Description and Characterization of a Surface Lectin from Giardia ...

INFECTION AND IMMUNITY, Feb. 1986, p. 661-667 0019-9567/86/020661-07$02.00/0 Copyright C) 1986, American Society for Microbiology

Vol. 51, No. 2

Description and Characterization of a Surface Lectin from Giardia lamblia MICHAEL J. G. FARTHING,t* MIERCIO E. A. PEREIRA, AND GERALD T. KEUSCH

Division of Geographic Medicine, Department of Medicine, Tufts-New England Medical Center, Boston, Massachusetts 02111 Received 3 April 1985/Accepted 22 October 1985

The mechanisms by which the human enteric pathogen Giardia lamblia colonizes the proximal small intestine poorly understood. Although the parasite possesses an attachment organelle on its ventral surface, the "sucking" disk, we considered that like many bacteria and some protozoa, G. lamblia might also have a surface membrane-associated modality for adherence to its host. Using an erythrocyte mixed-agglutination model, we demonstrated a parasite surface lectin with specificities for D-glucosyl and D-mannosyl residues. This lectin is soluble in Triton X-100, is calcium dependent, and is maximally active at pH 5.5 to 6.0. Partial purification was achieved by serial extraction of parasites in Triton X-100 followed by Sephadex G-150 affinity chromatography. The lectin could not be surface radiolabeled with 125I-Bolton-Hunter reagent, but radiolabeling of the hapten eluate from an affinity column produced four bands of 57,000 to 78,000 Mr on sodium dodecyl sulfatepolyacrylamide gels under reducing conditions. The biological function of this lectin is unknown. The presence of mannosyl residues on the luminal surface of human small intestinal epithelial cells suggests that there are receptors for Giardia lectin at the site of colonization. are

Diamond, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Md.) were cultivated axenically without antibiotics in Trypticase (BBL Microbiology Systems, Cockeysville, Md.)-Panmedeserum-iron medium (44) or in a modified Trypticase-yeastiron-serum medium and harvested as we have described previously (7, 11). A roller-bottle (Bellco Glass, Inc., Vineland, N.J.) culture technique was used when large numbers of parasites were required (10). Trophozoites were sedimented by centrifugation at 500 x g at 4°C for 10 min and, after removal of the supernatant, were washed three times in 10 mM phosphate-buffered saline (PBS), pH 7.2. Trophozoite numbers were determined by counting appropriately diluted suspensions in a Spencer Bright-Line hemacytometer. Preparation and treatment of erythrocytes. Mammalian (rabbit, sheep, goat, horse, pig, guinea pig, cow, and human types A, B, and 0) and pigeon erythrocytes were obtained fresh as whole blood and stored with an equal volume of Alsever's solution at 4°C for up to 10 days. Erythrocytes were washed three times in PBS before trypsin treatment or use in the attachment assay. Trypsinization was performed with a 10% suspension (vol/vol) of erythrocytes in PBS with trypsin (type III; Sigma Chemical Co., St. Louis, Mo.) (final concentration, 0.5 mg/ml) for 30 min at 37°C. Rabbit erythrocytes were fixed by exposure to 1% glutaraldehyde as described previously (26). Giardia-erythrocyte attachment assay. A mixed-agglutination microassay was developed to investigate physicochemical and other determinants of parasite attachment to erythrocytes. Agglutination assays were performed in 96well U-shaped microtiter plates (Dynatech Laboratories, Inc., Alexandria, Va.), using serial doubling dilutions of Giardia trophozoites (initial concentration, 1.0 x 107 to 2.0 x 107 trophozoites per ml) in PBS-0.5% bovine serum albumin. The same volume (25 ALl) of 4% erythrocyte suspension in PBS-bovine serum albumin was mixed thoroughly with trophozoites and incubated for 1 h at room temperature (23°C). The index of trophozoite-erythrocyte agglutination

The mechanisms by which the enteric protozoan pathogen Giardia lamblia colonizes the human small intestine are uncertain. An important step in the establishment of infection by luminal and atrial pathogenic microorganisms is adherence to host surface epithelium (3, 8, 43). G. lamblia is generally considered to utilize a specific attachment organelle on its undersurface, the ventral "sucking" disk (8, 15, 19). Hydrodynamic and also mechanical forces are thought to mediate attachment of the parasite to the substratum (12, 13, 20, 21, 38). There are several unexplained conceptual gaps in the proposed models of attachment. First of all, the parasite must be in the correct orientation before mechanical or hydrodynamic forces become operative. Second, the organism must be in close apposition to the substratum before a negative pressure can be developed or the proposed "grasping" movements of the ventrolateral flange become effective (8, 38). Third, it is also difficult to envisage such attachment mechanisms being effective when intestinal mucus is the target of adherence, although parasites are commonly seen penetrating and apparently locked in a layer of mucus over the surface of intestinal epithelial cells (42). In light of these considerations and the knowledge that surface membrane carbohydrate-mediated attachment mechanisms are widespread in nature (2), we investigated possible lectin-mediated attachment of G. lamblia in a model system with erythrocytes. We present evidence that G. lamblia attaches to erythrocytes via a mannose-glucose binding lectin associated with its surface membrane. MATERIALS AND METHODS Cultivation and harvesting of Giardia trophozoites. G. lamblia trophozoites (Portland 1 strain; obtained from L. * Corresponding author. t Present address: Department of Gastroenterology, St. Bartholomew's Hospital, West Smithfield, London EClA 7BE, United

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(a) Without temperature shift

(b) With temperature shift

128 64 32 a)

16

8 4

230C 370C

40C

37°C-4°C 37°C Shift Shift FIG. 1. Temperature dependence of G. lamblia-erythrocyte mixed agglutination with untreated (O) and trypsinized (1) rabbit erythrocytes, pH 7.2. (a) Single-phase temperature experiments were read after 1 h of incubation. (b) Two-phase experiments were read after 1 h and then again after a further 1-h incubation at the second incubation temperature.

40C

the titer, defined as the reciprocal of the highest trophozoite dilution which agglutinated an equal volume of erythrocyte suspension. This was determined by direct observation of the microtiter plate and confirmed by microscopic examination. This microassay was used to investigate erythrocyte species specificity and temperature, pH, and divalent-cation dependency of erythrocyte attachment to G. lamblia. Experiments with untreated and trypsinized rabbit erythrocytes were performed at 4 and 37°C in addition to those at room temperature. Two-phase temperature studies were also performed, with the initial incubation at 23°C followed by a second incubation at 4°C and vice versa. Assays were performed in the presence or absence of the divalent cations calcium, magnesium, and manganese (0.6 to 5.0 mM), using divalent-cation-free buffers and the chelating agents EGTA (ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid) and EDTA (0.6 to 5.0 mM). Optimal pH for G. lamblia-erythrocyte attachment was studied with fresh and glutaraldehyde-fixed rabbit erythrocytes through pH 4.5 to 9.0 Attachment inhibition studies were performed in the presence of a wide range of simple and complex sugars (100 mM) and glycoproteins (0.5 to 1.0%). Carbohydrate inhibition studies were performed with the concentration of Giardia trophozoites equivalent to 4 hemagglutinating units, that is, a trophozoite concentration four times that of the endpoint for agglutination (41). Serial doubling dilutions of sugars or glycoproteins were added to G. lamblia-erythrocyte incubations, and the minimum concentration causing inhibition was recorded. Solubilization of Giardia HA. Roller-bottle cultivation was used to produce up to 5.0 x 109 Giardia trophozoites for each experiment (10). After being washed in PBS, trophozoites were exposed to 2.0 ml of 0.05% Triton X-100 in 10 mM Tris-150 mM sodium chloride (pH 6.0) containing calcium chloride (2 mM), the protease inhibitors phenylmethylsulfonyl fluoride (5.0 ,uM), leupeptin (1.0 ,uM), and chymostatin (1.0 1xM), and sodium azide (0.02%). Triton X-100 was was

chosen because a previous study had shown that the Giardia cytoskeleton was insoluble in this detergent at concentrations of 10 column volumes)

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FIG. 2. Effect of divalent cations on G. Iamblia-erythrocyte mixed agglutination at pH 7.2 and 23°C. Calcium (0) but not magnesium (0) or manganese (data not shown) enhanced agglutination, and agglutination was inhibited in a dose-dependent manner by EDTA and EGTA (O). Reconstitution with calcium in the presence of 1.25 mM EGTA (U) restored agglutination titers.

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TABLE 1. Carbohydrate and glycoprotein inhibition of G. lamblia-erythrocyte mixed agglutination and Triton X-100-soluble MIC (mM)b

Carbohydrate or glycoprotein

G. Iamblia-erythrocyte

cx-Methyl-D-mannoside D-Glucose D-Mannose Ovomucoid

25 50 100 3.0

agglutination

TritonX-100soluble HA

25 0.78 25 0.31 a No inhibition was observed with 100 mM D-glucosamine, N-acetyl-Dglucosamine, D-galactose, N-acetyl-D-galactosamine, lactose, fucose, or 1% orosomucoid. bLowest concentration of carbohydrate or glycoprotein which completely inhibited mixed agglutination with untreated rabbit erythrocytes at pH 6.0 and 23°C. Values for ovomucoid are given as milligrams per milliliter.

and then eluted with 100 mM D-glucose or a-methyl-Dmannoside. After exhaustive dialysis against TEB, column fractions were again tested for HA. Column fractions which had HA but no radioactivity were radiolabeled by further exposure to 125I-Bolton-Hunter reagent or by the iodogen method (32). These affinity-purified column fractions were subjected under reducing conditions to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (27). Gels were stained with Coomassie brilliant blue and autoradiographed with Kodak X-Omat R-1 film (Eastman Kodak Co., Rochester, N.Y.).

RESULTS G. Iamblia-erythrocyte mixed agglutination. Giardia trophozoites agglutinated rabbit erythrocytes most avidly, particularly after trypsin treatment, and this erythrocyte species was therefore used in all further studies. Agglutination was not impaired by glutaraldehyde fixation of erythrocytes. Mixed agglutination was temperature, pH, and calcium 30

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TABLE 2. Triton X-100-soluble Giardia HA in serial extracts Extract'

Giardia HAa

9

10 11 12

TX-100 extracts

FIG. 3. Triton X-100 (TX-100)-soluble Giardia HA (L) and 125i radioactivity (0) of 12 serial extracts of '25I-Bolton-Hunter reagent surface-labeled Giardia trophozoites (-109 parasites).

663

1 2 3 4 5 6 7 8 9 10 11 12

Protein concn

(mg/ml) 0.85 3.40 4.23 2.90 2.90 0.72 0.76 0.54 0.58 0.42 0.77 0.61

Hemagglutination titer5 0 0 0 8 32 128 512 256 128 256 128 32

Sp actprotein titer/mg of

0 0 0 2.8 11.0 177.8 673.7 474.1 220.7 609.5 166.2 52.5

a Sequential Triton X-100 (0.05%) extracts of Giar-dia trophozoites. Hemagglutination performed with untreated rabbit erythrocytes at pH 6.0 and 23°C.

dependent. Incubation at 4°C markedly reduced agglutination titers compared with those of experiments performed at room temperature (23°C), but these titers were not increased further by incubation at 37°C (Fig. 1). Once agglutination had occurred at either 23 or 37°C, reducing the temperature to 4°C in a second incubation did not dissociate G. lambliaerythrocyte aggregates. Optimum pH for agglutination was pH 6.0 for both fresh and glutaraldehyde-treated erythrocytes. Fixed erythrocytes were used in these studies since at pH c5.0 fresh erythrocytes tended to autoagglutinate. Agglutination was enhanced at calcium concentrations of >0.625 mM, although neither EDTA nor EGTA (5 mM) was able to totally abolish agglutination (Fig. 2). Magnesium or manganese ions had no demonstrable effects on agglutination. G. lamblia-erythrocyte mixed agglutination was completely inhibited by D-glucose, D-mannose, a-methyl-Dmannoside, and the glycoprotein ovomucoid but not by more than 20 other sugars or glycoproteins of diverse structures (Table 1). Soluble Giardia HA. Serial TEB extraction of Giardia trophozoites yielded 50,000 x g supernatants with HA. In the representative experiment shown in Table 2 with 2.9 x 109 parasites, maximal HA was found in extracts 7 to 10 with a 350-fold increase in specific activity from the initial to peak extracts with HA. Unlike mixed agglutination with whole trophozoites, Triton X-100-soluble HA was temperature independent, the activity being unchanged from 4 to 37°C, but the optimum pH was similar to that found for trophozoite-erythrocyte interaction at pH 5.5 to 6.0. Soluble HA was similarly calcium dependent and increased fourfold by the addition of calcium (1.0 mM) but not magnesium ions, although neither EDTA nor EGTA (5 mM) could abolish hemagglutination completely. Hemagglutination was again inhibited by D-glucose, Dmannose, a-methyl-D-mannoside, and ovomucoid but at lower concentrations than were required to inhibit G. lamblia-erythrocyte interactions (Table 1). Soluble HA remained stable when stored at 4°C in the presence of sodium azide for at least 3 months. Surface labeling and analysis by SDS-PAGE. Lectin activity (specific activity, titer per milligram of protein) and radioactivity of 12 serial TEB extracts are shown in Fig. 3. Peak lectin activity in this experiment was not achieved until extract 11, unlike that shown in Table 2 when peak HA appeared earlier. The explanation for such variation is unknown, but the variation commonly occurred. SDS-

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MW

-24K

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18K 14K

FIG. 4. Autoradiograph of SDS-PAGE of 12 serial Triton X-100 extracts of G. lamblia from the experiment shown in Fig. 3. MW, X Molecular weight; K, _-.

PAGE of these 12 extracts indicated that this approach to lectin solubilization and isolation probably functioned as a preliminary purification step (Fig. 4). Early extracts (1 and 2) predominantly contained material of low Mr, whereas in later extracts (5 to 11) proteins of higher Mr were in evidence. A total of 18 major bands were identified in extracts with HA activity. Extract 11, which had the highest HA, was then subjected to Sephadex G-150 affinity chromatography. Some HA appeared in the column flowthrough. D-Glucose (100 mM) specifically eluted HA which was not associated with significant radioactivity (Fig. 5), indicating that this material was not surface radiolabeled. When this material was again iodinated (fraction 128; Fig. 5), SDSPAGE revealed two major polypeptides, 78,000 and 57,000 Mr, two minor polypeptides, 69,500 and 59,000 Mr, and additional low-molecular-weight material that did not resolve in the 11% SDS-PAGE (Fig. 6, lane B). The specific activity of the glucose eluate was 16,840 HA units per mg of protein, indicating approximately 40-fold purification. DISCUSSION We report here that axenically cultivated Portland 1 strain G. lamblia possesses a surface membrane-associated lectin with specificities for glucosyl and mannosyl residues on the experimental target, rabbit erythrocytes. Since mannosyl residues are present on the surface of mammalian intestinal epithelial cells (25, 47, 48) and there appear to be functional receptors for bacterial mannose-binding lectins (34), it is possible that this lectin may participate in the attachment of G. lamblia to intestinal epithelium.

As in previous studies of microbial surface lectins, an erythrocyte model system of adherence was employed to characterize Giardia lectin activity. Trypsinized rabbit erythrocytes were agglutinated most avidly, suggesting that Giardia lectin may be interacting with core mannosyl residues, an effect also described with ConA (45). Similar to other D-mannose-binding lectins such as ConA, Pisum sativum, and Lens culinaris (40), there was no evidence of blood-group specificity in the agglutination of human erythrocytes by the parasite (data not shown). Scanning electron microscopic examination of G. lamblia-erythrocyte attachment demonstrated that erythrocytes attach over the entire parasite surface, displaying no predilection for the ventral disk (M. J. G. Farthing, G. T. Keusch, and P. Lin, unpublished observations). G. lamblia-erythrocyte interaction was completely inhibited by some glucosyl- and mannosyl-containing saccharides although only at relatively high concentrations (Table 1). However, similar concentrations are reported for complete inhibition of Escherichia coli (type 1 pili)-epithelial cell interactions (37), although carbohydrate concentration required for inhibition generally falls markedly when purified lectin (9) rather than the intact cell is used for agglutination, as was observed in the present study (Table 1). We have clearly not attempted to fully define the intestinal receptor for this lectin, but when the binding site has been characterized we would anticipate that inhibition of binding will be observed at much lower saccharide concentrations. Our studies with whole organisms and Triton X-100soluble HA suggest that Giardia lectin activity is both pH

GIARDIA SURFACE LECTIN

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FIG. 5. Sephadex G-150 affinity chromatography of Triton X-100-soluble Giardia HA (extract 11, Fig. 3 and 4) in TEB with calcium (2 mM) at pH 6.0. Protein (0), 1251 radioactivity (A), and HA (O) appeared in the column flowthrough, but after extensive column washing, 100 mM D-glucose eluted fractions (127 to 129) which after dialysis against TEB-Ca2+ yielded HA (E) with high specific activity but which were devoid of 1251 radioactivity.

and calcium dependent. Physical and chemical modulation of lectin structure and activity is well recognized (35). ConA, for example, exists as a monomer at low pH, but at pH 5.6 the protein progressively dimerizes as the pH increases, with aggregate formation at pH 7.0 and above (23, 33). A recently isolated N-acetyl-D-glucosamine-binding lectin from Entamoeba histolytica is also pH dependent, being maximally active at pH 5.7 to 6.0 but inactive at pH 7.2 (26). G. lamblia-erythrocyte attachment and Triton X-100-soluble Giardia HA were maximal at pH 5.5 to 6.0. Luminal pH in the mammalian duodenum and jejunum is close to neutrality (14). The intestinal microclimate of the jejunum, the zone immediately adjacent to the luminal surface of the intestinal epithelium, however, has a pH 1.0 to 2.0 units below luminal pH (29, 30). Thus, lectin-mediated attachment of G. lamblia to the surface epithelium might be facilitated by having a pH optimum in this range. Our observation that Giardia-lectin activity is increased in the presence of calcium is not surprising since divalent-cation dependence has been described for a variety of plant and animal lectins of different saccharide specificity, notably ConA (22), lima bean lectin (16), rabbit hepatic lectin (24), and Ulex lectin II (39). Temperature dependence was only demonstrated in mixed-agglutination experiments with Giardia trophozoites and erythrocytes and not with soluble Giardia HA. Temperature reduction may alter subunit arrangement and activity of some lectins, whereas the agglutinating activity of others remains unchanged (17). Our findings, however, suggest that inhibition of G. lamblia-erythrocyte attachment at 4°C was most likely related to changes in membrane dynamics rather than an effect on lectin structure or conformation. We cannot, however, exclude the possibility in the soluble HA preparations that Triton X-100 exerted a stabilizing effect on lectin activity. Soybean lectin, for example, extracted from the plant root appears to have enhanced activity when solubilized and prepared in similar concentrations of Triton

X-100 (5). Similarly, inhibitory effects on activity of Giardia contractile proteins would also be apparent at low temperatures (36), although this is unlikely to be a major consideration since G. lamblia-erythrocyte agglutination remains stable once first completed at higher temperatures. Lectin solubilization, isolation, and purification were accomplished by serial Triton X-100 extraction followed by affinity chromatography. Serial Triton X-100 extraction resulted in a 350-fold increase in specific activity of HA from the first Triton-100 extract with HA to the peak fractions (Table 2), with a further 40-fold increase after Sephadex affinity column chromatography. However, despite employing physical and chemical conditions which our earlier experiments had indicated were optimal for Giardia lectin activity, affinity for Sephadex was modest, and there was a relatively poor yield of purified material. This may be related to the presence of Triton X-100, which has been reported to interfere with lectin purification by affinity chromatography

(2, 5).

The biological role of this lectin in the parasite life cycle and in its interaction with the small intestinal epithelium is a matter for speculation. Although D-glucosyl residues are not found on human intestinal epithelial cells, D-mannosyl residues are present on the luminal surface and may serve as possible receptor sites for Giardia lectin (25). In addition, the density of D-mannosyl residues is apparently greater in poorly differentiated crypt cells compared with villus cells (47). In giardiasis, intestinal epithelial cell turnover is increased (31), and the villus therefore becomes populated with relatively immature cells, potentially rich in such putative receptors for Giardia lectin. If true, this surface Giardia lectin could be important for colonization and may complement ventral disk-mediated attachment by providing a primary braking mechanism which operates over the entire surface of the parasite. At the same time, since some dietary plant lectins are injurious to the mammalian small intestine

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ing acylating agent. Biochem. J. 133:529-539. 5. Bowles, D., H. Lis, and N. Sharon. 1979. Distribution of lectins in membranes of soybean and peanut plants. I. General distribution in root, shoot and leaf tissue at different stages of growth. Planta 145:193-198. 6. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-259. 7. Diamond, L. S., D. R. Harlow, and C. C. Cunnick. 1978. A new medium for the axenic cultivation of Entamoeba histolytica and other entamoeba. Trans. R. Soc. Trop. Med. Hyg. 72:431-432. 8. Erlandsen, S. L., and D. G. Chase. 1974. Morphological alterations in the microvillous border of villous epithelial cells produced by intestinal microorganisms. Am. J. Clin. Nutr. 27:1277-1286. 9. Eshdat, Y., I. Ofek, Y. Yashouv-Gan, N. Sharon, and D. Mirelman. 1978. Isolation of mannose-specific lectin from Escherichia coli and its role in the adherence of the bacteria to epithelial cells. Biochem. Biophys. Res. Commun. 85: 1551-1559.

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FIG. 6. Autoradiograph of SDS-PAGE of postdialysis 1251labeled (iodogen method) D-glucose column eluate (lane B; column fraction 128, Fig. 5) and surface-radiolabeled material in column flow (lane A; fraction 3, Fig. 5). MW, Molecular weight; K, 103.

(28), it is possible that the Giardia lectin contributes to the functional or morphological damage or both in the small intestine found in some individuals with giardiasis. ACKNOWLEDGMENTS This work was supported by a grant in Geographic Medicine from the Rockefeller Foundation, M.J.G.F. performed these studies during the tenure of a Wellcome Tropical Lectureship from the Wellcome Trust, United Kingdom. LITERATURE CITED 1. Agrawal, B. B. L., and I. J. Goldstein. 1967. Protein-carbohydrate interaction. VI. Isolation of concanavalin A by specific adsorption to cross-linked dextran gels. Biochim. Biophys. Acta

147:262-271. 2. Barondes, S. H. 1981. Lectins: their multiple endogenous cellular functions. Annu. Rev. Biochem. 50:207-231. 3. Beachey, E. H. 1981. Bacterial adherence: adhesin-receptor interactions mediating the attachment of bacteria to mucosal surfaces. J. Infect. Dis. 143:325-345. 4. Bolton, A. E., and W. M. Hunter. 1973. The labelling of proteins to high specific radioactivities by conjugation to a '251-contain-

10. Farthing, M. J. G., M. E. A. Pereira, and G. T. Keusch. 1982. Giardia lamblia: evaluation of roller bottle cultivation. Exp. Parasitol. 54:410-415. 11. Farthing, M. J. G., S. R. Varon, and G. T. Keusch. 1983. Mammalian bile promotes growth of Giardia lamblia in axenic culture. Trans. R. Soc. Trop. Med. Hyg. 77:467-469. 12. Feely, D. E., and S. L. Erlandsen. 1982. Effect of cytochalasinB, low Ca++ concentration, iodoacetic acid and quinacrine-HCl on the attachment of Giardia trophozoites in vitro. J. Parasitol. 68:869-873. 13. Feely, D. E., J. V. Schollmeyer, and S. L. Erlandsen. 1982. Giardia spp.: distribution of contractile proteins in the attachment organelle. Exp. Parasitol. 53:145-154. 14. Fordtran, J. S., and T. W. Locklear. 1966. Ionic constituents and osmolarity of gastric and small intestinal fluids after eating. Am. J. Dig. Dis. 11:503-521. 15. Friend, D. S. 1966. The fine structure of Giardia muris. J. Cell Biol. 29:317-332. 16. Galbraith, W., and I. J. Goldstein. 1972. Phytohemagglutinin of the lima bean (Phaseolus lunatus). Isolation, characterization and interaction with type A blood group substances. Biochemistry 11:3976-3984. 17. Gordon, J. A., and M. D. Marquardt. 1974. Factors affecting hemagglutination by concanavalin A and soybean agglutinin. Biochim. Biophys. Acta 332:136-144. 18. Hahn, H. J., B. Hellman, A. Lernmark, J. Sehlin, and I. B. Taljedal. 1974. The pancreatic B-cell recognition of insulin secretagogues. J. Biol. Chem. 249:5275-5284. 19. Holberton, D. V. 1973. Fine structure of the ventral disc apparatus and the mechanism of attachment in the flagellate Giardia muris. J. Cell Sci. 13:11-41. 20. Holberton, D. V. 1974. Attachment of Giardia-a hydrodynamic model based on flagellar activity. J. Exp. Biol. 60:207-221. 21. Holberton, D. V., and A. P. Ward. 1981. Isolation of the cytoskeleton from Giardia. Tubulin and a low-molecular weight protein associated with microribbon structures. J. Cell Sci. 47:139-166. 22. Kalb, A. J., and A. Levitzki. 1968. Metal-binding sites of concanavalin A and their role in the binding of ct-methyl-Dglucopyranoside. Biochem. J. 109:669-672. 23. Kalb, A. J., and A. Lustig. 1968. The molecular weight of concanavalin A. Biochim. Biophys. Acta 168:366-367. 24. Kawasaki, T., and G. Ashwell. 1976. Chemical and physical properties of an hepatic membrane protein that specifically binds asialoglycoproteins. J. Biol. Chem. 251:1296-1302. 25. Kim, Y. S., A. Morita, S. Miura, and B. Siddiqui. 1984. Structure of glycoconjugates of intestinal mucosal membranes: role in bacterial adherence, p. 99-109. In E. C. Boedeker (ed.), Attachment of organisms to the gut mucosa, vol. 2. CRC Press, Inc., Boca Raton, Fla. 26. Kobiler, D., and D. Mirelman. 1980. Lectin activity in Entamoeba histolytica trophozoites. Infect. Immun. 29:221-225. 27. Laemmli, U. K. 1970. Cleavage of structural proteins during the

VOL. 51, 1986

28. 29. 30.

31. 32.

33.

34.

35.

36. 37.

assembly of the head of bacteriophage T4. Nature (London) 227:680-685. Lorenzsonn, V., and W. A. Olsen. 1982. In vivo responses of rat intestinal epithelium to intraluminal dietary lectins. Gastroenterology 82:838-848. Lucas, M. L., J. A. Blair, B. T. Cooper, and A. J. Matty. 1975. Further investigations with pH-microelectrodes into jejunal microclimate in rat and man. Gut 16:844. Lucas, M. L., F. H. Lei, and J. A. Blair. 1980. The influence of buffer pH, glucose and sodium ion concentration on the acid microclimate in rat proximal jejunum in vitro. Pfluegers Arch. 385:137-142. MacDonald, T. T., and A. Ferguson. 1978. Small intestinal epithelial cell kinetics and protozoal infection in mice. Gastroenterology 74:496-500. Markwell, M. A., and C. F. Fox. 1978. Surface-specific iodination of membrane proteins of viruses and eucaryotic cells using 1,3,4,6-tetrachloro-3-alpha,6-alpha-diphenylglycoluril. Biochemistry 17:4807-4817. McKenzie, G. H., W. H. Sawyer, and L. W. Nichol. 1972. The molecular weight and stability of concanavalin A. Biochim. Biophys. Acta 263:283-293. Nagy, B., N. W. Moon, and R. E. Isaacson. 1976. Colonization of porcine small intestine by Escherichia coli: ileal colonization and adhesion by pig enteropathogens that lack K88 antigen and by some acapsular mutants. Infect. Immun. 13:1214-1220. Nicholson, G. L. 1974. The interactions of lectins with animal cell surfaces. Int. Rev. Cytol. 39:89-190. Nicholson, G. L. 1976. Transmembrane control of the receptors on normal and tumour cells. I. Cytoplasmic influence over cell surface components. Biochim. Biophys. Acta 457:59-108. Ofek, I., D. Mirelman, and N. Sharon. 1977. Adherence of Escherichia coli to human mucosal cells mediated by mannose receptors. Nature (London) 265:623-625.

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38. Owen, R. L. 1980. The ultrastructural basis of Giardia infection. Trans. R. Soc. Trop. Med. Hyg. 74:429-43. 39. Pereira, M. E. A., F. Gruezo, and E. A. Kabat. 1979. Purification and characterization of lectin II from Ulex europaeus seeds and an immunological study of its combining site. Arch. Biochem. Biophys. 194:511-525. 40. Pereira, M. E. A., and E. A. Kabat. 1979. Immunochemical studies on lectins and their application to the fractionation of blood group substances and cells. Crit. Rev. Immunol. 1:33-78. 41. Pereira, M. E. A., M. A. Loures, F. Vilialta, and A. F. B. Andrade. 1980. Lectin receptors as markers for Trypanosoma cruzi. Developmental stages and a study of the interaction of wheat germ agglutination with sialic acid residues on epimastigote cells. J. Exp. Med. 152:1375-1392. 42. Poley, J. R., and S. Rosenfield. 1982. Malabsorption in giardiasis: presence of a luminal barrier (mucoid pseudomembrane). A scanning and transmission electron microscopic study. J. Pediatr. Gastroenterol. Nutr. 1:62-80. 43. Ravdin, J. I., and R. L. Guerrant. 1981. Role of adherence in cytopathogenic mechanisms of Entamoeba histolytica. J. Clin. Invest. 68:1305-1313. 44. Visvesvara, G. S. 1980. Axenic growth of Giardia lamblia in Diamond's TPS-1 medium. Trans. R. Soc. Trop. Med. Hyg. 74:213-215. 45. Vlodavsky, I., M. Inbar, and L. Sachs. 1972. Temperaturesensitive agglutinability of human erythrocytes by lectins. Biochim. Biophys. Acta 274:364-369. 46. Warren, L. 1959. The thiobarbituric assay of sialic acids. J. Biol. Chem. 234:1971-1975. 47. Weiser, M. M. 1972. Concanavalin A agglutination of intestinal cells from the human fetus. Science 177:525-526. 48. Weiser, M. M. 1973. Intstinal epithelial cell surface membrane glycoprotein synthesis. I. An indicator of cellular differentiation. J. Biol. Chem. 248:2536-2541.