Characterization of a galactose-specific lectin from Actinomyces ...

Vol. 38, No. 3

INFECTION AND IMMUNITY, Dec. 1982, p. 993-1002

0019-9567/82/120993-10$02.00/0 Copyright C 1982, American Society for Microbiology

Characterization of a Galactose-Specific Lectin from Actinomyces viscosus by a Model Aggregation System MARY J. HEEB, ANN H. COSTELLO,t AND OTHMAR GABRIEL* Department of Biochemistry, Georgetown University Schools of Medicine and Dentistry, Washington, D.C. 20007

Received 19 July 1982/Accepted 20 August 1982

A simple model system has been developed in which lectin-mediated aggregation of glycoprotein-coated beads can be monitored by following the decrease in light scattering at 650 nm. Aggregation has been characterized with the lectin of Actinomyces viscosus T14V. Its dependence on pH, temperature, and stirring rate was examined, and the number of bacterial cells in relation to the number of latex beads resulting in optimal aggregation was established. This system has the advantage of permitting the study of a single ligand of defined structure. The ligand density was determined with radiolabeled glycoproteins. Under the conditions of the assay, ligand leakage was less than 3%, and ligands were not displaced from the beads by various proteins, glycoproteins, or by other components present in the assay mixture. Latex beads coated with asialofetuin aggregate upon the addition of A. viscosus T14V cells. By contrast, when asialofetuin was first extensively treated with purified galactose oxidase, no aggregation occurred. Only after reduction with NaBH4 was aggregation restored, demonstrating that galactose termini of asialofetuin are essential for the binding of A. viscosus lectin. An absolute requirement for calcium was also demonstrated. Various sugars inhibited aggregation in the following order, starting with the most effective: lactose, methyl-p-D-galactopyranoside, galactose, N-acetylgalactosamine, methyl-ot-D-galactopyranoside. Beads coated with fimbriae from A. viscosus coaggregated with neuraminidase-treated human erythrocytes and with Streptococcus sanguis cells. In each instance the aggregation was inhibited by lactose, indicating that the A. viscosus lectin is located in the fimbriae. Cells grown under different conditions differed in their effectiveness in aggregating glycoprotein-coated beads, suggesting differences in lectin density or accessibility. Two different experimental designs were used to establish the minimum ligand density for aggregation to occur. In one type of experiment, a threshold concentration was found for asialo a1-acid glycoprotein, but not for asialofetuin. With an alternate approach in which a different population of galactose residues was exposed, a threshold phenomenon was also demonstrated for asialofetuin. The importance of structural ligand features in the aggregation assay is discussed in view of these findings. Various pathogenic bacteria possess lectins hemagglutination assays. In the case of the A. which allow adherence to host cells. Most com- viscosus lectin, hemagglutination is neuraminimonly, this adherence has been studied by test- dase dependent and lactose inhibitable (9), suging hapten inhibition of hemagglutination by gesting a specificity for galactose, but it has not bacteria such as Escherichia coli (13, 14), Vibrio been possible to abolish hemagglutination by cholerae (28), Pseudomonas aeruginosa (21), chemical or enzymatic modification of galactose and Actinomyces viscosus or Actinomyces naes- termini. This paper describes a simpler aggregalundii (9, 15, 39). It is often difficult to discern all tion system where chemically defined ligands of the factors contributing to the specificity of can be adsorbed to latex beads, resulting in the lectin because of the complexity of the specific interaction between bacterial lectin and erythrocyte membrane. In addition, other cell the carbohydrate-containing ligand adsorbed to surface-related interactions independent of car- the surface of the beads. Cellular adhesion has bohydrate-lectin recognition may further com- been studied previously with latex particles to plicate proper interpretation of data obtained by investigate leukocyte surface properties (3), platelet aggregation (22), the presence of specific antigens (19), and antibody binding to Streptot Present address: School of Dental Medicine, Tufts University, Boston, MA 02111. coccus mutans (38). The system described here 993

994

HEEB, COSTELLO, AND GABRIEL

has been characterized with A. viscosus, but it can be used to screen other potential lectincontaining bacteria, to test purified glycoproteins or glycolipids as ligands, and to investigate the role of carbohydrate-mediated adhesion in bacteria-host cell interaction in other ways. A. viscosus has been implicated in periodontal disease (2) and root caries (29). It adheres either to previously colonized organisms such as streptococci (4, 7) or to epithelial cells (16) by means by a lectin-like interaction. A. viscosus T14V secretes neuraminidase and agglutinates erythrocytes in a neuraminidase-dependent, lactoseinhibitable manner (9, 15, 35, 39) by two sequential steps: (i) action of neuraminidase to unmask galactose termini, and (ii) binding of exposed galactose residues to bacterial lectin. In spite of the indirect evidence for the involvement of galactose termini, it has not been possible to alter galactose residues of asialo erythrocyte membranes by treatment with galactose oxidase or P-galactosidase and thereby prevent agglutination (R P. Ellen, E. D. Fillery, K. H. Chan, and D. H. Grove, J. Dent. Res. 58A:341, abstr. no. 999, 1979). To overcome some of the problems in studying lectin interaction with complex membrane surface structures, we explored the model system to determine the more detailed specificity requirements of the A. viscosus lectin and to confirm that the lectin is located on the bacterial fimbriae (6). We also sought to compare the characteristics of this lectin to those of other lectins which have been studied. (Preliminary reports of this work have been presented in A. H. Costello and 0. Gabriel, J. Dent. Res. 60A:548, 1981; M. J. Heeb and 0. Gabriel, Proc. Am. Chem. Soc. Div. Carbohydr. Chem. Abstr. 17, 1982; and M. J. Heeb and 0. Gabriel, Fed. Proc. 41:abstract 4456, 1982.) MATERIALS AND METHODS Materials. A. viscosus T14V was obtained from B. Hammond, University of Pennsylvania School of Dentistry, Philadelphia, Pa. Streptococcus sanguis 34 was

obtained from Floyd McIntire, University of Colorado, Denver. Glycophorin was purified from human erythrocytes by published methods (20). a1-Acid glycoprotein was a gift from Milan Wickerhauser, American Red Cross Blood Research Laboratories, Bethesda, Md. Bovine submaxillary mucin and antifreeze glycoprotein were kindly donated by Gilbert Ashwell, National Institutes of Health, Bethesda, Md. Bovine serum albumin (BSA) coupled to lactoneotetraose was donated by David Zopf, National Institutes of Health, Bethesda, Md. Isolated fimbriae from A. viscosus were a generous gift from John Cisar of the National Institute of Dental Research, Bethesda, Md. Outdated human erythrocytes (type 0) were obtained from the Georgetown University Hospital blood bank, Washington, D.C. Crystalline BSA was from Miles Laboratories, Inc., Elkhart, Ind.; radioisotopes were obtained from Amersham Corp., Arlington Heights, Ill.;

INFECT. IMMUN.

and pronase was from Calbiochem, LaJolla, Calif. Sodium borohydride was from Eastman Chemical Products, Inc., Rochester, N.Y., and disodium EDTA was from Fisher Scientific Co., Pittsburgh, Pa. Sugars, galactose oxidase, latex beads, fetuin type IV, Nacetylneuraminic acid (NeuNAc), chloramine T, and Clostridium perfringens neuraminidase type IX were obtained from Sigma Chemical Co., St. Louis, Mo. Galactose oxidase was further purified by chromatography on DE-52 cellulose (Whatman) in 0.05 M Trishydrochloride, pH 8.0. The eluted enzyme was precipitated with ammonium sulfate and stored at 4°C as an ammonium sulfate suspension. Bacterial culture. A. viscosus T14V was grown at 37°C in static aerobic cultures and, unless otherwise noted, harvested by centrifugation at 10,000 x g for 10 min at mid-log phase (about 21 h, or 50 U on a Klett colorimeter with 640 nm filter). The medium contained 0.5% each of yeast extract, tryptone (Difco Laboratories, Detroit, Mich.) and K2HPO4; 0.05% Tween 80; and 0.15% glucose. Harvested cells were washed three times in phosphate-buffered saline (PBS), pH 7.4, and were stored in PBS containing 0.02% NaN3 and 0.1% BSA. The stock suspension was adjusted to an absorbance at 650 nm (A650) of 2.0, or about 1010 cells per ml as determined in a Coulter Counter. For experiments comparing various growth phases of A. viscosus, parallel cultures were harvested at different times. Slant cultures (48 h) were gently scraped and rinsed from the agar surface. Aggregation system. Latex beads (diameter, 5.7 ±m) were suspended at 1:50 (vol/vol) in Tris-glycine saline (7.3 g of glycine and 10 g of NaCl per liter), pH 8.2, containing 0.1 mM CaC12, 0.02% NaN3, and 0.5 mg of glycoprotein per ml. The mixture was incubated at 37°C for 2 h with gentle agitation every 30 min. The suspension was then stored at 4°C. Just before use, the beads were washed three times in the same buffer, substituting 0.1% BSA for the glycoprotein. Beads were suspended in 2 ml of the wash buffer and were adjusted to an A650 of 0.7 to 1.0 with constant stirring. Bacteria were added in a small volume (10 to 40 i±l), and the progress of aggregation was followed by measuring the decrease in absorbance with time. Unless otherwise noted, the following conditions were employed: temperature, 23°C; pH, 8.2; initial A650 of beads, 0.7 to 1.0 (about 4 x 106 beads per ml); concentration of bacteria, 1.5 x 108 cells per ml. A Gilford recording spectrophotometer with a Beckman DU monochromator and a temperature-controlled cuvette compartment was used to measure the progress of aggregation. Four round cuvettes (1-dram vials; Wheaton Scientific, Millville, N.J.) containing one 5-mm stirring bar each were placed in the cuvette holder. A magnetic stirrer was placed above the sample compartment. The rate of stirring was set to 300 rpm. Radiolabeling. Specific labeling of NeuNAc residues of glycoproteins by chemical modification results in the formation of a seven-carbon analog, NeuNAc7. [3H]NeuNAc7-fetuin was prepared by mild periodate oxidation followed by treatment with NaB3H4 as previously described for ceruloplasmin (46). The specific activity was found to be 2.0 x 104 dpm/nmol of NeuNAc. Tritiated asialoglycoproteins such as [3H]asialo al-acid glycoprotein were prepared by labeling terminal galactose residues as described in the

CHARACTERIZATION OF A. VISCOSUS LECTIN

VOL. 38, 1982 1.

E 0

to 0

0 co 0 c

.02 0 0

Time

In

min

FIG. 1. Neuraminidase-dependent, lactose-inhibitable aggregation of latex beads coated with fetuin by A. viscosus T14V. Latex beads were coated with asialofetuin (A and C) or native fetuin (B) as described in the text. Washed A. viscosus cells were added to each sample (Av). In B, a sample of C. perfringens neuraminidase (N) was added to initiate aggregation. In C, lactose (L) at a final concentration of 20 mM inhibited the aggregation reaction, but in B, a higher concentration of lactose (200 mM) was required to reverse the aggregation completely.

literature for ceruloplasmin (34). Specific activities in the range of 2 x 104 dpm/nmol of galactose. For the preparation of "251-labeled materials, 20 ,.g of asialoglycoprotein was reacted with 13 p.g of chloramine T (27) and 400 ,uCi of Na125I in 65 ,ul of 0.4-M potassium phosphate, pH 7.5, for 30 s at room temperature followed by 26 ,ug of sodium metabisulfite in 20 ,ul of potassium phosphate. Low-molecular-weight reactants were removed by chromatography on a column (0.6 by 16 cm) of G-50 which had been previously treated with 0.5 ml of 20%o BSA and then washed in 0.05 M potassium phosphate, pH 7.8. The iodinated glycoprotein was collected in tubes previously coated with 20%o BSA and rinsed extensively. Specific activities in the range of 1 x 109 dpm/nmol of were

995

glycoprotein were obtained. Serum glycopeptides. Whole serum (5 ml) was treated at 37°C with 5-mg portions of pronase at 12-h intervals in 0.1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, pH 7.2, containing 10 mM calcium chloride. After 48 h of digestion, the incubation was subjected to centrifugation at 12,000 x g for 20 min. The supernatant was applied to a Sephadex G-50 column and was eluted with PBS. Phenolsulfuric acid-positive (12) fractions which were retarded by the gel were pooled, lyophilized, and dissolved in PBS. Removal of sialic acid from erythrocytes and glycoproteins. Human erythrocytes were washed three times in PBS, pH 7.4, followed by centrifugation at 300 x g for 10 min. A 50%o packed cell suspension was treated with neuraminidase (0.7 U/ml of suspension) at 37°C for 2 h with gentle agitation every 30 min. Approximately 0.5 ,umol of NeuNAc was removed per ml of packed cells, as determined by a thiobarbituric assay procedure (42). The cells were washed three times in PBS containing 0.1 mM CaCl2-0.02% NaN3 and were stored at 4°C as a 50%o suspension. Glycoproteins were treated with neuraminidase (0.3 U/10 mg of glycoprotein) for 2 h at 37°C. NeuNAc was measured by the thiobarbituric acid assay procedure to establish that between 76 and 100%o of available NeuNAc had been removed. For experiments where beads were coated with mixtures of native and asialoglycoprotein, the experiments were also performed with asialoglycoprotein prepared by treatment at 80°C for 1 h in 0.1 N H2SO4 followed by neutralization. Enzymatic modification of asialofetuin. Asialofetuin (1 mg) was treated for 66 h at room temperature with 4.5 U of purified galactose oxidase, and half of this preparation was used to coat beads. The other half was reacted for 30 min with NaB3H4, brought to pH 5 with acetic acid, and dialyzed. j-Galactosidase was added, and the mixture was dialyzed until 87% of the counts had been released. The modified glycoprotein was used to coat latex beads. Aggregation of neuraminidase-treated [3HINeuNAc7fetuin-coated beads. Beads coated with [3H]NeuNAc7fetuin were prepared and washed as usual. A sample was treated with 0.1 N NaOH to remove bound ligand, which was counted to determine ligand density (1.9 x 107 molecules of NeuNAc per bead). Beads were suspended at 1:50 (vol/vol) in the aggregation buffer,

TABLE 1. Determination of ligand density for 5.7-,um beads Ligand

Residue labeled

Molecules pH

bead (Xl1O6ya

per

Galactose 7.4 1.1C [3H]asialo a,-acid glycoprotein Galactose 8.2 0.6c [3H]asialo a,-acid glycoprotein 8.2 0.5c III-labeled asialo al-acid glycoprotein Tyrosine 7.4 0.15 l25l-labeled asialo a,-acid glycoproteinb Tyrosine NeuNAc7 8.2 1.6d [3H]fetuin Each molecule of al-acid glycoprotein contains approximately 15 galactose residues (11), and each molecule of fetuin contains approximately 12 residues (46). b Protein mixture contained unlabeled serum proteins. c Calculated effective ligand concentration on latex bead surface is 0.6 + 0.2 mM, with a concentration of (0.7 - 0.3) x 106 molecules in the volume occupied by a hollow shell, 10 nm thick, surrounding 5.7-,um diameter a

beads. d Calculated effective ligand concentration on latex bead surface is 1.3 mM.

996


and a sample was tested for counts in the supernatant and for aggregation of the beads. Neuraminidase (0.02 U/ml) was added at room temperature, and samples were taken periodically for aggregation tests and counting of the supernatant. Since this particular NeuNAc analog is a poor substrate for neuraminidase (44), its release occurred over a conveniently long period of time. Kinetic experiments. Beads were coated with defined mixtures of native and asialoglycoprotein varying from 0 to 100%o in the asialo form. From the ligand density per bead determined in other experiments (see below), an effective concentration of the asialoglycoprotein (lectin substrate) in a monomolecular layer on the surface of the beads was calculated. For 100%o asialofetuin and 100% asialo al-acid glycoprotein, these concentrations were approximately 1.3 and 0.6 mM, respectively. The rate of aggregation was determined in the presence and absence of inhibitors as a function of asialoglycoprotein concentration under standardized conditions as noted for the aggregation assay.

RESULTS Aggregation of coated beads. Beads coated with appropriate asialoglycoproteins aggregated upon the addition of washed A. viscosus cells, but beads coated with the corresponding native glycoprotein did not, unless neuraminidase was also added (Fig. 1). Lactose at 20 mM inhibited aggregation. The rate of aggregation could be determined from the slope in the linear portion of the absorbance curve. The greatest extent of aggregation occurred in 5 to 20 min. The light scattering data are similar to data reported for hemagglutination by various Actinomyces strains (9), which also demonstrated neuramini-

INFECT. IMMUN.

1.0

CaCI 2

oE 0.8 0

* EDTA .6 Av

o

I

.n .40

a

.2 AB 8 12 16 Timo In min FIG. 3. Calcium-dependent aggregation of latex beads coated with asialofetuin by A. viscosus T14V added as shown (Av). (A) Standard assay conditions; (B) addition of 5 x 10-4 M EDTA followed by 1.2 x 10-2 M CaC12. 4

dase-dependent, lactose-inhibitable aggregation. The two systems differ in that, for the hemagglutination reaction, it is possible to reverse aggregation within seconds by adding 20 mM lactose, but in the latex bead assay, reversal requires higher concentrations of lactose and occurs over a period of 4 min or longer. Ligand density. Beads coated with ligand of known specific radioactivity were washed well, and samples were counted in a gamma counter (1251) or a liquid scintillation counter (3H). The NaBH4 1.0. number of beads was determined in duplicate at E .9 Av two dilutions in a Petroff-Hauser counting chamber. From these figures, the ligand density for o .8 asialo al-acid glycoprotein averaged (7 ± 3) x Co.7 B 105 molecules per bead (Table 1, lines 1 to 3), and the density for fetuin was calculated as 1.6 o.5 x 106 molecules per bead (Table 1, line 5). When .4 D .4 serum proteins were mixed with the labeled 0 .3 glycoprotein before coating to beads, the density of radioligand decreased (Table 1, line 4). These < .2 beads were still aggregated by A. viscosus cells, \l A although at a slower rate. .1 Test for ligand leakage and exchangeability. 8 12 16 4 Beads coated with 125I-labeled asialo a1-acid Time in min glycoprotein were used to determine ligand leakFIG. 2. Effect of galactose oxidase and P-galacto- age after washing. Leakage was 3% in 3 h and sidase treatment of asialofetuin upon aggregation of 10% in 48 h at pH 8.2 and room temperature. coated latex beads by A. viscosus T14V. Galactose Leakage was greater at pH 7.4 (11% in 3 h and oxidase-treated asialofetuin was adsorbed onto latex 25% in 48 h). Beads could be stored at 4°C in the beads (A). Upon adding washed A. viscosus cells (Av), no aggregation occurred until the addition of NaBH4. glycoprotein solution used for coating and In another experiment (B), P-galactosidase-treated washed just before use. Leakage was not signifiasialofetuin was adsorbed onto latex beads and tested cantly altered by omitting BSA from the wash buffer; but since A. viscosus cells can bind to under standard aggregation assay conditions.


VOL. 38, 1982

TABLE 2. Coaggregation of fimbriae-coated beads with equal volumes of erythrocytes or S. sanguis cells Rate of aggregation

Beads mixed witha:

Human erythrocytes, type 0 ...... Neuraminidase-treated human

......

(4A65dmin) 0.002

erythrocytes ......................... 0.109 S. sanguis cells ........................ 0.080 S. sanguis + 0.15 M lactose ............. 0.006 No addition ...... 0.000 a Initial A650, 0.69 to 0.81 for all suspensions. and cross-link uncoated beads, the beads were kept saturated with protein during the aggregation tests to abolish very small artifacts owing to exposed sites on the beads. Once adsorbed to the beads, radioligands were not detectably displaced from the beads by exposure to BSA, fetuin, asialofetuin, lactose, or components present in the aggregation mixture. Optimal conditions for the model system. Aggregation occurs between pH 5 and 11, with a broad optimum around pH 8.2. Asialofetuincoated beads 1 ,um in diameter aggregated poorly, but coated beads of 5.7 to 25 gxm aggregated well, with an optimum diameter of 5.7 ,um. The rate of aggregation depended on the concentrations of both bacteria and beads. The rate increased as the initial A650 of beads was varied from 0.25 to 1.0. At a constant bead A650 of 1.0, aggregation was detectable at about 3 bacteria per bead, and the rate of aggregation increased to a maximum at about 30 bacteria per bead. Aggregation rates were approximately equal at ionic strengths of 0.05 and 0.15, but the rate was significantly less at an ionic strength of 0.55. Increase in the stir rate up to about 300 rpm promoted a faster rate of aggregation; the stir rate was maintained constant with a powerstat. Aggregation occurred between 10 and 41°C, with an optimum at 25°C. General applicability of the model. Glycoproteins terminating with beta-linked galactopyranose, either in the native form (antifreeze glycoprotein) or after treatment with neuraminidase (asialofetuin, asialoglycophorin, asialo a1-acid glycoprotein) served as ligands promoting aggregation by A. viscosus. A synthetic glycoprotein terminating in galactopyranose (BSA coupled to lactoneotetraose) served as a ligand. Alphalinked N-acetylgalactosamine termini were also recognized (asialo bovine submaxillary mucin), but glycoproteins not terminating in these sugars (ovalbumin) and a protein containing no carbohydrate (BSA) were not recognized. These results, together with the previous data, indicate that this model system is analogous to hemagglu-

997

tination by A. viscosus, demonstrating lactoseinhibitable, neuraminidase-dependent aggregation. Effect of carbohydrate modification. Although lactose-inhibitable, neuraminidase-dependent aggregation was demonstrated in the experiments just described, this cannot be considered as conclusive proof that A. viscosus lectin recognizes galactose termini. Additional independent evidence was provided by converting galactose termini to the corresponding 6-aldehydo derivative. For this purpose, asialofetuin was extensively treated with purified galactose oxidase. Electrophoresis of a sample revealed that no detectable proteolysis had occurred. Latex beads coated with this preparation did not aggregate with bacteria; however, when NaBH4 was added to the reaction mixture, restoring the galactose termini of the glycoprotein, the ability to aggregate was fully recovered (Fig. 2, curve A). This is direct evidence for galactose-dependent aggregation. In another experiment, asialofetuin treated with 3-galactosidase (87% of galactose removed) did not promote aggregation when adsorbed to beads (Fig. 2, curve B). Divalent metal requirement. Washed A. viscosus cells were mixed with beads coated with asialofetuin, and as expected, aggregation occurred (Fig. 3, curve A). When 5 x 10- M EDTA was added to the reaction mixture, aggregation was abolished. Aggregation was restored upon adding CaC12 to a concentration of 10-3 M or greater (Fig. 3, curve B). When magnesium ions were substituted for calcium ions, aggregation took place at a slower rate and to a lesser extent; manganese or zinc ions did not replace calcium (data not shown). Role of A. viscosus fimbriae. The role of fimbriae in lectin-like activity was established in various bacterial systems such as E. coli (17, 41). To investigate the possibility of fimbrial involveTABLE 3. Effect of bacterial growth conditions upon latex bead aggregation assay Growth conditions

Liquid medium Mid-log phase Late log phase Stationary phase Solid medium

Cell density at harvest (Klett U, 640 nm)

37°C

50 80 135

atie

Aggregation

(h)

(AA/min)a

21 23 40

0.075 0.089 0.133

rate

48 0.303 120 0.270 a Aggregation was carried out with 1.5 x 108 cells per ml and 4 x 106 asialo a,-acid glycoprotein-coated beads per ml. Assay conditions are described in the text.

998


0.25A a 0.20-

B

*

E 0.15A

< .10.05-

20

40

60

80

Io0

%Asialofetuin

V

1

(arbitrary

units)

FIG. 4. Effect of ligand density on rate of aggregation of fetuin-coated beads by A. viscosus T14V. Beads were coated with various mixtures of asialofetuin and native fetuin. Each type of bead was tested for the rate of aggregation (AA/min) in the standard assay with 5.0 x 107 bacteria per ml (A) and with 1.5 x 108 bacteria per ml (B). The data in a were replotted in b as a double reciprocal Lineweaver-Burk type plot, where [ is the reciprocal of the percentage of IS] 1 asialofetuin and V is the reciprocal of aggregation velocity. ment, beads were coated with isolated fimbriae from A. viscosus. These beads aggregated with human erythrocytes in a neuraminidase-dependent manner (Table 2). Fimbriae-coated latex beads also aggregated in a lactose-inhibitable manner with S. sanguis cells, which are known to coaggregate with A. viscosus cells (31, 33). This confirms that the A. viscosus lectin is located on the fimbriae, consistent with evidence obtained by Cisar et al (6), and provides an additional approach for studying or detecting the lectin. Like the aggregation of glycoproteincoated beads by whole bacteria, the aggregation of fimbriae-coated beads with S. sanguis was inhibited by lactose. Again, it was more difficult to reverse aggregation by addition of lactose than to reverse aggregation between A. viscosus T14V cells and asialo erythrocytes. Several experiments were carried out to investigate possible differences in lectin activity as a function of bacterial growth conditions. For this purpose, A. viscosus cells were harvested from liquid medium at various growth phases. Cells in stationary phase were more effective in

INFECT. IMMUN.

promoting aggregation than were cells harvested earlier (Table 3). Also, cells grown on solid medium promoted aggregation better than cells grown in liquid medium, suggesting differences in the density, specificity, or accesibility of the fimbriae. Structural differences of ligands and kinetics of aggregation. There are several obstacles to a complete understanding of the binding of A. viscosus lectin to glycoprotein receptors. First, multivalent binding has a cooperative effect upon binding, and at present little is known about how many lectin binding sites interact with how many ligands. A. viscosus fimbriae do not aggregate S. sanguis cells unless the fimbriae are cross-linked with anti-A. viscosus antibodies before incubation with S. sanguis cells (5). This could occur either because the fimbriae contain only one binding site each or because they contain a number of low-affinity sites; in either case, cross-linking would enhance multivalency. Second, the valency of a particular glycoprotein such as asialofetuin is unknown, and it is uncertain whether all galactose termini are equivalent in their ability to interact. Recent experiments with the hepatic binding protein present in intact hepatocytes (Y. C. Lee, R. R. Townsend, M. R. Hardy, D. J. Connolly, W. R. Bell, J. Lonngren, J. Arnarp, M. Haraldsson, and H. Lonn, Proc. Am. Chem. Soc. Div. Carbohydr. Chem. Abstr. 18, 1982) indicate strong dependence of lectin binding upon the clustering of galactosyl residues. To examine some of the differences that structural features of glycoproteins may have on the progress of aggregation, the kinetics of aggregation were studied with ligands containing various structures. Beads were coated with defined mixtures of native fetuin and asialofetuin, and the rate of aggregation with A. viscosus increased in proportion to the asialofetuin concentration. The rate data obtained look like simple classical kinetic data for a saturable system (Fig. 4). As can be seen, for asialofetuin there is no requirement for a minimum ligand density (threshold) for aggregation to occur. A double reciprocal plot for rate data taken in the presence and absence of 5 mM lactose follows the pattern of classical competitive inhibition, with the curves intersecting at the 1/V axis, where V = Vmax (Fig. 5). When analogous experiments were performed with mixtures of asialo a1-acid glycoprotein and native a1-acid glycoprotein, a threshold in ligand density was observed below which no aggregation occurred (Fig. 6). When calculated estimates for asialo a1-acid glycoprotein concentration on the beads (Table 1) were used in combination with the 1I[S] intercept data from


VOL. 38, 1982

999

cosamine, and glycerol did not inhibit at 50 mM, and the Ki for other sugars, tested at concentrations of 5 to 50 mM, were: lactose, 0.79 mM; methyl-beta-galactopyranoside, 1.2 mM; galactose, 3.6 mM; N-acetylgalactosamine, 6.8 mM; and methyl-alpha-galactopyranoside, 17.5 mM. This is consistent with the observation that glycoproteins terminating in either beta-linked galactose or alpha-linked N-acetylgalactosamine serve equally well as bead receptors for the A. viscosus lectin.

I (arbitrary units,

asialofetuin)

FIG. 5. Lactose inhibition of aggregation of asialofetuin-coated beads by A. viscosus T14V as a function of ligand density. Experiments were conducted as for Fig. 4, curve B in the presence (0) and absence (O) of 5 mM lactose. For abbreviations, see legend to Fig. 4.

DISCUSSION The utility of latex beads (3, 19, 22, 38) and other plastic surfaces (10, 24, 36) for investigating surface-related phenomena has been demonstrated by many investigators. Our first objective was to establish the use of adsorbed glycoproteins and derivatives for the study of lectin recognition and to determine optimal con0.20 i-a

Fig. 5 and 6, the approximate Km value for asialo

al-acid glycoprotein was 1.1 mM, and the Km

value for asialofetuin was 0.14 mM. This represents an eightfold difference, even though the number of molecules per bead and the number of galactose residues per bead as previously calculated (Table 1) did not vary by more than a factor of two. Galactose termini in asialofetuin were varied by a different experimental approach. In this instance, latex beads were coated with [3H]NeuNAc7-fetuin, and when they were mixed with A. viscosus cells, no aggregation occurred (Fig. 7). Another sample of [3H]NeuNAc7-fetuin beads was treated with a limiting amount of neuraminidase, and no aggregation was observed. The progress of exposure of galactose termini was followed by measuring the [3H]NeuNAc7 released. Surprisingly, no aggregation occurred until 6 to 8% of galactose residues were unmasked by neuraminidase. The apparent Km (calculated from a double reciprocal plot similar to Fig. 6) was about five times higher than in the experiment with mixtures of native fetuin and its asialo derivative. The observed difference in the aggregation assay with asialofetuin as ligand (Fig. 4 and 7) must be a consequence of structural variations of exposed galactose residues. Competition for the lectin-binding site. Various sugars were tested for their ability to inhibit the aggregation, and inhibition constants were calculated from double reciprocal plots. Ki = [I]/ (Km app/Km) - 1, where [I] is the concentration of inhibitor tested, and Km app is the apparent Km in the presence of that particular concentration of inhibitor. Sucrose, glucose, N-acetylglu-

*cE

0.15

.4 0.10.

0.05

0.05-

0

r

20 40 60 80 100

% Asialo- a1I acid glycoprotein

1

V

I

is-]

(arbitrary units)

FIG. 6. Effect of ligand density on rate of aggregation of a,-acid glycoprotein-coated beads by A. viscosus T14V. Beads were coated with various mixtures of asialo and native a1-acid glycoprotein. Each type of bead was tested for the rate of aggregation (AA/min) in the standard assay in the presence of (0) and absence (O) of 5 mM lactose. The data in a were replotted in b as a double reciprocal Lineweaver-Burk type plot where [ is the reciprocal of the percentage asialo [S] 1 a-acid glycoprotein and is the reciprocal of aggregation velocity. Points with less than 12% asialo alacid glycoprotein were omitted from b. -

1000

0


16

*08

12

8

~~~~~~.06 -

.04

z

Z

4.

.02

2

4 6 8 10 Time in hours FIG. 7. Rate of aggregation (,AA/min) of [3H]NeuNAc7-fetuin-coated beads by A. viscosus T14V (0) compared with the percentage of [3H]NeuNAc7 released during neuraminidase treatment (0). Beads were coated with [3H]NeuNAc7fetuin and treated with a limiting amount of neuraminidase as described in the text.

ditions. The model system described, by which glycoprotein-coated beads were aggregated by the T14V strain of A. viscosus, provided aggregation data (Fig. 1) similar to those previously obtained by hemagglutination assay. The major advantage of the described system is that a single ligand with a defined structure can be selected to study cell surface interaction, with the expectation of studying the effect of chemical or enzymatic modification of carbohydrate termini upon aggregation. The method is simple, and the optimal conditions of ionic strength, pH, and temperature are convenient for several experimental designs. Unwashed beads can be stored refrigerated in the coating solution for at least 2 months, and A. viscosus cells can also be stored for months without significant deterioration of their ability to aggregate coated beads. Bacteria can be added to the aggregation mixture in a small volume, in numbers which do not appreciably alter the A650- It is interesting that beads about the same size as erythrocytes (diameter, 5.7 ,um) are most effective. Another observation is that although faster stirring rates accelerate aggregation, drawing the aggregates into a Pasteur pipette disrupts them. A significant difference between hemagglutination and bead aggregation is that the latter is more difficult to reverse by adding lactose. A similar observation was reported by other researchers who used plastic surfaces (36). Inhibitors of cell attachment to fibronectin-coated microtiter wells were effective only when added before the cells or during the first few minutes of

INFECT. IMMUN.

incubation with cells. The authors suggested that the initial specific recognition was followed by an active cell-dependent attachment process. One alternate explanation might be that hydrophobic interactions between the plastic surface and the cell take place once the cell is brought close to the surface. Lactose-inhibitable, neuraminidase-dependent aggregation has been demonstrated for glycoproteins with beta-linked galactose or alphalinked N-acetylgalactosamine as the penultimate sugar residues, suggesting recognition of these two carbohydrates by A. viscosus lectin. However, it is possible that cleavage of NeuNAc removes a charge restriction for some other recognition phenomenon. This type of misinterpretation was reported in the determination of MN blood group specificity (40). The experiments with galactose oxidase and ,B-galactosidase (Fig. 2) exclude this possibility and provide strong evidence that the galactose residues per se are essential for recognition by the A. viscosus lectin. We consider the demonstration of a strict dependence of the progress of aggregation upon the presence of galactose termini in the latex bead aggregation assay as strong evidence that we are indeed studying a carbohydrate-mediated recognition phenomenon and not other types of molecular interaction. It should be noted here that previous attempts to modify galactose termini on the surface of neuraminidase-treated erythrocytes by the action of galactose oxidase or ,B-galactosidase failed to abolish aggregation with A. viscosus (Ellen et al., J. Dent. Res. 58A:341, abstr. no. 999, 1979). We have found that even extended exposure to enzymatic treatment of erythrocytes does not result in quantitative modification or removal of galactose termini. By contrast, the quantitative enzymatic modification of fetuin in solution was accomplished before the modified ligand was adsorbed to the latex beads, resulting in abolished aggregation. Inhibition studies demonstrated that betalinked galactose is a more effective inhibitor than galactose and that alpha-linked galactose is a less effective inhibitor. Yet the alpha-linked Nacetylgalactosamine termini of asialo bovine submaxillary mucin serve as effective lectin receptors (glycoproteins or inhibitors terminating with beta-linked N-acetylgalactosamine were not tested). It is possible that A. viscosus possesses more than one lectin, but aggregation of beads coated with asialofetuin can be inhibited by N-acetylgalactosamine almost as well as by galactose, and aggregation of asialomucin-coated beads can be completely inhibited by lactose (data not shown). This tends to suggest that there is only one lectin with a specificity for both galactose and N-acetylgalactosamine residues.

VOL. 38, 1982


Also, when monoclonal antibodies were produced against fimbrial antigens, only one antigen was demonstrated to be associated with lectin activity (37). Unfortunately, attempts to isolate the lectin or lectins from A. viscosus fimbriae were not successful. Examples of lectins with specificity for more than one sugar have been described, such as concanavalin A (1) and hepatic binding protein (25). In all of these cases, the specificity may be directed toward structural features which are common to two or more sugars. The A. viscosus lectin is somewhat similar to the hepatic binding protein (25) and to some of the plant lectins, particularly peanut lectin (23), in its recognition of D-galactose or N-acetylgalactosamine residues. The species of origin and the cellular location are quite different in each case, and there are other obvious differences, such as the failure of EDTA to inhibit hemagglutination by peanut lectin (unpublished observation) and a high carbohydrate content for hepatic binding protein (30) but not for A. viscosus fimbriae (8). The fact that A. viscosus fimbriae-coated beads aggregate with S. sanguis cells as well as with human asialo-erythrocytes (Table 2) strengthens previous evidence (6) that A. viscosus fimbriae contain the lectin. Other bacteria, such as E. coli, contain fimbriae-associated lectins with specificity for mannose or other sugars (17, 41), and latex beads coated with E. coli fimbriae have been used to demonstrate hemagglutination (18, 32). Recent evidence strongly suggests that there are two types of fimbriae on A. viscosus cells, only one of which is involved in lactose-sensitive adherence (37). Like E. coli mannose-sensitive lectins (41), those of A. viscosus are more evident at later growth stages, and like the E. coli mannose-resistant hemagglutinins (13), they are more evident in cells grown on solid medium (Table 3). Further experiments are required to investigate the molecular basis for the dependence of lectin activity upon bacterial growth conditions, especially with respect to the availability of nutrients. The importance of this phenomenon has practical implications when surface properties of bacterial systems are studied and bacteria grown under standard laboratory conditions in a liquid medium are compared with cells grown on host tissues in vivo. Examination of binding phenomena such as fibroblast adhesion (26), hepatocyte adhesion (47), and cold agglutination (45) reveals a requirement for a certain minimal ligand density or threshold. To address this question, experiments were carried out with defined mixtures of glycoproteins having known ligand densities. The experiments in which beads were coated with mixtures of asialofetuin and native fetuin

1001

indicated no threshold and a small apparent Km (Fig. 4). By contrast, beads coated with [3H]NeuNAc7-fetuin and incubated with neuraminidase under limiting conditions displayed a threshold for ligand density and a larger apparent Km (Fig. 7). In the former type of experiment, all galactose residues in a given asialofetuin molecule are available for recognition by the lectin. In the latter type of experiment, the galactose residues exposed by neuraminidase action are more randomly distributed, with less opportunity for multivalent binding with the lectin. It is also possible that the small number of galactose residues exposed are not those most preferred for lectin recognition. The same reasoning applies to the experiment illustrated in Fig. 2, curve B, where the galactose termini remaining after P-galactosidase treatment (approximately 13%) were not sufficient to support bead aggregation. A higher apparent Km for asialo a,-acid glycoprotein than for asialofetuin (Fig. 4 and 6) may indicate that the less complex 0-linked oligosaccharides are more easily recognized by the lectin than the N-linked structures. Asialo al-acid glycoprotein contains five N-linked oligomers, mostly triantennary, and no 0-linked oligomers (11), and fetuin contains three of each type of oligosaccharide (43). This is not the only possible explanation for the differences in aggregation behavior since the distribution of oligosaccharides on the surface of the two glycoproteins and the possibilities for multivalency may differ significantly. Work is in progress to use this model system to gain more specific information about the role of glycoprotein structure in lectin recognition. ACKNOWLEDGMENTS We thank John Cisar of the National Institute of Dental Research, Bethesda, Md., for his helpful suggestions and Ann Marini of the University of Massachusetts Medical School, Worcester, Mass., for her assistance in the "25I-labeling experiments. LITERATURE CITED 1. Agrawal, B. B. L., and I. J. Goldstein. 1967. Proteincarbohydrate interaction. VI. Isolation of concanavalin A by specific adsorption on cross-linked dextran gels. Biochim. Biophys. Acta 147:262-271. 2. Bowden, G. H., and J. M. Hardie. 1973. Commensal and pathogenic Actinomyces species in man, p. 227-299. In G. Sykes and F. A. Skinner (ed.), Actinomycetales: characteristics and practical importance. Academic Press, Inc., New York. 3. Bubenik, J., M. Malkovsky, and E. Suhajova. 1978. The latex particle adherence (LPA) assay for detection of leukocytes with adhesive surface properties. Cell. Immunol. 35:217-225. 4. Cisar, J. 0. 1982. Coaggregation reactions between oral bacteria: studies of specific cell-to-cell adherence mediated by microbial lectins, p. 121-131. In R. J. Genco and S. E. Mergenhagen (ed.), Host-parasite interactions in periodontal diseases. American Society for Microbiology, Washington, D.C. 5. Cisar, J. O., E. Barsumian, S. Curl, A. E. Vatter, A.

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