©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Biochemical Properties of the Ligand-binding 20-kDa Subunit of the -Glucan Receptors on Human Mononuclear Phagocytes (*)

(Received for publication, August 17, 1994; and in revised form, November 14, 1994)

Tamás Szabó Julian L. Kadish Joyce K. Czop (§)

From the Department of Medicine, Harvard Medical School and the Department of Rheumatology and Immunology, Brigham and Women's Hospital, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

beta-Glucan receptors are present on mammalian leukocytes and initiate phagocytosis of particulate yeast beta-glucans, such as zymosan particles. Human monocytes and U937 cells express two membrane proteins of 180 and 160 kDa, each of which binds particulate yeast glucan through a 20-kDa polypeptide constituent. In this report, the structural composition of the two beta-glucan receptors and the biochemical properties of their polypeptide constituents were examined. The 180-kDa receptor was composed of three disulfide-linked polypeptides of 95, 60, and 20 kDa, whereas the 160-kDa receptor was a multimer of two polypeptides of 27 and 20 kDa. Unlike other receptor constituents, the 20-kDa polypeptide was nonglycosylated and focused at two distinct isoelectric points. Immunoblots of the focused polypeptides showed the two 20-kDa variants and the 95-kDa subunit to be constitutively tyrosine-phosphorylated, a feature not previously reported for receptors on human mononuclear phagocytes. Dephosphorylation of the receptor proteins resulted in the loss of antigenic phosphotyrosine without affecting the antigenicity of either 20-kDa variant for the anti-idiotypic antibody to beta-glucan receptors. Separate analysis of the 160-kDa receptor showed it contained both variants of the 20-kDa polypeptide. Thus, the 20-kDa subunit constituent of the two beta-glucan receptors is a functionally and chemically unique polypeptide with apparent microheterogeneity in its primary structure.


INTRODUCTION

Yeast beta-glucan is a carbohydrate polymer derived from the cell wall of Saccharomyces cerevisiae and composed of beta-D-glucopyranosyl residues with consecutive 1,3 and branched 1,6 linkages(1) . beta-Glucan is a major constituent of yeasts and fungi (2) and is the active pharmacological agent of zymosan particles(3, 4) . The administration of yeast beta-glucans to laboratory animals rapidly gives rise to a state of increased resistance to a variety of biologic insults through mechanisms initiated by macrophages (5, 6, 7) . Recent studies with the soluble yeast beta-glucan, Betafectin, have shown it to protect against lethal peritonitis in the rat model for intra-abdominal sepsis (8) and have demonstrated its efficacy as an anti-infective agent against postoperative infections in high risk surgical patients(9) . Unlike nearly all ligands thus far reported for mammalian cells, the yeast beta-glucans are molecules which are totally foreign to animals and, yet, are nonimmunogenic(10, 11) .

The particulate yeast beta-glucans have been extensively used in isolated cell systems because of their potent and pleiotropic immunomodulatory activating capacities. Glucan and zymosan particles stimulate rapid phagocytic responses in human monocytes, and these are inhibited selectively by soluble yeast beta-glucans(12, 13) . Ligation of monocyte beta-glucan receptors by particulate yeast glucans induces production of a broad spectrum of cellular metabolites (14) and phosphorylation of a substantial number of cellular proteins(15, 16) , some of which appear to be linked to early secretory and synthetic responses(15) . During serum opsonization, the dominant complement fragment deposited onto zymosan particles is iC3b(17) , which binds to the I domain of the alpha-chain subunit of complement receptor type 3 (CR3; CD11b)(^1)(18) . The deposition of C3 fragments on particles already displaying an independent phagocytic signal augments phagocytosis by improving contact between particle and cell(19, 20, 21, 22, 23) . A role for a glucan-binding site on CR3 has been suggested(24) . However, ligand binding studies with purified CR3 show no binding by this complement receptor to unopsonized zymosan particles or to soluble beta-glucan(25) , and functional studies with monocytes from patients genetically deficient in CR3 show no impairment in the zymosan-induced production of platelet-activating factor by a mechanism inhibitable by soluble beta-glucans(26) .

beta-Glucan receptors have been best described for human monocytes in terms of ligand specificity. Monocyte beta-glucan receptors do not recognize homopolysaccharides of mannose and galactose, or homoglucosyl polysaccharides with alpha-anomeric or beta-1,4 linkages(12) . The smallest functional ligand has been isolated and shown by mass spectrometry to be a yeast heptaglucoside(27) . Structural conformations inherent to the yeast glucan polymers are required for monocyte recognition, a feature not exhibited by mammalian receptors for other carbohydrate molecules, such as mannose (28) and galactose (29, 30) . Steps to prepare an antibody to monocyte beta-glucan receptors have focused on the ligand specificity of the receptors so as to obtain an antibody to the ligand-binding domain(31) . As schematically shown in Fig. 1, an anti-idiotypic antibody was prepared to the antigen-combining site or idiotype (Id) of a monoclonal antibody (mAb OEA10) that recognizes the biologically active epitopes on particulate yeast glucans and the purified yeast heptaglucoside. The idiotypic specificity of the anti-Id is apparent by its capacity to compete with beta-glucan ligand for mAb OEA10 and by its selective capacity to bind and block monocyte ingestion of particulate yeast glucans. Two beta-glucan receptors of 180 and 160 kDa have been isolated from human myelomonocytic U937 cells and normal monocytes by immunoaffinity purification with the anti-Id(32) . On both types of cells, the two receptor proteins contain a 20-kDa polypeptide subunit which expresses the anti-Id epitope and glucan-binding domain, and the 160-kDa protein presents as the immunodominant beta-glucan receptor. In this report, we present the structural composition of the 180- and 160-kDa beta-glucan receptor proteins on U937 cells and characterize the polypeptide subunit chains of the two multimeric proteins. Each polypeptide subunit exists as a distinct structural domain maintained by disulfide bonds, and the 20-kDa glucan-binding domain presents as two allelic variants differing in isoelectric points.


Figure 1: Schematic representation of a binding site-specific antibody for beta-glucan receptors. Soluble and particulate yeast beta-glucans are recognized by plasma membrane receptors (A) and by mouse mAb OEA10 (B). An antibody, prepared to the antigen-combining region or Id of the mouse monoclonal, binds to the ligand-binding region of beta-glucan receptors (C).




EXPERIMENTAL PROCEDURES

Materials

Acrylamide, SDS, glycine, ampholytes, nitrocellulose, and all other electrophoretic supplies were obtained from Bio-Rad; cystatin from Aldrich; pepstatin, leupeptin, phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, Nonidet P-40, sodium thioglycollate, periodic acid solution, Schiff's reagent, and general chemicals from Sigma; and the human myelomonocytic U937 cell line was from the American Type Culture Collection (Rockville, MD).

Cell Culture

U937 cells were cultured in tissue culture flasks (Costar Corp., Cambridge, MA) containing RPMI 1640 medium (Life Technologies, Inc.) and 10% heat-inactivated (56 °C for 30 min) calf serum (Life Technologies, Inc.). The cell cultures were incubated at 37 °C in a humidified atmosphere of 5% CO(2) and either harvested during logarithmic growth by centrifugation or used as inoculum for scale-up production of cells in spinner flasks. The cells were extensively washed in 0.01 M phosphate-buffered saline (PBS), pH 7.4, counted on a Coulter counter (Coulter Electronics, Hialeah, FL), and assessed for viability by trypan blue exclusion, which was >95%.

Antibodies

Nonimmune rabbit IgG (Miles, Inc., West Haven, CT), rabbit IgG anti-phosphotyrosine (Upstate Biotechnology Inc., Lake Placid, NY), and goat IgG anti-rabbit F(ab`)(2) (Cappel Laboratories, Westchester, PA) were purchased; rabbit IgG anti-Id (31) and mouse IgG1 mAb BD4 anti-fibronectin (33) were prepared and purified as described elsewhere(31, 33) . The preparation of mouse IgG1 mAb ACIA12 was carried out as previously described(31) . BALB/c mice were immunized with 25 µg of purified U937 cell beta-glucan receptor proteins, hybridomas were prepared by polyethylene glycol fusions with single cell suspensions of spleen cells and plasmacytoma Sp 2/0, and positive antibody-secreting hybridomas were identified with purified receptor in a solid phase radioimmunoassay. The antibodies were raised in spinner cultures and purified by affinity column chromatography with rat mAb AHF5 anti-mouse light chain antibodies that had been coupled to Sepharose 4B (Pharmacia Biotech Inc.) by cyanogen bromide activation (34) . mAb ACIA12 immunoprecipitated the 160-kDa but not the 180-kDa beta-glucan receptor from detergent lysates of surface-radioiodinated U937 cells (Fig. 2) and recognized conformational determinants in the receptor that were lost by reduction of disulfide bonds.


Figure 2: Immunospecificity of mAb ACIA12 for the 160-kDa beta-glucan receptor. Detergent-soluble proteins from surface-radioiodinated U937 cells were sequentially immunoprecipitated for 1 h at 4 °C with mouse IgG1 (mAb BD4), mouse IgG1 mAb ACIA12, and the rabbit IgG anti-Id (left panel), or were preadsorbed with rabbit IgG and the anti-Id before immunoprecipitation with mAb ACIA12 (right panel). The immunoadsorbed proteins were eluted, resolved in nonreduced samples by SDS-PAGE in 5-15% acrylamide gradient gels, dried, and detected by exposure to x-ray film. Variable amounts of residual 180-kDa protein were present in cell samples heavily surface-labeled and immunoprecipitated by mAb ACIA12, suggesting a close association between the two beta-glucan receptor proteins in the cell membrane. Mobility and size of prestained protein standards are indicated.



Protein-coupled Sepharose Beads

Bovine serum albumin (BSA), nonimmune rabbit IgG, anti-Id, mAb BD4, and mAb ACIA12 were each coupled in 0.1 M phosphate buffer, pH 7.0, at a concentration of 4 mg of protein/g of activated CH-Sepharose beads (Pharmacia) with coupling efficiencies of 80-90%.

Radiolabeling of Proteins and Cells

Antibodies were radiolabeled to specific activities of 1-2 times 10^6 cpm/µg by the chloroglycoluril method (35) in tubes coated with 50 µg of IODO-GEN (Pierce) and containing 100-200 µg of IgG protein and 1 mCi of carrier-free NaI (NEN-DuPont). Iodinated antibody was separated from free iodide by filtration through a 5-ml Excellulose desalting column (Pierce) in PBS and 0.02% NaN(3).

The surface-specific method described for lactoperoxidase (Sigma) and H(2)O(2)(36) was used to radiolabel 2 times 10^7 U937 cells in 1 ml of PBS with 1 mCi of NaI at 4 °C for 15 min. The labeled cells were washed, lysed at 4 °C for 1 h in PBS containing 1% Nonidet P-40, 5 mM diisopropyl fluorophosphate, 2 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin, and 1 µM leupeptin (lysis buffer), and centrifuged at 12,000 times g for 30 min at 4 °C. The detergent-soluble fractions were precleared with BSA-Sepharose beads for 18 h at 4 °C and subjected to sequential immunoprecipitation(32) .

beta-Glucan Receptor Purification

Scale-up purification of the 180- and 160-kDa beta-glucan receptors was performed by immunoaffinity chromatographic procedures previously published for smaller preparations(32) . U937 cells were raised in spinner flasks, washed, lysed at a cell density of 5 times 10^6 cells/ml in lysis buffer, and centrifuged to remove detergent-insoluble materials. The detergent-soluble fractions were supplemented with 0.02% NaN(3) before sequential passage through columns of Sepharose 4B, nonimmune rabbit IgG-Sepharose, and rabbit IgG anti-Id-Sepharose at a flow rate of 20 ml/h at 10 °C. The columns were washed in PBS with azide until baseline levels were established. The anti-Id column was then washed in azide-free PBS, and the bound materials were eluted with 0.1 M glycine, pH 2.5. The eluted proteins were concentrated under nitrogen by positive pressure filtration on a PM-10 membrane (Amicon Corp., Danvers, MA), dialyzed against 0.1 M NaCl in 1 mM phosphate buffer, pH 7.0, and stored in aliquots at -70 °C. The average yield of receptor protein from two large preparations was about 500 µg/10 lysed U937 cells as measured by the Bio-Rad microassay for protein with BSA as the reference standard.

SDS-Polyacrylamide Gel Electrophoresis (PAGE)

Protein samples for SDS-PAGE were electrophoresed in 1.5-mm discontinuous slab gels with a 3% gel stacked on a 5-15% acrylamide gradient resolving gel(37) . The protein samples were denatured by heating at 100 °C for 5 min in Laemmli sample buffer containing 1% SDS with and without 100 mM dithiothreitol. The mobilities of mixtures of prestained protein standards (Life Technologies, Inc.) containing myosin (200 kDa), phosphorylase b (97 kDa), BSA (68 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), beta-lactoglobulin (18 kDa), and lysozyme (14 kDa) were used as markers.

For nonreducing/reducing two-dimensional SDS-PAGE, nonreduced samples of affinity-purified proteins were resolved by electrophoresis and gel strips, 11 times 2 cm, containing the resolved proteins were excised from the gel. The gel strips were incubated at 21 °C for 20 min in sample buffer containing 2% SDS and 10 mM dithiothreitol and inserted into a 13-cm sample well of the second gel. Prestained standards were loaded into a separate 7-mm well, and the running buffer for the second gel contained 0.1 mM sodium thioglycollate. The separated proteins were detected by silver staining as recommended by the manufacturer (Bio-Rad).

Immunoblotting

Proteins resolved by two-dimensional gel analysis were electrophoretically transferred onto nitrocellulose and analyzed by the Western blot method described (33) with 10 µg/ml anti-Id or 4 µg/ml anti-phosphotyrosine and 10^5 cpm/ml goat anti-rabbit F(ab`)(2). The primary and secondary antibodies were diluted in 0.01 M Tris, 0.15 M NaCl, 0.02% NaN(3), 2% BSA, pH 7.4, and blots of receptor proteins probed with nonimmune rabbit IgG and the labeled secondary antibody were negative by radioautography.

Isoelectric Focusing

Two-dimensional electrophoresis with isoelectric focusing in the first dimension and SDS-PAGE in the second was carried out in slab gels as previously described(38) , except that the final concentration of ammonium persulfate was 2.4% (w/v) and the gel overlay was distilled water. The focusing gels contained 5% acrylamide, 8 M urea, 1% 3/10 ampholytes (w/v), and 0.5% Nonidet P-40. The protein samples were prepared in 8 M urea, 1% Nonidet P-40, 1% ampholytes, 0.5 M 2-mercaptoethanol, and 0.18% phenol red. The cathode (upper) and anode (lower) compartments contained 0.02 N NaOH and 0.085% phosphoric acid, respectively; electrophoresis was carried out at a constant voltage of 400 for 18-20 h. Strips (11 times 2 cm) containing the focused proteins were excised from the gel, rinsed, and treated for 20 min in Laemmli sample buffer containing 2% SDS and 10 mM dithiothreitol before insertion into the troughlike well of a 5-15% acrylamide gradient slab gel. A sample-free strip was cut into 1-cm segments, and the ampholytes in each were eluted with distilled water to determine the pH gradient. The gel-separated focused proteins were either transferred onto nitrocellulose for immunoblot analysis or fixed and stained with Coomassie Blue.

Carbohydrate Analyses

For deglycosylation of Asn-linked oligosaccharides, 12 µg of purified receptor protein were denatured by boiling for 5 min in 0.1% SDS, 20 mM 2-mercaptoethanol, 0.08 M Tris buffer, pH 8.0, and digested at 37 °C for 18 h in the presence of 1.3% Nonidet P-40 and 0.8 unit of recombinant N-glycanase enzyme (Genzyme Corp., Cambridge, MA) per 50 µl of reaction mixture. Reaction mixtures of receptor protein without enzyme and parallel samples of 10 µg of ovalbumin with and without enzyme were treated in a similar fashion. The sample proteins were denatured in 8 M urea, 1% SDS, and 0.2 M 2-mercaptoethanol, incubated for 5 min in a boiling water bath, and subjected to SDS-PAGE.

For carbohydrate analysis by periodic acid-Schiff (PAS) reagent, reduced samples of purified receptor protein were separated by SDS-PAGE, electroblotted onto nitrocellulose, treated for 15 min with 1% periodic acid in 3% acetic acid, and washed with several changes of distilled water. The oxidized samples were stained for 15 min in the dark with fresh Schiff's reagent, treated for 5 min with 0.5% sodium m-bisulfite, washed, and stored in the dark. By this procedure, the carbohydrate in ovalbumin (4%) and rabbit IgG (3%) was detectable in blots of 0.1 µg of electrophoresed protein. To assess for transfer efficiency, concurrent samples of electrophoresed receptor protein were electroblotted onto nitrocellulose and stained for protein with 0.1% Ponceau S in 0.1% acetic acid.

Amino Acid Composition

Receptor proteins in reduced samples were resolved by SDS-PAGE, transferred to Immobilon polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) in 0.01 M 3-cyclohexylamino-1-propanesulfonic acid, pH 11.25, containing 10% methanol, stained briefly with Coomassie Blue, and destained. The stained bands were excised and submitted to William S. Lane, Harvard Microchemistry Facility, Cambridge, MA, for acid hydrolysis and analysis of phenylthiocarbamyl-amino acids. Analysis was carried out for 1.5, 3.8, 16.6, and 23.2 pmol of the 95-, 60-, 27-, and 20-kDa polypeptides, respectively.


RESULTS

Subunit Composition of the beta-Glucan Receptor Proteins

By SDS-PAGE under reducing conditions, the 180- and 160-kDa beta-glucan receptors present as a complex mixture of smaller reduction products (32) . To identify the subunit constituents of each receptor molecule, the receptor proteins were purified from detergent-lysed U937 cells by affinity column chromatography with the anti-Id, resolved by two-dimensional SDS-PAGE under nonreducing conditions in the first dimension and reducing conditions in the second, and detected by staining with silver. Electrophoretic separation of 5 µg of purified protein showed both receptors consisted of several disulfide-linked polypeptides (Fig. 3). The 180-kDa receptor contained polypeptides of 95, 88, 60, and 20 kDa, whereas the 160-kDa receptor consisted of two polypeptides of 27 and 20 kDa at molar concentrations of 2 and 5, respectively. Analysis of four different preparations showed variable levels of the 88-kDa polypeptide, which was absent in smaller batches, suggesting that this molecule represented a degraded form of the 95-kDa polypeptide. The concentrations of two apparent aggregates of high molecular weight also varied from preparation to preparation, and these were most prominent when large batches of cells were processed for receptor purification. Doubling the concentrations of protease inhibitors during column loading, including them in the wash buffers prior to receptor elution, and adding another thiol protease inhibitor, cystatin, to the mixture of protease inhibitors during cell lysis failed to alter the molecular nature or concentrations of the 95-(88-), 60-, 27-, and 20-kDa subunit constituents of the 180- or 160-kDa receptor. Similar two-dimensional analysis of receptors immunoprecipitated from detergent lysates of surface-radioiodinated U937 cells by the anti-Id revealed the same polypeptide constituents in radioautographs (not shown), indicating that each subunit of the 180- and the 160-kDa beta-glucan receptors was surface-expressed. The amino acid composition of the four subunit constituents of the two beta-glucan receptors is shown in Table 1. The 95- and 27-kDa polypeptides had amino acid compositions nearly identical to each other and these were more similar to the 60-kDa than the 20-kDa polypeptide subunit.


Figure 3: Subunit constituents of the 180- and 160-kDa beta-glucan receptor. Two-dimensional SDS-PAGE in 5-15% acrylamide gradient gels was performed with 5 µg of affinity-purified U937 cell receptor protein under nonreducing (NR) conditions in the first dimension and reducing (R) conditions in the second. The reference parent molecules and their protein subunits were detected by staining with silver. Analyses of four preparations of purified beta-glucan receptors showed variations in the proportions of the apparent aggregates of high molecular size and the 88-kDa subunit. The mobility of a BSA contaminant falls midpoint on the diagonal and was detectable in lanes with and without loaded sample. Mobility and size of prestained protein standards are indicated.





Charge Characteristics and Glycosylation of beta-Glucan Receptor Subunit Constituents

Two-dimensional electrophoresis with isoelectric focusing in the first dimension and SDS-PAGE in the second was carried out with 12 µg of the immunoaffinity-purified U937 cell beta-glucan receptor proteins and the separated polypeptides detected by staining with Coomassie Blue. The subunit polypeptides each focused as discrete molecules with different isoelectric points (Fig. 4). The 95-kDa polypeptide focused at pI 5.0-5.2, the 60-kDa at pI 5.6-5.7, and the 27-kDa at pI 4.8-5.1. The 20-kDa polypeptide focused as two species: a major one with a pI of 6.0-6.2 and a slightly more negative molecule with a pI of 5.7-5.9. Extending the electrophoretic separation on the focusing gel from 8,000 to 12,000 V-h did not alter the migration of the subunit constituents or result in comigration of the two 20-kDa moieties.


Figure 4: Isoelectric points of the beta-glucan receptor subunit constituents. Two-dimensional electrophoresis with isoelectric focusing in the first dimension and SDS-PAGE in the second was carried out under reducing conditions with 12 µg of immunoaffinity purified U937 cell beta-glucan receptors. The pH gradient in the focusing gel was determined by measuring the pH of ampholytes eluted into distilled water from 1-cm^2 segments of the 5% slab gel, and the focused receptor polypeptides were identified by staining the 5-15% acrylamide gradient sizing gel with Coomassie Blue. Each of the subunit polypeptides exhibited the same pI when focusing was increased from 8,000 to 12,000 V-h, indicating that equilibrium had been reached.



Charge heterogeneity of otherwise identical proteins is frequently attributable to the content of sialic acid residues in oligosaccharide side chains, many of which are covalently linked to N-glycosylation sites. The contribution of glycosylation as a source of charge heterogeneity of the 20-kDa polypeptide was approached in two ways: enzymatic removal of N-linked glycosylated units and chemical oxidation of monosaccharides followed by staining with Schiff's reagent. By the first method, 12 µg of purified beta-glucan receptor protein or 10 µg of ovalbumin were incubated for 24 h at 37 °C in the absence and presence of N-glycanase, and the resulting products were resolved by SDS-PAGE. Under these enzymatic and electrophoretic conditions, the ovalbumin control exhibited a downward shift in electrophoretic mobility of about 1,000 daltons after treatment with N-glycanase (Fig. 5). The 95-kDa polypeptide contained substantial amounts of N-linked carbohydrate, with the predominant N-deglycosylated form of this molecule having a molecular size of 76 kDa. Digestion with N-glycanase resulted in less dramatic decreases in the apparent molecular weights of the 60- and 27-kDa polypeptides and produced no detectable change in the 20-kDa polypeptide. A likely dimer of incompletely reduced 20-kDa polypeptide (32) presented as a band of 40 kDa and, as with the fully reduced 20-kDa subunit, showed no shift in electrophoretic mobility after enzymatic digestion. Carbohydrate analysis by PAS staining can potentially detect any oligosaccharide side chain of mammalian proteins irrespective of linkage or sugar modification. For this analysis, 20 µg of purified beta-glucan receptor protein were resolved by SDS-PAGE under reducing conditions, transferred onto nitrocellulose, and assessed for carbohydrate by the PAS assay. A concurrent sample of 12 µg of receptor protein was separated on the same gel, transferred onto nitrocellulose, and stained for protein. The rank order of glycosylation for the four receptor constituents was the same by PAS staining as that detected by N-deglycosylation (Fig. 6). The 95-kDa polypeptide had the highest content of oligosaccharide, whereas the 20-kDa polypeptide had no detectable carbohydrate despite its being the most prominent transferred polypeptide subunit.


Figure 5: N-Glycosylation of the beta-glucan receptor constituent polypeptides. Reaction mixtures (50 µl) of reduced samples containing 12 µg of purified receptor protein were incubated without (Control) and with 0.8 unit of N-glycanase (N-g'ase) for 24 h at 37 °C. The resulting products were resolved by SDS-PAGE and stained with Coomassie Blue. Parallel samples containing 10 µg of ovalbumin (OA) were treated in a similar fashion. Under these conditions of hydrolysis, the ovalbumin control exhibited a downward shift in electrophoretic mobility of about 1,000 daltons after treatment with N-glycanase. Mobility and size of prestained protein standards are indicated.




Figure 6: Chemical analysis of the oligosaccharide content of the beta-glucan receptor polypeptides. Samples of 20 µg of purified receptor protein were resolved under reducing conditions in 5-15% acrylamide gradient gels by SDS-PAGE, transferred onto nitrocellulose, and stained for carbohydrate (CHO) by treatment with PAS reagent. Concurrent samples of 12 µg of purified receptor proteins were stained for protein with Ponceau S. The lower level of sensitivity of the PAS method was 5-10 ng of CHO for glyco-protein standards electrophoresed and immobilized on nitrocellulose.



Constitutive Phosphorylation of the beta-Glucan Receptor Subunit Polypeptides

Constitutive phosphorylation has been described for a number of proteins, among which are several nonglycosylated proteins of the CD3 complex(39) . To determine whether the charge heterogeneity exhibited by the 20-kDa polypeptide was associated with differences in phosphorylation, 12 µg of purified receptor protein were subjected to two-dimensional separation by isoelectric focusing and SDS-PAGE, and the resolved polypeptides were probed in immunoblots with anti-phosphotyrosine. The 95-kDa subunit and both species of the 20-kDa polypeptide presented as tyrosine phosphoproteins, whereas the 60- and 27-kDa polypeptides did not (Fig. 7). Similar analysis of purified receptor proteins pretreated with alkaline phosphatase (40) before electrophoretic separation and immunodetection with anti-phosphotyrosine showed no immunoreactive molecules (not shown). The negativity of this control blot provided a convenient sample for determining whether the two species of 20-kDa polypeptide exhibited the same differential electrophoretic mobilities as their tyrosine phosphorylated counterparts. For this analysis, the blot bearing the dephosphorylated sample was washed and probed with the anti-Id. Both species of dephosphorylated 20-kDa polypeptide were detectable with the anti-Id and both were slightly shifted to more basic isoelectric points, but the charge difference between the two variants was unchanged (not shown).


Figure 7: Constitutive tyrosine phosphorylation of beta-glucan receptor polypeptides. Two-dimensional gel analysis by isoelectric focusing followed by SDS-PAGE was carried out under reducing conditions with 12 µg of purified beta-glucan receptors. The polypeptide subunits were transferred onto nitrocellulose, probed with anti-phosphotyrosine, and detected by radioautography with I-labeled goat anti-rabbit F(ab`)(2). Control blots on Immobilon treated with 1 M KOH before incubation with the primary antibody revealed reactivity by each of the three polypeptide subunits with anti-phosphotyrosine. The pH gradient of the focusing gel was determined as described in Fig. 4.



Receptor Protein Distribution of the 20-kDa Constituents

Purification of the 180- and 160-kDa beta-glucan receptors is achieved with the anti-Id by its binding the 20-kDa polypeptides in both receptor molecules. To determine whether the receptors were homogeneous in their content of 20-kDa polypeptide variants, the 160-kDa receptor was purified by immunoadsorption with mAb ACIA12 (Fig. 2) from a preparation of receptors isolated with the anti-Id. The eluted proteins were subjected to two-dimensional gel analysis by isoelectric focusing followed by SDS-PAGE and the focused constituents of the 160-kDa receptor detected by Coomassie Blue staining. The purified preparation was rendered free of the 180-kDa beta-glucan receptor by adsorption with mAb ACIA12 and the resulting 160-kDa receptor was found to be heterogeneous in its content of 20-kDa molecules (Fig. 8). Approximately 80% of the 20-kDa polypeptides focused at a pI of 6.1-6.3 and 20% at a pI of 5.9-6.0. Both of these polypeptides presented as more discrete and intense bands than the 27-kDa subunit, which focused as a more diffuse band with a pI of 4.8-5.2. By densitometry, the relative proportion of the 20- and 27-kDa polypeptides of the 160-kDa parent protein was 2:1.


Figure 8: Localization of the two species of 20-kDa polypeptides within the 160-kDa beta-glucan receptor. The 160-kDa receptor was isolated by affinity purification with mAb ACIA12 and reduced samples with 20 µg of purified protein were subjected to two-dimensional gel electrophoresis by isoelectric focusing followed by SDS-PAGE. The protein constituents in the sizing gel were detected by staining with Coomassie Blue, and the pH gradient in the focusing gel was determined as described in Fig. 4.




DISCUSSION

The present studies demonstrate the structural composition of two beta-glucan receptors on human myelomonocytic U937 cells and investigate the biochemical properties of their disulfide-linked subunit constituents. Immunologic studies on human monocyte and U937 cell beta-glucan receptors have shown both receptors to be multimeric and have in common a 20-kDa polypeptide subunit which contains the glucan binding domain(32) . The 20-kDa polypeptide is a key subunit constituent for receptor function and contains epitopes for the anti-idiotypic antibody previously shown to bind human monocytes and selectively block their phagocytosis of particulate yeast glucans(31) . As with anti-idiotypic antibodies to other receptors(41) , ligand rather than membrane protein is used to generate antibody, thereby limiting the antigenic specificity of the final product to an antibody directed against ligand-binding sites in the receptor (Fig. 1). The capacity of mAb OEA10 anti-beta-glucan to recognize a haptenic ligand (31) , the yeast heptaglucoside(27) , greatly increased the likelihood that the anti-idiotypic antibody would be reactive and specific for human beta-glucan receptors. The availability of U937 cells made it possible to prepare the large amounts of purified beta-glucan receptor protein needed for biochemical analyses. By two-dimensional analysis, each of the two nonreduced receptors was reduced to its constituent subunits, and each polypeptide constituent was detected by a sensitive silver staining technique. The 180- and 160-kDa beta-glucan receptors both presented as complex dulfide-linked structures with the composition of one, indicating it to be multivalent (Fig. 3). The 180-kDa receptor was composed of three disulfide-linked polypeptides of 95, 60, and 20 kDa and was estimated to contain these subunits at equimolar ratios. The composition of the 160-kDa receptor was limited to two polypeptides of 27 and 20 kDa, which were linked to each other by disulfide bonds at molar ratios of about 2 and 5, respectively. The presence of as many as five 20-kDa subunits in each 160-kDa receptor molecule may relate to the homopolymeric nature of the beta-glucan ligands and effect high avidity binding between beta-glucan receptors and ligands. No significant differences in the number or sizes of the receptor subunits were apparent by the inclusion of additional protease inhibitors at various points during purification, indicating that both beta-glucan receptors were cleaved and bridged by disulfide bonds in their native state. The anti-Id recognizes primary rather than conformational determinants in the 20-kDa polypeptide, making it possible to enrich for denatured or native forms of the 20-kDa subunit during receptor purification. Scale-up preparations of isolated receptor proteins, however, contained no monomeric or partially assembled 20-kDa polypeptide subunits (Fig. 3), suggesting that each of the two beta-glucan receptors is derived from a proreceptor by proteolytic processing at regulated cleavage sites rather than by post-translational assembly.

The composite biochemical properties of the 20-kDa polypeptide subunit showed it to be an unusual polypeptide and a unique constituent of beta-glucan receptors. Compositional analysis of the four polypeptide subunits from the two receptors revealed high similarities between the amino acids of the 95-kDa subunit of the 180-kDa receptor and the 27-kDa subunit of the 160-kDa receptor protein, whereas relatively little similarity in amino acid composition was apparent between the 20-kDa subunit and the three other subunit constituents (Table 1). Except for the 20-kDa polypeptide, each subunit constituent of the 180- and 160-kDa beta-glucan receptors exhibited a single isoelectric point, with the 95- and 27-kDa polypeptides focusing at nearly the same pI (Fig. 4). The 20-kDa polypeptide focused as 2 molecules with pI more basic than those of the other subunits, which may reflect its higher content of lysine residues. An average charge differential of 0.3 pH unit was expressed by the two variants of the 20-kDa subunit, the more minor of which had the lower pI and accounted for about 15-20% of the two 20-kDa molecules. Since charge heterogeneity often resides with the quantities of sialic acid residues in oligosaccharide side chains or modifications by sulfation or phosphorylation of sugar residues, glycosylation of the beta-glucan receptor constituents was the first parameter examined. The molecular size of the 20-kDa polypeptide was unaffected by hydrolysis with N-glycanase, suggesting the absence of N-linked oligosaccharides in this molecule (Fig. 5). A sensitive and more general assay for carbohydrates was devised based on PAS staining. By this method, monosaccharides containing vincinal hydroxyls are required for positivity, a criterion met by nearly all oligosaccharide chains of mammalian glycoproteins. Blots containing the 20-kDa polypeptide in amounts 80-100-fold greater than the lower level of detection of the typically glycosylated reference proteins showed no carbohydrate by PAS staining (Fig. 6), indicating that the primary structure and/or folding of the 20-kDa polypeptide precluded glycosylation. Unlike the 20-kDa polypeptide, each of the other receptor constituents was glycosylated as assessed by the enzymatic and chemical assays. In both assays, the 95-kDa polypeptide presented as the most glycosylated subunit, the 60-kDa as the least, and the 27-kDa as the most complex. These data suggested that much of the carbohydrate detected by PAS staining in these receptor constituents consisted of N-linked oligosaccharides.

Studies on phosphorylation were initiated to determine whether phosphorylation was a factor contributing to the charge heterogeneity of the 20-kDa polypeptide. Even when consideration was given to the fact that unopsonized zymosan particles induce protein tyrosine phosphorylation in human monocytes(16) , the finding that beta-glucan receptors from unstimulated cells were phosphorylated on tyrosine residues of the 95- and 20-kDa constituent subunits was unexpected (Fig. 7). As indicated in the text, dephosphorylation with alkaline phosphatase rendered these molecules free of phosphates but had little if any effect on their overall electrophoretic differences. That both variants of the 20-kDa polypeptide retained reactivity with the anti-Id suggested that phosphoamino acids were not located within the glucan-binding domain. Although constitutive phosphorylation has been reported for a number of receptors, the vast majority of these protein phosphorylations occur through serine or threonine residues. For receptors on human mononuclear phagocytes, cross-linking of FcRI (CD64) and FcRII (CD32) on U937 (42) and THP-1 (43) cells by receptor antibodies stimulates tyrosine phosphorylation, but neither phagocytic receptor appears to be constitutively phosphorylated. Constitutive rather than stimulus-inducible phosphorylation is a feature of the alpha-chains (CD11a, b, and c) of the beta2 integrin proteins (CD11/CD18), for which the phosphorylated substrate is serine (40) . Conversely, the common beta-chain (CD18) is not phosphorylated in its basal state, but treatment of human monocytes with soluble agents, such as phorbol esters, rapidly and dramatically stimulates serine phosphorylation of CD18. To our knowledge, no receptor on human mononuclear phagocytes has been shown to be constitutively tyrosine phosphorylated prior to this report.

The preparation of monoclonal antibodies to purified 180- and 160-kDa beta-glucan receptors resulted in a mAb specific for the 160-kDa receptor and provided a means for separating the two receptors (Fig. 2). Analysis by isoelectric focusing clearly showed the purified 160-kDa receptor to contain two variants of the 20-kDa polypeptide (Fig. 8). Comparison of the 20-kDa variants before and after purification of the 160-kDa protein from receptor preparations revealed no significant differences in their isoelectric points or relative proportions, thereby diminishing the possibility that each of the two receptors contained a different type of 20-kDa polypeptide with a distinguishing isoelectric point. Since isoelectric focusing is sensitive to a difference of a single changed amino acid, the charge heterogeneity observed for the 20-kDa polypeptide was a likely reflection of microheterogeneity in its primary structure. That the compositions and relative concentrations of each 20-kDa variant were comparable regardless of whether samples were stained with protein dyes or antibodies indicated them to be highly homologous constituents and occurring at fixed frequencies in the beta-glucan receptors. These data were supported by those obtained with the purified 160-kDa receptor protein, all of which are consistent with the 20-kDa polypeptides being allelic variants.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant AI23542 and by a grant from Alpha-Beta Technology, Inc., Worcester, MA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Longwood Medical Research Center, 221 Longwood Ave., Boston, MA 02115. Tel.: 617-278-0631; Fax: 617-739-7095.

(^1)
The abbreviations used are: CR3, complement receptor type 3 (CD11b/CD18); BSA, bovine serum albumin; Id, idiotype; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PAS, periodic acid-Schiff reagent; PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank Drs. David S. Adams, Gary Ostroff, and Spiros Jamas for their helpful discussions and support.


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