Introduction

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Airway mucus plays an important role in host defense against airborne particles through its viscoelastic property. This physicochemical property of the mucus, which is crucial for its proper mucociliary function, is contributed mainly by mucus glycoproteins or mucins present in the mucus. Abnormalities in either the quality or quantity of these mucins therefore could lead to the pathology of the airway. In the airway, mucins are secreted by two types of cells: goblet cells on the surface epithelium and mucous cells in the submucosal gland. Although a great deal of information is now available regarding the regulation of mucin secretion by these cell types (1, 2), very little is known about either the structure or the subcellular origin of these mucins.

The presence of high molecular-mass mucin-like glycoproteins (HMGP) was demonstrated on the apical surface of hamster tracheal surface epithelial (HTSE) cells in primary culture based on lectin binding studies (3, 4). These cell-surface mucins had a slow turnover rate compared with that of the mucins secreted spontaneously, and were releasable by human neutrophil elastase by a proteolytic cleavage (3). These cells have also been shown to express messenger RNAs of Muc-1 (5, 6)the cell-surface mucins that were originally discovered in some carcinomas (7). In the present study, we intended to isolate and characterize these HMGP from the plasma membrane of the primary HTSE cells and then to determine whether or not these membrane HMGP are Muc-1 mucins. Here we report that the plasma membrane of primary HTSE cells contain HMGP that seem to be integral glycoproteins but different from the cell-surface Muc-1 mucins, and that both the size and charge of these HMGP appear to be similar to those of secreted mucins. Thus the present results indicate that there are at least two different types of mucins on the surface of HTSE cells.

	Materials and Methods

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Materials

³H-Hyaluronic acid was purified from spent media of primary rabbit tracheal epithelial cells grown on plastic dishes, which had been metabolically radiolabeled with [6-³H]glucosamine as described previously (8). All other materials were obtained from Sigma Chemical Company, Inc. (St. Louis, MO) unless stated otherwise.

Cell Culture and Metabolic Radiolabeling

Tracheas were obtained from male Golden Syrian hamsters of 6-7 wk of age (Harlan Sprague-Dawley, Indianapolis, IN). Primary HTSE cells were prepared and cultured as previously described (4). Briefly, cells were cultured on a thick collagen gel and maintained in a medium supplemented with various factors and 5% fetal bovine serum (4). The culture medium was changed on Days 1, 3, and 5 following the plating. Metabolic radiolabeling of cells was performed by incubating confluent cultures in 100-mm dishes for 24 h with the medium containing one or a combination of the following materials: [6-³H]glucosamine (10 µCi/ml), [1-¹⁴C]glucosamine (5 µCi/ml), and [³H]palmitic acid (40 µCi/ml) (New England Nuclear, Boston, MA). Following incubation, the spent media were collected and the floating cells were removed by centrifugation at 200 × g at 4°C for 5 min.

Dissociation and Homogenization of HTSE Cells

Radiolabeled cells in 100-mm dishes were rinsed with phosphate-buffered saline (PBS), pH 7.4, and then incubated in 5 ml of Medium 199, pH 7.2, containing 4,000 U of collagenase (type IV; Worthington Biochemical Corp., Freehold, NJ) for 4 h inside a 37°C CO₂ incubator to dissociate the cells from the gel. The suspension was pelleted by centrifugation at 200 × g for 10 min at 4°C. The pellet was washed twice by centrifugation with 5 ml of a homogenization buffer before resuspending in 1 ml of the same buffer. The homogenization buffer contained 0.28 M sucrose, 10 mM MgCl₂, 150 mM NaCl, 50 mM 2-(N-Morpholino)- ethanesulfonic acid, and 1 mM ethyleneglycol-bis-(-aminoethyl ether)-N,N'-tetraacetic acid, pH 6.0. Immediately before homogenization, 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.1 mg/ml soybean trypsin inhibitor were added to the cell suspension. Homogenization was performed at 4°C inside a 50-ml polypropylene tube by sonication twice for 15 s at power setting 6 using a micro-ultrasonic cell disruptor (Model W-10; Mansonics, Plainview, NY).

Subcellular Fractionation

Subcellular fractionation was carried out with a slight modification of the protocol described by Langridge and colleagues (9) (Figure 1). Briefly, the crude homogenate diluted to 10 ml in the homogenization buffer was transferred to a 50-ml propylene tube and centrifuged at 700 × g for 15 min at 4°C. The pellet (P1) was discarded and the resulting supernatant (S1) was then centrifuged at 10,000 × g for 30 min at 4°C to sediment a crude membrane fraction (P2). The resulting supernatant (S2) was diluted to 38.5 ml with 50 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (Hepes), pH 7.5, transferred to polyallomer tubes, and centrifuged at 100,000 × g for 1 h at 4°C in an ultracentrifuge (Beckman LS-80; Beckman Instruments, Inc., Palo Alto, CA) to precipitate a second membrane fraction (P3). The resulting supernatant (S3) was discarded. Resolution of P2 and P3 fractions into purified membrane fractions was achieved by the following steps. Both fractions were resuspended separately in 3 ml of 50 mM Hepes, pH 7.5, and loaded over a 2-ml sucrose cushion (48% wt/ vol) in Ultra Clear tubes (Beckman Instruments) fitted onto an SW 55Ti rotor (Beckman Instruments) and centrifuged at 100,000 × g for 1 h at 4°C. Following centrifugation, each tube displayed four regions: a soluble fraction at the upper end of the tube (fraction I), a membrane band floating above the sucrose cushion (fraction II), the sucrose cushion itself (fraction III), and a hard membrane pellet (fraction IV). Fractions II, III, and IV were resuspended separately with 50 mM Hepes, pH 7.5, to make 38.5 ml, and centrifuged at 100,000 × g to remove sucrose. Each pellet fraction was diluted with 50 mM Hepes, pH 7.5, and used immediately for marker enzyme assays.

View larger version (18K): [in this window] [in a new window]   Figure 1.   Flow chart of subcellular fractionation of hamster tracheal epithelial cells. Confluent HTSE cells were homogenized and subjected to differential centrifugation as described in MATERIALS AND METHODS. After the sucrose density gradient, four fractions were collected: P2-I from the top supernatant, P2-II from the interface between the top supernatant phase and the sucrose phase, P2-III from the sucrose phase, and P2-IV from the pellet. These fractions were subjected to the marker enzyme assay as seen in Figure 2.

View larger version (16K): [in this window] [in a new window]   Figure 2.   Identification of subcellular fractions. The four different fractions obtained after the sucrose density gradient centrifugation as described in Figure 1 were analyzed for the presence of the following enzymes as described in MATERIALS AND METHODS: SDH (succinic dehydrogenase) as a marker for mitochondria; ALP (alkaline phosphatase) and 5-Nu (5'-nucleotidase) for the plasma membrane; ACP (acid phosphatase) for the lysosome; TPP (thiamine pyrophosphatase) for the Golgi; and G6P (glucose-6-phosphatase) for the rough endoplasmic reticulum. Fractions 2, 3, and 4 represent fractions P2-II, P2-III, and P2-IV, respectively. Fraction P2-I was free of any of these enzyme activities, whereas fraction P2-II was enriched with the plasma membrane marker enzymes and will be referred to as the plasma membrane preparation or fraction throughout the text.

Identification of the Plasma Membrane Fraction (Marker Enzyme Assay)

Both the identity and purity of the plasma membrane fraction were monitored by analysis of various marker enzymes in the fraction. The marker enzymes used for different subcellular fractions and the analytical assays used for the enzymes were: alkaline phosphatase (10) and 5'-nucleotidase (11) for the plasma membrane, acid phosphatase (12) for the lysosomal membrane, thiamine pyrophosphatase (13) for the Golgi, glucose-6-phosphatase (14) for the rough endoplasmic reticulum, and succinate dehydrogenase (15) for mitochondria. Protein was quantitated by the method of Bradford (16) with bovine serum albumin as standard.

Enzymatic Digestions

All of the digestions, except with phosphatidylinositol-phospholipase C (PI-PLC) and glycosylphosphatidylinositol-phospholipase D (GPI-PLD) were carried out as previously described (17). Briefly, the enzyme digestions were performed as follows: hyaluronidase (Sigma type VI-S, 200 U/ml) at 37°C for 16 h in PBS adjusted to pH 5.5 by the addition of 0.1 M citric acid; chondroitinase ABC (0.4 U/ml) at 37°C for 5 h in 0.1 M Tris acetate, pH 7.3; heparitinase (5 U/ml) at 42°C for 5 h in 0.1 M sodium acetate, 1 mM CaCl₂, pH 7.0; and pronase (Type XIV protease, 5 mg/ml; Sigma) at 37°C for 16 h in PBS, pH 7.2. At the end of each enzyme digestion, the reaction mixture was boiled for 2 min to denature the enzyme and then centrifuged at 12,000 × g for 2 min, and the supernatant was applied to a Sepharose CL-4B column.

Phase Transition Using Triton X-114 and Digestion with PI-PLC and GPI-PLC

Triton X-114 was precondensed as described by Bordier (18). The purified plasma membrane fraction was lyophilized and then mixed with a 1.5% Triton X-114 solution containing 10 mM Tris-HCl, pH 7.5, and 0.5 M NaCl. The mixture was equilibrated in ice for 30 min to solubilize the membrane lipids, then incubated at 37°C for 10 min to precipitate the detergent. The two phases were separated by centrifugation at 2,000 × g for 10 min at 37°C. The precipitate was extracted twice with 100 µl of 0.006% Triton X-114 and the supernatant was pooled with the original supernatant (the aqueous phase) and lyophilized. The "pellet" (the detergent phase) was solubilized with 1 ml cold absolute acetone (80°C) to dissolve the detergent while precipitating the proteins. Both the lyophilized aqueous phase and the acetone pellet were dissolved in 50 mM sodium acetate, pH 7.0, containing 0.1% sodium dodecyl sulfate (SDS), and boiled for 2 min before applying to Sepharose CL-4B columns. Digestions with PI-PLC and GPI-PLD were performed as previously reported (19). Briefly, the detergent "pellet" obtained from the Triton X-114 phase separation (see below) was dissolved with 200 µl of the above Triton X-114 buffer, and kept at 15°C for 5 h in the presence of either 15 mU/ml of PI-PLC (Bacillus cereus; Sigma) or 50 mU/ml of GPI-PLD (Streptomyces species, Type VII; Sigma). In the case of GPI-PLD, CaCl₂ was added to 5 mM and the reaction mixture was adjusted to pH 7.0 with Tris. After the reaction, the phases were separated and subjected to Sepharose CL-4B column chromatography as described previously.

Chemical Digestions

Reductive -elimination was performed as previously described (17). Briefly, the sample was incubated in 50 mM NaOH/1 M NaBH₄ at 37°C for 16 h, then neutralized with glacial acetic acid at the end of the reaction, and the borate was removed by repeated evaporation with methanol containing 1% acetic acid. The sample was then lyophilized and redissolved in the elution buffer before being subjected to Sepharose CL-4B chromatography.

Chromatography

Column chromatography procedures used in this experiment have been described previously (17) and gel-filtration columns were eluted with a buffer containing 50 mM sodium acetate, pH 7.2, and 0.1% SDS unless otherwise specified. Briefly, for diethylaminoethyl (DEAE)-Sephacel anion exchange chromatography, the ³H-void volume (V_o) sample was first subjected to a buffer exchange by passing through a Sephadex G-50 column that had been equilibrated with a buffer containing 8 M urea, 0.15 M NaCl, 0.5% (vol/vol) Triton X-100, and 0.05 M sodium acetate, pH 6.0. The V_o fractions were pooled and then applied to a DEAE-Sephacel column (1.0 × 2.5 cm) that had been equilibrated with the same buffer. After washing with 5 ml of this buffer, elution was continued by applying a gradient of NaCl from 0.15 to 1.4 M over 46 ml. Fractions of 0.8 ml were collected at a flow rate of 3.0 ml/h. The recoveries of radioactivity after column chromatography were greater than 80% in gel-filtration columns, and about 90% in the anion-exchange column.

CsCl Equilibrium Density Gradient Centrifugation

CsCl density gradient centrifugation was performed as previously described (23). Briefly, 100 µl of each sample was added to a CsCl solution prepared in PBS, pH 7.2, with a final density of 1.5 g/ml that contained 4 M guanidine HCl and 0.2% SDS. The CsCl sample solution was boiled for 5 min, transferred into polyallomer tubes, loaded into a SW 55Ti rotor, and centrifuged at 105,000 × g for 48 h at 10°C.

Immunoprecipitation with an Anti-Muc-1 Mucin Antibody

The immunoprecipitation was carried out with the polyclonal antibody CT-1 as previously described (24). Briefly, confluent HTSE cells grown on 100-mm dishes were first starved for 2 to 4 h in a glucose-free Dulbecco's modified Eagle's medium, and then metabolically radiolabeled for 24 h by incubating the cells in the complete medium (2 ml/ dish) which contained a "low" level of glucose (0.4 g/liter) and [6-³H]glucosamine (100 µCi/ml). At the end of the labeling, cells were washed with PBS and lysed using a buffer containing 40 mM sodium phosphate (pH 7.2), 250 mM NaCl, 50 mM NaF, 5 mM ethylenediaminetetraacetic acid, 1% Triton X-100, 1% deoxycholate, 10 mM benzamidine, 25 µg/µl leupeptin, 110 µg/µl PMSF, and 10 µg/µl aprotinin. The cell lysate was incubated with rabbit preimmune sera for 1 h at 4°C to minimize the nonspecific binding to the antisera. At the end of the incubation, 1/10 volume of protein A-agarose beads (10% in the lysing buffer, vol/vol) was added and mixed for 1 h on a rotating wheel at 4°C, followed by centrifugation to remove any nonspecific binding to the beads. The same procedures were applied to this "precleaned" lysate, namely, incubation first with an anti-Muc-1 antiserum (CT-1) in the presence or absence of a 17-mer blocking peptide (0.1 µg/ml) consisting of the C-terminal region of human Muc-1 mucin (24), and then with the protein A-agarose beads. The immune complex was precipitated by centrifugation at 10,000 × g for 2 min at 4°C. The precipitate was washed 4 times, each time using a different buffer: (1) the lysis buffer in 0.25 M NaCl, (2) the lysis buffer in 0.5 M NaCl, (3) the lysis buffer in 0.25 M NaCl, and (4) double-distilled water. The extensively washed precipitate was then mixed with 2× Laemmli's sample buffer and boiled for 2 min, and its total volume was adjusted for gel-filtration using the elution buffer. In the case of the membrane ³H-HMGP, the ³H-radioactivity of the final precipitate was too low (< 50 cpm) to run the CL-4B column and therefore the supernatant samples were applied to the column.

SDS-Polyacrylamide Gel Electrophoresis

SDS-polyacrylamide gel electrophoresis (PAGE) was carried out at 100 V through a 3% stacking gel and a 5% separating gel using a Minigel apparatus (Bio-Rad Laboratories, Hercules, CA). At the end of the electrophoresis, the gel was dried and exposed to a sheet of X-ray film (Eastman Kodak, Rochester, NY) with intensifying screens for 10 d.

	Results

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Subcellular Fractionation of HTSE Cells

Sequences involved in subcellular fractionation and activity profiles of various enzyme markers in individual fractions are indicated in Figures 1 and 2, respectively. The plasma membrane markers alkaline phosphatase and 5'-nucleotidase were recovered mostly in fraction P2-II with recoveries of 40 and 35% from the original crude homogenate, respectively. This fraction contained relatively low activities of other marker enzymes: 0.9% in succinate dehydrogenase (mitochondria), 5.5% in acid phosphatase (lysosome), 0.75% in thiamine pyrophosphatase (Golgi), and 0% in glucose-6-phosphatase (rough endoplasmic reticulum). Therefore, the enzyme profile of the P2-II fraction indicated that this fraction was highly enriched with the plasma membrane. In contrast, the P3-II fraction was enriched with other microsomal enzymes (51.3% in acid phosphatase, 53% in thiamine pyrophosphatase, and 45% in glucose-6-phosphatase, calculated from the total activity in the original homogenate) but had relatively low activities in the plasma membrane markers (16% in 5'-nucleotidase and 5.5% alkaline phosphatase) compared with the P2-II fraction. Fractions P2-I and P3-I were free of any of these enzymes. On the basis of the total protein concentration, the P2-II fraction had a 61-fold enrichment in 5'-nucleotidase activity and a 55-fold increase in alkaline phosphatase activity over the original crude homogenate. On the other hand, ³H-radioactivity in the P2-II fraction constituted 0.7% of the total [³H]glucosamine-labeled glycoconjugates and 4.0% of the total crude homogenate.

Purification of HMGP Synthesized by HTSE Cells

Both spent (conditioned) media and P2-II fractions that were obtained from [³H]glucosamine-labeled HTSE cells were subjected to Sepharose CL-4B column chromatography. The resulting ³H-V_o fractions (Figure 3) were pooled and examined for the presence of proteoglycans by enzyme digestion. The ³H-V_o from the plasma membrane fraction was totally resistant to chondroitinase ABC (Figure 4A), hyaluronidase (data not shown), or heparitinase (data not shown), indicating the absence of hyaluronic acid, dermatan sulfate, chondroitin sulfate, or heparan sulfate proteoglycans. However, the ³H-V_o was completely digested with both pronase and alkaline borohydride treatment, with the resulting digests included in the column with Kd of 0.59 (Figure 4B) and 0.74 (Figure 4C), respectively. (Kd is the distribution coefficient defined as the fraction of the stationary phase which is available for diffusion of a given solute species, and calculated here using the equation Kd = [V_e  V_o]/[V_t  V_o], where V_e, V_o, and V_t represent elution volume, void volume, and total volume, respectively.) The results indicate that the ³H-V_o glycoconjugates are O-linked glycoproteins. The V_o from P2-II will henceforth be referred to as HMGP of the plasma membrane, or simply the membrane HMGP. Similarly, the V_o obtained following digestion of the spent media with hyaluronidase will be defined as HMGP of the spent media, or the secreted HMGP.

View larger version (9K): [in this window] [in a new window]   Figure 3.   Sepharose CL-4B gel-filtration column chromatography of the plasma membrane fraction. Confluent HTSE cells were metabolically radiolabeled with [3H]glucosamine for 24 h, and the membrane fraction (P2-II) was prepared from their homogenates as seen in Figures 1 and 2. The membrane fraction was then subjected to Sepharose CL-4B gel-filtration column chromatography eluting with 50 mM sodium acetate, pH 7.0, containing 0.1% SDS. Vo and Vt represent void volume and total volume, respectively. Three Vo fractions (#22-24) were pooled (Vo sample) and subjected to further characterization.

View larger version (13K): [in this window] [in a new window]   Figure 4.   Digestion of Vo fractions from the plasma membrane with (A) chondroitinase ABC, (B) pronase, and (C) alkaline borohydride. The Vo sample obtained from the plasma membrane preparation (Figure 3) was treated with either chondroitinase ABC, pronase, or alkaline borohydride as described in MATERIALS AND METHODS. At the end of the reaction, the reaction mixture was applied to a Sepharose CL-4B column under the conditions described in Figure 3. Open circles: before treatment; closed circles: after treatment. Vo and Vt represent void volume and total volume, respectively.

Extraction of HMGP with Triton X-114

Both the P2-II fraction and the spent media were extracted with Triton X-114, and the resulting aqueous and detergent phases of Triton X-114 from each sample were chromatographed separately on Sepharose CL-4B columns. Extraction of the plasma membrane fraction resulted in a distribution of 13 and 87% of the ³H-activity in the aqueous and detergent phases, respectively. When samples recovered from the detergent and aqueous phases were chromatographed on Sepharose CL-4B columns, 72 and 28% of ³H-V_o were contributed by the detergent and aqueous phases, respectively (Figure 5A). On the other hand, after extraction of the spent media, 98 and 2% of the ³H-activity were found in the aqueous and the detergent phases, respectively. Sepharose CL-4B column chromatography of these samples revealed that 90% of ³H-V_o in the spent media was from the aqueous phase and the remaining 10% from the detergent phase (Figure 5B). Thus the result indicates that most of the HMGP in the plasma membrane are integral membrane glycoproteins.

View larger version (14K): [in this window] [in a new window]   Figure 5.   Sepharose CL-4B column chromatography following partitioning in Triton X-114 of (A) the plasma membrane fraction and (B) the spent medium. Confluent HTSE cells were metabolically radiolabeled with [3H]glucosamine for 24 h, and both the P2-II fraction and the spent media were subjected to phase separation by Triton X-114 as described in MATERIALS AND METHODS. The resulting detergent and aqueous phases were individually applied to Sepharose CL-4B eluting with 50 mM sodium acetate, pH 7.0, containing 0.1% SDS. Open circles: aqueous phase; closed circles: detergent phase.

Digestion of the Plasma Membrane with PI-PLC and GPI-PLD

To see if the membrane HMGP are anchored to the plasma membrane via a GPI linkage, the detergent phase obtained following the Triton X-114 phase separation was treated with either PI-PLC or GPI-PLD, then subjected again to the Triton X-114 phase partitioning. If digested, HMGP are expected to be released into the aqueous phase because of their loss of the diacylglycerol moiety. Figure 6 shows that all ³H-radioactivity is found in the detergent phase and virtually no ³H-activity detected in the aqueous phase, suggesting that ³H-HMGP are not likely anchored to the plasma membrane via the GPI anchor. A similar result was obtained after treatment with 50 mU/ml of GPI-PLD, as described in MATERIALS AND METHODS (data not shown).

View larger version (12K): [in this window] [in a new window]   Figure 6.   Treatment of the plasma membrane fraction with PI-PLC. Confluent HTSE cells were metabolically radiolabeled with [3H]glucosamine for 24 h, and the P2-II fraction was purified as described in Figure 1. The plasma membrane was then subjected to phase separation using Triton X-114 as described in Figure 7. The pellet recovered from the detergent phase was dissolved in the same Triton X-114 solution and incubated at 15°C for 5 h in the presence of 15 mU/ml of PI-PLC as described in MATERIALS AND METHODS. At the end of incubation the solution was phase-separated at 37°C, and individual phases were adjusted to 0.1% SDS, heat-boiled, and finally applied to a Sepharose CL-4B column pre-equilibrated with 50 mM sodium acetate containing 0.1% SDS. Open circles: aqueous phase; closed circles: detergent phase.

View larger version (13K): [in this window] [in a new window]   Figure 7.   CsCl equilibrium density gradient centrifugation of the membrane HMGP. The membrane HMGP or Vo metabolically double-radiolabeled with [3H]palmitic acid and [14C]glucosamine were obtained as described in Figure 3. The radiolabeled HMGP preparation was mixed with CsCl (a starting density of 1.5 g/ml) and centrifuged as described in MATERIALS AND METHODS. Note that both 3H (closed circles) and 14C (open circles) radioactivities comigrate, suggesting the presence of covalent binding of lipids to the membrane HMGP. Open triangles represent the density of CsCl.

CsCl Equilibrium Density-Gradient Centrifugation

After CsCl equilibrium density-gradient centrifugation of the membrane HMGP (double-radiolabeled with [³H]palmitic acid and [¹⁴C]glucosamine), radioactivity was found in two major peaks, one with a density of 1.5 g/ml and another with 1.3 g/ml, and the profile of ³H-radioactivity was virtually superimposable on that of ¹⁴C-radioactivity (Figure 7).

Sepharose CL-2B and DEAE-Sephacel Column Chromatography

When ³H-HMGP from spent media and the plasma membrane were subjected to chromatography on a Sepharose CL-2B column, 47% of the secreted and 14% of the membrane ³H-HMGP were excluded (Figure 8). Thus, HMGP in the plasma membrane are also heterogeneous in size and their average size seems to be smaller than that of secreted HMGP. The elution profile of DEAE-Sephacel anion exchange column chromatography (Figure 9) shows that 59% of the total ³H was a pass-through (peak I), whereas 41% was eluted with NaCl concentrations ranging from 0.15 to 0.5 M (peak II) with a peak fraction at 0.3 M. 

View larger version (14K): [in this window] [in a new window]   Figure 8.   Sepharose CL-2B column chromatography of HMGP. The 3H-Vo sample obtained from Figure 3 was applied to a Sepharose CL-2B column eluting with 50 mM sodium acetate, pH 7.2, containing 0.1% SDS. Open circles: spent medium Vo sample; closed circles: membrane Vo sample.

View larger version (12K): [in this window] [in a new window]   Figure 9.   DEAE-Sephacel ion exchange chromatography of the membrane HMGP. The membrane 3H-HMGP obtained from Figure 3 was applied to the anion exchange column as described in MATERIALS AND METHODS. The second peak appeared with NaCl concentrations of 0.15 to 0.5 M, centering at 0.3 M. The dotted line represents the concentrations of NaCl.

Desialylation

Treatment of ³H-HMGP with 0.1 N sulfuric acid released ³H-radioactivity, which accounted for 44% of the total ³H radioactivity.

Immunoprecipitation with an Anti-Muc-1 Antibody

When the cell lysate was immunoprecipitated with an anti-Muc-1 mucin antiserum (CT-1) and the precipitate subjected to Sepharose CL-4B column chromatography, most of the ³H-radioactivity was included in the column (Figure 10A). The presence of the blocking peptide (0.1 µg/ml) during the immunoprecipitation, however, resulted in disappearance of a major peak with Kd of 0.22 with no significant change in the size of ³H-V_o, which constitutes less than 4% of the total counts/min of all the eluted fractions (Figure 10A); this profile remained the same with higher concentrations (0.2 µg/ml and 0.4 µg/ml) of the blocking peptide. On the other hand, when the membrane ³H-HMGP was immunoprecipitated in the presence or absence of the blocking peptide, most of the ³H activity was found in the supernatants (Figure 10B) with little or no ³H in the precipitate of either sample (data not shown). This result was reproduced in two additional experiments.

View larger version (12K): [in this window] [in a new window]   Figure 10.   Sepharose CL-4B gel-filtration column chromatography of the cell lysate or the plasma membrane HMGP following immunoprecipitation with an anti-Muc-1 mucin antibody. Both the cell lysate and the membrane HMGP of confluent HTSE cells were incubated with an anti-Muc-1 mucin antibody (CT-1) in the presence or absence of a blocking peptide followed by immunoprecipitation with Protein A-agarose beads as described in MATERIALS AND METHODS. The resulting immunoprecipitate sample from the cell lysate (A) and the supernatant sample from the membrane HMGP (B) were applied to Sepharose CL-4B columns. The amount of 3H activity immunoprecipitated from the 3H-HMGP was negligible. Open circles: with the blocking peptide; closed circles: without the blocking peptide.

SDS-PAGE

As seen in Figure 11A, the glycoconjugates immunoprecipitated with the CT-1 antibody showed a smear starting from the near origin down to about 170 kD. On the other hand, the membrane HMGP remained at the origin. Extension of PAGE from 2 h (Figure 11A) to 6 h (Figure 11B) revealed very little, if any, overlap between the immunoprecipitated sample and the HMGP, indicating that most of the Muc-1 mucins, if not all, are smaller in size than the HMGP of the plasma membrane.

View larger version (62K): [in this window] [in a new window]   Figure 11.   Comparison of the electrophoretic mobility of 3H-HMGP and 3H-Muc-1 mucins on SDS-polyacrylamide gels. Both the immunoprecipitated 3H sample from Figure 10 (lane 1) and the membrane 3H-HMGP obtained from Figure 3 (lane 2) were subjected to 5% SDS-PAGE with a 3% stacking gel at 100 V for 2 h (A) and 6 h (B) as described in MATERIALS AND METHODS. 3H activities of the samples applied in A were 28,000 and 15,000 cpm for lanes 1 and 2, respectively, whereas those in B were 16,000 and 15,000 cpm. Myosin of rabbit muscles was used as a prestained size marker for 205 kD.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

To identify the HMGP on the cell surface, we first purified the plasma membrane by sequential and differential centrifugations (Figure 1). The purity of the plasma membrane was assessed by three criteria: (1) the presence of marker enzymes, (2) their specific activity, and (3) the degree of cross-contamination with other membrane markers. Most of the activities of the plasma membrane enzymes (alkaline phosphatase and 5'-nucleotidase), the markers used in this experiment, were found in the P2-II fraction (Figure 2), indicating that this fraction is highly enriched with the plasma membrane. Because 5'-nucleotidase is present in both the plasma and the nuclear membranes, whereas alkaline phosphatase is found only in the plasma, the latter has been chosen to be a better marker for the plasma membrane (11). The specific activity of alkaline phosphatase (848.5 nM × min¹ × mg protein¹) in the P2-II fraction of HTSE cells was within the range of those reported for the apical membrane of bovine tracheal epithelial cells (9). This value was 55 times greater than that of the crude homogenate, which is already 10- to 20-fold higher than the values reported for renal and intestinal luminal membranes (25, 26). The P2-II fraction contained very little or no enzyme activities of the Golgi, the rough endoplasmic reticulum, and the mitochondria, but about 5% of the lysosomal enzyme activity (Figure 2). On the basis of this purity data, we conclude that the P2-II fraction is highly enriched with the plasma membrane and contains relatively small degrees of contamination with other membranes.

We then examined whether P2-II fraction contains HMGP. Mucins are generally defined as HMGP with O-glycosidic linkage, which are excluded from the Sepharose CL-4B gel filtration column (17). Therefore, we first applied the P2-II fraction to a Sepharose CL-4B column eluting with a detergent-containing buffer; the presence of a detergent in the buffer has been shown to be crucial for dissociation of hydrophobic macromolecules from mucins (23). Figure 3 shows the profile of Sepharose CL-4B column chromatography of the P2-II fraction. To determine whether V_o fractions contain some proteoglycans, we treated the V_o with various proteoglycan-digesting enzymes using the same protocol we used previously for secreted V_o fractions (17): chondroitinase ABC for hyaluronic acid, dermatan sulfate, and chondroitin sulfate proteoglycans; heparitinase for heparan sulfate proteoglycan. The V_o fractions were totally resistant to either chondroitinase ABC (Figure 4A) or heparitinase (data not shown). Thus, total resistance to these enzymes indicates that they are not those proteoglycans. On the other hand, complete degradation of these glycoconjugates by pronase (Figure 4B) indicates that these are glycoproteins.

To see whether the V_o fractions contain O-linked glycoproteins, the V_o sample was subjected to the reductive mild alkaline hydrolysis, which was shown to deglycosylate O-linked oligosaccharides of mucins (27). Figure 4C shows that ³H-glycoconjugates in the V_o fractions were completely digested by the alkaline borohydride treatment. Thus the result indicates that the plasma membrane contains HMGP.

The plasma membrane ³H-HMGP were further characterized as follows. We first determined whether these ³H-HMGP are integral membrane glycoproteins. Triton X-114 is frequently used to distinguish various membrane glycoproteins between integral and external glycoproteins (18). Figure 5A shows that most of the membrane HMGP are partitioned in the detergent phase, indicating that they are mostly integral glycoproteins. In contrast, secreted mucins were found mostly in the aqueous phase (Figure 5B). We then examined whether they are anchored to the membrane through GPI linkage. The GPI anchor has been shown in a number of macromolecules and is generally susceptible to enzymes such as PI-PLC or GPI-PLD (19). If the membrane HMGP is bound to the plasma membrane through the GPI linkage, treatments with these enzymes should result in a decrease in the ³H-V_o of the detergent phase with a concomitant increase in the ³H-V_o of the aqueous phase. Figure 6 shows that the enzyme treatment did not affect the partitioning of the ³H-V_o fractions, indicating that ³H-HMGP in the plasma membrane are not anchored via GPI. We thus concluded that the plasma membrane HMGP are very likely integral or transmembrane glycoproteins.

The plasma membrane ³H-HMGP were then compared with secreted mucins in some of the characteristics previously reported on secreted mucins (17, 23)the distributions of the buoyant density, the size, and the charge. The distribution of the buoyant density of the plasma membrane ³H-HMGP (Figure 7) was almost identical to that of secreted mucins (23). The low-density HMGP is likely due to the association of lipids and therefore might be similar to the ones which are releasable by dirhamnolipid, a toxin from Pseudomonas aeruginosa, as recently reported by Fung and coworkers (28). On the other hand, both the size (Figure 8) and charge distribution (Figure 9) seem somewhat different between these two mucins. The overall greater size of secreted mucins, however, does not seem to be a result of aggregation because a highly dissociative condition was used for the column elution (23). The slightly greater acidity of the membrane HMGP is likely due to a difference in ³H-sialic acid contents between the membrane HMGP and secreted mucins44 and 20-30% (17) of the total ³H activity, respectivelyand/or a difference in sulfation (data not available). These biochemical characteristics of the plasma membrane and secreted mucins are summarized in Table 1.

In light of the fact that these membrane HMGP are not only the cell-surface mucins but also highly sialylated, it was suspected that they might be the product of the Muc-1 mucin gene which was originally identified in breast and pancreatic cancer cells (27, 29). The gene is expressed not only in the cancer cells but also in various secretory epithelial cells, including the airway (5, 24). We have recently shown that the present HTSE cells also express the Muc-1 gene at confluence (6), and the deduced amino-acid sequence of the Muc-1 complementary DNA revealed the presence of a transmembrane domain (6, 30, 31). To identify Muc-1 mucins from HTSE cells, the whole cell lysate was immunoprecipitated with an anti-Muc-1 mucin antibody (CT-1) (24) in the presence or absence of a blocking peptide (24), and the immunoprecipitated sample was subjected to Sepharose CL-4B column chromatography. This antibody recognizes the cytoplasmic tail of Muc-1 mucins and has been shown to cross-react with Muc-1 mucins from various species (24). Figure 10A shows that Muc-1 mucins are included in the column but not excluded. To confirm that the V_o fractions or ³H-HMGP are not Muc-1 mucins, we performed two additional experiments as follows. First, we examined whether the membrane ³H-HMGP bind specifically to CT-1. When the plasma membrane ³H-HMGP sample (3,000 cpm) was immunoprecipitated with CT-1 in the presence and absence of the blocking peptide, the ³H-radioactivity of the final precipitates were almost negligible (less than 50 cpm) in both cases. Lack of ³H-activity in the precipitates was not due to the final washing step with water because the result was exactly the same without the final wash (data not shown). On the other hand, V_o profiles of the resulting supernatant samples were virtually superimposable (Figure 10B). Therefore, it appears that the small amount of counts per minute present in the V_o in Figure 10A might be a result of nonspecific binding. Second, we compared the sizes of these two samples using SDS-PAGE. Figure 11 shows that, while HMGP are at the origin of the running gel, Muc-1 mucins are distributed from about 170 kD to near the origin. The extended PAGE seems to show little or no overlap between the HMGP and Muc-1 mucins. These results, however, do not conclusively rule out the presence of some Muc-1 mucins in the HMGP because "high molecular-mass" Muc-1 mucins as previously reported in a human breast cancer cell line (139H2) (32) that are not immunoprecipitable with the CT-1 antibody might be present. Nevertheless, the present data seem to indicate that most, if not all, of the membrane HMGP are not Muc-1 mucins.

In view of all of the biochemical and physical characteristics examined here, the plasma membrane HMGP appear to be somewhat similar to the secreted mucins (17), albeit with some differences in both the size and the charge of the molecules. It is possible that the membrane HMGP are released from the cell surface, perhaps by some unknown proteolytic mechanisms (3, 33), to become part of the secreted mucins. It is also possible that they derive from the secretory granules as previously proposed (1). In this model, mucins are packaged inside the secretory granules or vesicles in either free or membrane-bound forms. During exocytosis, mucins in the free form are released into the extracellular fluid, whereas mucins in the membrane-bound form remain bound to the apical surface of the cells. Further work is necessary, however, to confirm this possibility. Given that these are extremely large glycoproteins on the cell surface that are not only highly glycosylated but also negatively charged, it might be possible that they play an important role in the epithelial cell physiology and pathology. Possible functions of these cell-surface HMGP are currently under investigation.