation for Health Research (MSFHR) and Canadian Institutes of Health Research (CIHR). S. R. White is supported by National Institute of Allergy and Infectious Diseases Grant NIH-AI-053562. D. R. Dorscheid is supported by a Scholar Award from the MSFHR, operating grants from CIHR (CIHR/Allergen 79632 and CIHR/NHP 78381), a New Investigator award, the Canadian Intensive Care Foundation, and National Institutes of Health Grant NIH-66026.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. R. Dorscheid, iCAPTURE Centre, St. Paul's Hospital, Rm. 166, 1081 Burrard St., Vancouver, BC, Canada V6Z 1Y6 (e-mail: ddorscheid{at}mrl.ubc.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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