Departments of 2Surgery, 3Pharmacology, 4Medical Education, 5Microbiology, and 6Immunopharmacology Research Group, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 376140575, USA and 7Department of Chemistry, Tulane University, New Orleans, LA 70115, USA
Received on December 21, 1998; revised on November 1, 1999; accepted on November 7, 1999.
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Abstract |
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Key words: binding/glucan/macrophage/receptor/polymer
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Introduction |
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Several reports suggest that specific physicochemical parameters, such as primary structure, solution conformation, molecular weight and/or polymer charge may play a role in determining whether and with what affinity (13)-ß-D-glucans bind to macrophage receptor(s) and modulate immune function. However, the relationship between (13)-ß-D-glucan physicochemical parameters and receptor ligand interaction has not been defined. This was due in part to the lack of well-characterized (13)-ß-D-glucan polymers with varying molecular weights and conformational structures.
We have demonstrated that aqueous SEC/MALLS/DV can be employed to establish molecular mass moments, r.m.s. radii and polydispersity of water soluble (13)-ß-D-glucan biological response modifiers (Mueller et al., 1995). In addition, we have reported on the application of SEC/MALLS/DV to establish the relationship between molecular mass and polymer size (Mark-Houwink and
values) in order to gain insights into the solution structure of (13)-ß-D-glucans (Mueller et al., 1995
). The purpose of this investigation was to characterize a variety of (13)-ß-D-glucan and non-glucan polymers in order to accurately establish molecular mass moments and to gain insights into the solution conformation and compare and contrast the effect of glucan polymer structure and conformation on receptor binding affinity.
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Results |
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13C-n.m.r. analysis of polymers
Figure 1 shows representative 13C-n.m.r. analyses of three (13)-ß-D-glucans employed in the competition studies. Carbon assignments are given above each of the major glucose peaks. We observed six major peaks associated with the glucose polymer backbone of the (1
3)-ß-D-glucans (Figure 1). Curdulan, a nonbranched single helical (1
3)-ß-D-glucan, was selected as the glucan polymer control. The curdulan spectra show six clearly defined carbon peaks which are diagnostic for (1
3)-ß-D-glucans (Williams et al., 1991a
). Glucan phosphate, a predominantly nonbranched single helical polyelectrolyte, also shows the characteristic (1
3)-ß-D-glucan pattern (Williams et al., 1991a
). In agreement with previous results (Pretus et al., 1991
), the 13C-n.m.r. spectrum of scleroglucan shows two distinct patterns of glucosyl signals. The oligosaccharide backbone appears as a set of five broad signals, which correspond well with the curdulan spectrum (Figure 1). The prominent C-3 triplet of scleroglucan is indicative of a highly branched (1
3)-ß-D-glucan polymer. The triplet indicates C-3 carbons in three different magnetic environments, i.e., the C-3 carbon in the glucose monomers of the backbone, the C-3 carbon in the glucose monomers of the backbone which have (1
6)-ß-linked branches and the C-3 carbon in the (1
6)-ß-linked glucose branch. The set of narrow line-width peaks correspond to the C-2 and C-4 carbons of the side chain glucosyl units (Pretus et al., 1991
). The scleroglucan spectrum is consistent with a (1
6)-ß side chain branch on average every third glucose subunit along the polymer backbone (Pretus et al., 1991
). Laminarin is a well-characterized (1
3)-ß-D-glucan with (1
6)-ß-side chain branching on average every tenth glucose subunit along the polymer backbone (Williams et al., 1991a
). The laminarin 13C-n.m.r. spectrum is in close agreement with previous reports on this polymer (Williams et al., 1991a
).
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Discussion |
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Glucan sulfate and glucan phosphate are polyelectrolytes (Williams et al., 1991a,b). Laminarin, schizophyllan and scleroglucan are neutral polysaccharides (Pretus et al., 1991
; Mueller et al., 1995
; Williams et al., 1991a
). Laminarin, schizophyllan and scleroglucan exhibit varying degrees of (1
6)-ß side chain branching (Pretus et al., 1991
; Williams et al., 1991a
b). Even though we were dealing with a small number of well-characterized polymers there were several distinct observations that emerged. The data are characterized by very complex receptor ligand interactions which are indicative of multiple glucan binding sites. The binding/uptake of radiolabeled glucan phosphate was characterized by a single dissociation constant. Unlabeled glucan phosphate and schizophyllan competed for binding with characteristics of a single ligand-receptor interaction. Competition of radiolabeled glucan phosphate by scleroglucan and glucan sulfate could only inhibit ~40% of control binding/uptake, while laminarin inhibited ~60%. The results of the competition experiments are consistent with a (1
3)-ß-D-glucan binding interaction occurring at two different sites. Glucan phosphate and schizophyllan interact nonselectively with the two sites. Scleroglucan and glucan sulfate interact preferentially with one site (40% of the glucan phosphate binding) while laminarin appears to interact with the other site (60% of the glucan phosphate binding). These sites may represent separate extracellular carbohydrate receptors, binding sites or different saturable intracellular compartments to which carbohydrates are directed.
Glucan sulfate and glucan phosphate showed significantly lower binding affinities than did the neutral polysaccharides, laminarin, schizophyllan, and scleroglucan. This suggests that the presence of the charged species on the polymer may alter binding affinity. All of the neutral polysaccharides exhibited branching frequencies that were greater than the polyelectrolyte glucans. In general, the receptor showed significantly greater affinity for the branched neutral polysaccharides. Schizophyllan and scleroglucan are similar in that both of these polymers have a branching frequency of approximately one branch per every third glucose subunit along the (13)-ß-D-linked polymer backbone (Pretus et al., 1991
). A number of reports suggest the bioactivity of (1
3)-ß-D-glucans is related to the degree of side chain branching (Suzuki et al., 1988
; Kurachi et al., 1990
; Kiho et al., 1992
; Nemoto et al., 1993
; Chiba et al., 1996
). However, we observed dramatic differences between the binding affinity of schizophyllan (IC50 = 11 µM) and scleroglucan (IC50 = 23 nM) even though their branching frequency was very similar. When we compared the binding affinity of laminarin (1:10 branching) versus schizophyllan (1:3 branching), we observed a modest (21 µM vs. 11 µM) but significant difference. Thus, we conclude that branching frequency may enhance the affinity of the polymer for the U937 glucan receptor. We also noted that the polymers with the greatest molecular weight (i.e., schizophyllan and scleroglucan) exhibited higher binding affinities. Kojima et al. (Kojima et al., 1986
) have reported that the anti-tumor activity of schizophyllan is molecular weight dependent. This may reflect differences in pharmacokinetics rather than binding affinity. In our study the contribution of molecular weight cannot solely account for the differences since the binding affinity of schizophyllan was 11 µM and scleroglucan was 23 nM. By far the most significant difference in binding affinity appeared to strongly correlate with solution conformation. In this case, solution conformation refers to the tertiary structure which the polymer assumes in aqueous media. We examined the solution conformation by establishing the linear scaling relationships for each glucan as described by Mueller et al. (1995)
. Scleroglucan was unique in that it had a highly ordered solution conformation. This indicates that the predominant solution conformation of scleroglucan is a rigid triple helix as compared to the other glucans which show scaling relationships that suggest a single helical solution conformation (Mueller et al., 1995
). Thus, the dramatic difference between the binding affinity of the receptor for scleroglucan and the other glucan polymers seems to involve the more rigid solution structure. However, we cannot discount the effects of molecular mass on this polymer system.
Kulicke et al. (1997) investigated the correlation between immunological activity, molar mass and molecular structure of various (1
3)-ß-D-glucans. They studied the effect of glucans on superoxide anion production and TNF
release from human peripheral blood mononuclear cells (Kulicke et al., 1997
). Interestingly, they reported that low molar mass glucans increased TNF
release and superoxide anion release when compared to high molar mass glucans (Kulicke et al., 1997
). Further they stated that "helical structures were not essential" or advantageous for induction of immunologic activity (Kulicke et al., 1997
). However, the molar mass ranges of the glucans studied by Kulicke et al. were narrow, i.e., 1 x 105 to >2 x 106 g/mol, the glucan concentrations employed in the in vitro experiments was higher than reported elsewhere (Pretus et al., 1991
; Lowman et al., 1998
), and the parameters evaluated were not specific. We examined a broader molecular weight range of water soluble glucan polymers (103 to 106). We also examined neutral and polyelectrolyte glucans and the effect of branching frequency. More importantly, we employed a specific ligand receptor interaction and noted that the triple helical solution conformation of scleroglucan may be an important determinant with regard to glucan ligand macrophage receptor interaction.
The nature of the glucan receptor(s) is unknown. Our data clearly demonstrate the existence of at least two specific glucan binding site(s) on undifferentiated CR3-U937 (Mueller et al., 1996; Battle et al., 1998
). We confirmed the lack of CR3 expression on the U937 cells employed in the present study. In addition, we have shown that glucan ligand binding to the non-CR3-U937 receptor(s) will stimulate intracellular signaling pathways which culminate in the activation, translocation and nuclear binding of immunoregulatory and pro-inflammatory transcriptional activator proteins (Battle et al., 1998
). This indicates that ligation of a non-CR3 receptors by glucan ligand has functional consequences that are consistent with modulation of immune function. Michalek et al. have confirmed and extended this observation by reporting that a proprietary glucan (PPG-glucan) also binds to a site distinct from CR3 (Michalek et al., 1998
). Whether PPG-glucan and the glucans described in this study bind to the same site(s) is not known. Thornton et al. (1996)
, Vetvicka et al. (1996)
, and colleagues have reported a CR3 (CD11b/CD18) glucan binding site on macrophages, neutrophils and NK cells. The glucan binding is reported to be through one or more lectin sites located outside the CD11b I domain (Thornton et al., 1996
; Vetvicka et al., 1996
). Duan et al. (1994)
have also reported a ß-glucan binding lectin on NK cells which contributes to NK cell mediated cytotoxicity. Zimmerman et al. reported that lactosylceramide binds PPG-glucan and that this glycosphingolipid may be a leukocyte glucan binding moiety (Zimmerman et al., 1998
). Dushkin et al. (1996)
, Vereschagin et al. (1998)
, and colleagues have reported that a carboxymethylated glucan binds to the macrophage scavenger receptor. Thus, there may be multiple glucan binding sites on macrophages, neutrophils, and NK cells. Additional studies are required to determine the nature of the glucan receptor(s) and which receptor(s) are essential to the expression of the various immunobiological effects ascribed to (1
3)-ß-D-glucans.
Previous reports in the literature indicate that FITC-labeled glucans can be employed to assess receptor binding by flow cytometry (Ainsworth, 1994). We examined a commercially available FITC-labeled glucan ligand and were unable to document specific binding not only to U937, but also to HL-60, K562, and J774a.1 cells (data not presented). Therefore, we conclude that this FITC-glucan does not specifically stain/bind to the glucan receptor(s). This is also supported by the fact that we did not observe any competition by unlabeled glucans following flow cytometric analysis (data not shown). In an attempt to further address this issue, we prepared several FITC derivatives of glucan. None of the fluoresceinated glucans specifically bound to U937. The precise reasons for this failure to document binding of the FITC-labeled glucans are not clear. We speculate that FITC derivatization of glucan polymers results in extensive substitutions along the polymer backbone, probably at the C-6 hydroxyl, which may block specific binding sites along the polymer.
Stahl (1992) and Fearon and Locksley (1996)
have reviewed the role of carbohydrate recognition by immunocyte receptors as an important component of innate immune recognition. These authors speculate that carbohydrate recognition may have evolved as a component of innate immunity because complex carbohydrates are common constituents of microbial cell walls and membranes and are distinct from carbohydrates produced in mammalian cells (Fearon and Locksley, 1996
). Indeed, Stahl has stated that complex carbohydrates may be "ideal for specific recognition" in the induction of innate immune responses (Stahl, 1992
). In agreement with the concepts of Stahl (1992)
and Fearon and Locksley (1996)
, we propose that (1
3)-ß-D-glucan receptors may be important components of the human innate immune response recognition system. In support of this concept, Obayashi (Obayashi et al., 1992
; Obayashi, 1997
) and others (Tamura et al., 1994
, 1997; Miyazaki et al., 1995
) have reported high levels of glucans in the serum of patients with systemic fungemia and deep tissue mycoses. Further, they observed significant differences in serum glucan levels between patients with fungal infections and patients infected with bacteria which do not produce glucans (Obayashi et al., 1992
; Tamura et al., 1994
, 1997; Miyazaki et al., 1995
; Obayashi, 1997
). Since (1
3)-ß-D-glucans are not produced in mammalian systems it is assumed that the glucans detected in these patients was released from the fungal cell wall. Okamoto et al. (1998)
have recently reported the presence of circulating glucans in patients with Pneumocystis carinii infections. From the perspective of innate immune response to infection these observations suggest that the (1
3)-ß-D-glucan receptor may be involved in the recognition of circulating (1
3)-ß-D-glucan and may play a role in the immune response to systemic fungal and perhaps parasitic infections.
In conclusion, these data indicate the existence of at least two (13)-ß-D-glucan receptors on undifferentiated CR3-U937. We also observed that scleroglucan, glucan sulfate and laminarin appear to interact selectively with a single site, while other glucan phosphate and schizophyllan interact non-selectively with both sites. The data also indicate that the human U937 (1
3)-ß-D-glucan receptor(s) are specific for (1
3)-ß-linked glucopyranoses and the receptor(s) exhibit differential affinities for glucans with varying physical properties. Whether the differences in binding affinity of the receptor(s) relate to differences in expression of biological activity is not presently known. However, we now have the well-characterized ligands to investigate this intriguing question.
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Materials and methods |
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Carbohydrate polymers
We evaluated eight water soluble carbohydrate polymers. Glucan phosphate and glucan sulfate were prepared from water insoluble (13)-ß-D-glucan, isolated from S.cerevisiae as previously described (Williams et al., 1991a
). Schizophyllan (SPG, derived from S.commune) was obtained in sterile water (10 mg/ml) from Kaken Chemical Co. (Tokyo, Japan). Laminarin and mannan were purchased from Sigma Chemical Co. (St. Louis, MO). Water soluble scleroglucan was prepared according to the protocol of Pretus et al. (1991)
. Dextran was obtained from Pharmacia (Piscataway, NJ). Barley glucan was kindly provided by Dr. Peter Wood and Dr. Barry McCleary (Megazyme Ltd., Sydney, Australia). The curdulan which was used as a (1
3)-ß-D-glucan polymer control was a gift from Dr. Ragnar Rylander (Dept. of Environmental Medicine, University of Gothenburg). SEC/MALLS/DV analysis of the curdulan showed linear scaling relationships that were consistent with a single helical conformation. Table I lists the predominant glycosidic linkage and monosaccharide composition of the polysaccharides. The primary structure of each carbohydrate polymer was confirmed by variable temperature FT- 13C-n.m.r. in DMSOd6 at a concentration of 50 mg/ml as described previously (Ensley et al., 1994
; Lowman et al., 1998
). For the competitive displacement studies, stock solutions of the polysaccharides were prepared in RPMI 1640 cell culture media, filtered, and subsequently diluted over a concentration range. An FITC labeled ß-glucan (Molecular Probes, Eugene, OR) was employed for the flow cytometry experiments.
Determination of the specific refractive index increment (dn/dc)
The dn/dc values were determined with an Optilab 903 interferometric refractometer (Wyatt Technology, Santa Barbara, CA) at 25°C in 50 mM sodium nitrite mobile phase.
Characterization of water soluble polysaccharides by size exclusion chromatography/multi-angle laser light scattering/differential viscometry (SEC/MALLS/DV)
To establish molecular mass and size, polydispersity, (weight-average molecular mass Mw/number-average molecular mass Mn) and intrinsic viscosity, the polysaccharides were analyzed by SEC/MALLS/DV as previously reported (Mueller et al., 1995). The samples (~3 mg/ml) were dissolved in 50 mM sodium nitrite mobile phase. Three Ultrahydrogel SEC columns (2000, 500, and 120 Waters Corp.) were connected in series and the columns were maintained at 30°C with continuous flow of mobile phase. The system was calibrated using narrow-band pullulan and dextran standards. The weight-average molecular mass and the z-average radius of the center of gravity as an index of molecular size of the samples were determined by on-line MALLS photometry employing a DAWN-DSP-argon-ion (488 nm) MALLS photometer (Mueller et al., 1995
). Intrinsic viscosity was determined by in-line differential viscometry with a Viscotek model 200 differential viscometer (Viscotek, Houston, TX) (Mueller et al., 1995
). The Mark-Houwink or
-value for each polymer system was established with Unical software (v. 4.03, Viscotek, Houston, TX). The
-value for each polymer system was established with EASI software (v. 7.02, Wyatt Technology, Santa Barbara, CA) (Mueller et al., 1995
).
Radiolabeling of a water soluble (13)-ß-D-glucan phosphate
Water soluble (13)-ß-D-glucan phosphate was radiolabeled as previously described by our group (Mueller et al., 1996). Briefly, ~100 mg (13)-ß-D-glucan phosphate was dissolved in 1.5 ml DMSO overnight at 45°C. This solution is added to a vial containing tritiated NaB3H4 (ICN Biomedicals Inc., Irvine, CA; 25 mCi, 718 mCi/mmol). Since the tritium was introduced by reduction of the reducing terminus of the glucan phosphate polymer, a maximum of one tritium per glucan phosphate polymer was introduced.
Receptor-binding assays
Receptor binding was evaluated using the Millipore Multiscreen Assay System with 96-well-GF/C glass fiber filter plates (Millipore Corp., Bedford, MA). Displacement binding was determined in the present of a constant amount of radiolabeled ligand (15 µg/well) and increasing concentrations of unlabeled polysaccharide. The total volume was 200 µl/well. This volume was employed for all binding studies. After incubation at 37°C for 90 min, the plates were vacuum filtered and washed five times with warm serum-free RPMI 1640. The filters were then harvested and dried, and the radioactivity was determined by liquid scintillation counting (LSC 1409 Wallac Inc., Gaithersburg, MD) with a typical counting efficiency for tritiated glucan phosphate of 4550%.
Flow cytometry
U937 (1 x 106) cells were incubated with 25 µg of a commercially available FITC labeled water soluble ß-glucan (Molecular Probes, Eugene, OR) for 45 min at 37°C, 5% CO2 tension in a humidified environment. The cells were washed either three or five times with isothermic RPMI-1640 without serum protein. The control groups consisted of cells that were not treated with labeled ß-glucan or cells which were incubated with the labeled glucan and not washed with media. Competitive displacement binding was determined in the present of a constant amount of FITC-labeled ligand (25 µg) and increasing concentrations of unlabeled polysaccharides. The cells were harvested by centrifugation and suspended in isothermic RPMI-1640 without serum proteins. Fluorescence was excited using an argon-ion laser (488 nm); FITC emissions were distinguished by passage of emitted light through a 560 nm dichroic mirror and a 530/25 bandpass filter. Detection was triggered by forward-angle light scatter signals.
Data analysis
Binding displacement data for (13)-ß-D-glucans were analyzed by unweighted non-linear regression using models of one and two site competitive displacement (GraphPad Prism v. 2.1, San Diego, CA). Maximum (100%) binding was fixed to that seen in the absence of competing carbohydrates and nonspecific binding
0. Models were used to estimate the apparent binding parameters (% binding, IC50) for each site. Sequential F-testing was used to decide if a more complex model (e.g., two vs. one site) was justified. Following a significant F-test in a 1-way ANOVA, IC50 values for different glucan groups (Table III) were compared with the least significant difference procedure. A p value of
0.05 was considered significant.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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