INFECTION AND IMMUNITY, June 2002, p. 3259–3263 Vol. 70, No. 60019-9567/02/$04.00�0 DOI: 10.1128/IAI.70.6.3259–3263.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

NOTES

Entamoeba histolytica Lectins Contain Unique 6-Cys or 8-CysChitin-Binding Domains

Katrina Van Dellen,1 Sudip K. Ghosh,1 Phillips W. Robbins,2 Brendan Loftus,3 and John Samuelson1*Department of Immunology and Infectious Diseases, Harvard School of Public Health,1 and Department of Cell Biology, Boston

University School of Dental Medicine,2 Boston, Massachusetts, and The Institute for Genomic Research, Rockville, Maryland3

Received 3 August 2001/Returned for modification 1 October 2001/Accepted 22 February 2002

The Jacob lectin, the most abundant glycoprotein in the cyst wall of Entamoeba invadens, contains five unique6-Cys chitin-binding domains (CBDs). We identified Entamoeba histolytica and Entamoeba dispar genes encod-ing Jacob homologues, each of which contains two predicted 6-Cys CBDs. A unique 8-Cys CBD was found atthe N termini of the E. histolytica chitinase and three other predicted lectins, called Jessie 1 to Jessie 3. TheCBDs of four E. histolytica lectins (Jacob, chitinase, and Jessies 2 and 3) were expressed in secretory vesiclesof transfected amebae and shown to bind to particulate chitin.

The diagnostic and infectious stage of Entamoeba histolyticais the quadranucleate cyst, which has a wall composed in partof chitin (2). While E. histolytica causes dysentery and liverabscess, Entamoeba dispar (formerly known as nonpathogenicE. histolytica) is morphologically identical and does not causedisease (7). Because E. histolytica does not encyst in axenicculture, the reptilian pathogen Entamoeba invadens is used tomodel amebic encystation (10). The most abundant E. inva-dens cyst wall protein (Jacob) is a unique lectin composed offive tandemly arranged chitin-binding domains (CBDs), eachof which has six Cys residues and conserved aromatic aminoacids (11). The five CBDs in the amebic Jacob lectin presum-ably cross-link and stabilize chitin fibrils in the amebic cyst wallin the same way that CBDs of insect peritrophins cross-linkchitin fibrils in the wall that surrounds the insect blood meal(11, 24). Although CBDs of peritrophins and insect chitinasesalso contain six Cys residues and are related to each other bya common ancestry, the 6-Cys CBDs of the E. invadens Jacoblectin are not related to them by a common ancestry (conver-gent evolution) (9, 11, 24).

Although neither chitin synthase nor chitinase is present inE. invadens trophozoites, both enzymes are expressed by en-cysting amebae (5, 28). Amebic chitinases have a series ofhydrophilic heptapeptide repeats between an N-terminal signalsequence and a C-terminal glycohydrolase domain (6, 14, 16).The chitinase heptapeptide repeats, which vary from 4 to 18repeats in different isolates of E. histolytica and Entamoebadispar, have an amino acid composition similar to that of spac-ers or hinges present between CBDs of the Jacob lectin (11,13). This similarity led us to identify a putative 8-Cys CBD nearthe N terminus of the E. histolytica and E. invadens chitinases.Plant chitinases also have 8-Cys CBDs at their N termini,

which presumably help the chitinases bind to the chitin fibrilsthat the glycohydrolase domain will degrade (3).

We expressed the 8-Cys CBD of E. histolytica chitinase introphozoites and showed that it does bind to chitin. A survey ofshotgun sequences of the E. histolytica genome showed thatthis 8-Cys CBD is also present at the N termini of three hy-pothetical proteins (Jessie 1 to Jessie 3). The CBDs of Jessie 2and Jessie 3 were expressed and shown to bind chitin. In thisstudy, we also identified homologues of the E. invadens Jacoblectin in E. histolytica and E. dispar and showed that the E.histolytica Jacob is a chitin-binding protein.

A predicted E. histolytica Jacob lectin has two 6-Cys, chitin-binding domains. The peptide sequence of the E. invadensJacob lectin and TBLASTN were used to search 49,000 E.histolytica HM-1:IMSS strain genomic DNA sequences, whichwere deposited in the Genome Survey Sequences database ofthe National Center for Biotechnology Information by one ofthe coauthors (B. Loftus) (1, 11). A predicted E. histolyticaJacob lectin, which was 151 amino acids long with a predicted21-amino-acid signal sequence at its N terminus, showed 23%amino acid identity with a corresponding region of the E.invadens Jacob lectin (Fig. 1A) (11, 22, 26). The two predictedCBDs of the E. histolytica Jacob each contained six Cys resi-dues, which might form three disulfide bonds as described forCBDs of insect peritrophins and insect, nematode, and fungalchitinases (18, 24, 27). Like the E. invadens Jacob lectin, thepredicted E. histolytica Jacob lectin did not appear to containany transmembrane domains, which might anchor the peptidein the plasma membrane (25). The predicted E. histolyticaJacob CBDs did contain numerous aromatic amino acids (Tyr,Phe, and Trp), which have been implicated in carbohydratebinding by plant lectins (3, 29). There was a predicted N-linkedglycosylation site in the second CBD of the E. histolytica Jacoblectin, as well as two predicted N-linked glycosylation sites inthe hydrophilic spacer between the two CBDs (17). TheseN-linked glycosylation sites suggest that the E. histolytica Jacoblectin may be a glycoprotein like the E. invadens Jacob lectin

* Corresponding author. Mailing address: Department of Immunol-ogy and Infectious Diseases, Harvard School of Public Health, 665Huntington Ave., Boston, MA 02115. Phone: (617) 432-4670. Fax:(617) 738-4914. E-mail: jsamuels@hsph.harvard.edu.

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(11). Northern blot analysis suggested that mRNAs for the E.histolytica Jacob lectin are not transcribed by E. histolyticatrophozoites, which do not encyst in axenic culture (data notshown). We were unable to obtain fresh E. histolytica cysts forNorthern blot analysis. Jacob mRNAs are also absent from E.invadens trophozoites but are present in E. invadens cysts (11).

To test the function of the putative 6-Cys CBDs of the E.histolytica Jacob lectin, E. histolytica trophozoites, which do notexpress the Jacob lectin (above), were transfected with a vectorthat contained a modified E. histolytica Jacob gene between 5�and 3� untranslated regions of the E. histolytica actin gene. Thevector and methods were the same as those used previously to

express and localize intact and modified chitinases in E. histo-lytica trophozoites (14). The modified Jacob lectin, which wasdetected with a monoclonal antibody to a c-myc epitope tagplaced at its C terminus, was present in small vesicles of trans-fected amebae (Fig. 1B). These small vesicles resemble secre-tory vesicles, which contain the Jacob lectin in encysting E.invadens (11). When transfected trophozoites were lysed in0.1% Triton X-100, the myc-tagged Jacob lectin bound tightlyto particulate chitin and could be eluted only by boiling in 1%sodium dodecyl sulfate–5% 2-mercaptoethanol (Fig. 1E). Incontrast, Coomassie blue-stained gels showed that the vastmajority of trophozoite proteins do not bind to chitin (Fig.

FIG. 1. (A) Primary structure in single letter code of the putative E. histolytica (Eh) Jacob lectin aligned with homologous regions of the E.dispar (Ed) and E. invadens (Ei) Jacob lectins (residues 107 to 233). Sequence differences between E. histolytica and E. dispar Jacob lectins areshown in purple, while signal sequences, identified with SignalP (22), are shown in gray. Conserved Cys residues in the putative CBDs are shownin red, while other residues identical in all Jacob lectins are shown in blue. Spacers between putative CBDs are shown in green, while potentialsites of N-linked glycosylation are shown in light orange. (B) Confocal micrograph of the E. histolytica Jacob expressed with a myc tag in transfectedE. histolytica and identified with anti-myc monoclonal antibodies and Alexa Fluor 488-labeled anti-mouse IgG antibodies (staining methods aredescribed in reference 14). (C) Nuclear staining is seen when the cells in panel B are stained with the Alexa Fluor488-labeled anti-mouse IgGantibodies alone. (D) Coomassie blue-stained gel of total proteins from nontransfected E. histolytica trophozoites, trophozoite proteins bound tochitin beads, and trophozoite proteins that did not bind to chitin beads. (E) Western blot analysis of chitin binding by E. histolytica Jacob, detectedwith anti-myc antibodies and chemiluminescence.

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1D). These results demonstrate that the E. histolytica Jacoblectin binds chitin, most likely through both of its 6-Cys CBDs(although we cannot rule out the possibility that one of the6-Cys CBDs is not functional). While the E. invadens Jacoblectin has five CBDs and could theoretically cross-link fivechitin fibrils, the E. histolytica and E. dispar (next section)Jacob lectins each have two CBDs and could cross-link twochitin fibrils. In the same way, the number of CBDs in insectperitrophins, which cross-link chitin fibrils in the wall around ablood meal, have also been shown to vary between species (24).

An E. dispar Jacob lectin also has two putative 6-Cys, chitin-binding domains. The E. histolytica jacob gene was used toidentify an E. dispar jacob gene from a genomic DNA library,which was a generous gift of Michael Duchene (23). The pre-dicted E. dispar Jacob lectin, which was 150 amino acids longwith a predicted 21-amino-acid signal sequence, showed 85%amino acid identity with the E. histolytica Jacob lectin (Fig.1A). Most of the amino acid differences between the E. histo-lytica and E. dispar Jacob lectins were clustered in the firstCBD, while two of three predicted N-linked glycosylation siteswere conserved in the E. dispar Jacob lectin. No other homo-logues of the Jacob lectins were identified in GenBank NR,dBEST, Genome Survey Sequences, and unfinished microbialdatabases, using BLASTP or TBLASTN (1). These resultssuggest the Entamoeba Jacob lectins are unique, although theyserve the same function (cross-linking chitin fibrils) as insectperitrophins (24). Whether monoclonal antibodies to Jacoblectins may be used to discriminate cysts of E. histolytica fromthose of E. dispar in patients’ stools remains to be determined.

The E. histolytica chitinase has a unique 8-Cys chitin-bind-ing domain at its N terminus. An alignment of the N terminiof E. histolytica and E. invadens chitinases suggested the pres-ence of a putative CBD containing eight Cys residues, whichmight form four disulfide bonds as in plant lectins (Fig. 2A) (3,6, 19, 29). Although the signal sequences and hydrophilic re-peats of the E. histolytica and E. invadens chitinases were notalignable, the putative CBDs shared 42 of 64 amino acids(66%) (6, 22). Further, the CBDs have eight positionally iden-tical aromatic amino acids (Tyr, Phe, and Trp), which might beinvolved in carbohydrate binding (3, 29). Although plant lec-tins and chitinases also have 8-Cys CBDs, the amebic chitinase8-Cys CBDs were not related to them by a common ancestry(1, 3, 19, 29). This is another example of convergent evolution(9), as has been argued for the 6-Cys CBDs of the Jacob lectinand insect peritrophins (11, 24). The heptapeptide repeats ofthe E. histolytica and E. dispar chitinases, which vary so muchfrom one clinical isolate to the next (13), are likely spacers orhinges between the CBD and the catalytic domain.

To test the function of the putative chitinase 8-Cys CBD, E.histolytica trophozoites, which do not express chitinase (6),were transfected with an E. histolytica gene encoding a trun-cated chitinase, which included the signal sequence, putativeCBD, and heptapeptide repeats. The vector and methods werethe same as those used previously to express and localize intactand modified chitinases in E. histolytica trophozoites (14). Thetruncated chitinase, which was detected with polyclonal rabbitantibodies to the heptapeptide repeats, was present in smallsecretory vesicles that were similar in appearance to thoseassociated with intact chitinase (14) (Fig. 2B). When trans-fected trophozoites were lysed in 0.1% Triton X-100, the trun-

cated chitinase bound tightly to particulate chitin and could beeluted only by boiling it in 1% sodium dodecyl sulfate–5%2-mercaptoethanol (Fig. 2F). A positive control for chitin bind-ing was the full-length chitinase protein (Fig. 2F). A negativecontrol for chitin binding was a modified amebic Fe-hydroge-nase, which was epitope tagged with the chitinase heptapeptiderepeats (data not shown) (12). The truncated chitinase did notbind to cellulose or GlcNAc conjugated to agarose (data notshown), suggesting that the chitinase 8-Cys CBD is specific forchitin. It is possible that the 8-Cys CBD increases the activityof the amebic chitinase, in the same way that an 8-Cys CBDincreases the activity of a plant chitinase (30).

Three hypothetical E. histolytica lectins (Jessie 1 to Jessie 3)contain 8-Cys CBDs like those of chitinases. The hypotheticallectins Jessie 1 to Jessie 3, which were identified by searchingthe E. histolytica shotgun sequences with the chitinase CBDand TBLASTN, also contained an 8-Cys putative CBD justdistal to a predicted signal sequence (Fig. 2A) (1). Twentyamino acids (31%), including the eight Cys residues, werestrictly conserved in the 64-amino-acid CBDs of chitinases andJessies (26). An additional 24 amino acids (38%), includingnumerous aromatics, were conserved in the majority of chiti-nase and Jessie CBDs. Jessie 1 and Jessie 2, which were 90 and97 amino acids long, respectively, each contained a single sitefor N-linked glycosylation and little else C-terminal to theCBD. Jessie 3, which was 621 amino acids long, contained aseries of hydrophilic amino acids C-prime to the 8-Cys CBD,similar to the hydrophilic repeats that precede the catalyticdomain of amebic chitinases (6). However, the C-terminaldomain of Jessie 3, which was 469 amino acids long, showed nohomology to the catalytic domains of chitinases or any otherproteins in GenBank (1). Northern blots with probes specific toeach hypothetical Jessie gene suggested that mRNAs for all ofthe Jessie lectins are absent from E. histolytica trophozoites(data not shown).

To test the function of the 8-Cys CBDs of the Jessie lectins,the entire Jessie 1 and Jessie 2 lectins and the putative CBD ofJessie 3 were expressed with a c-myc epitope tag in transfectedE. histolytica trophozoites by methods described above for theJacob lectin. While all three Jessie lectins were present in smallvesicles of transfected amebae (Fig. 2C to E), only Jessie 2 andthe Jessie 3 CBD bound to particulate chitin (Fig. 2F). It is notclear why Jessie 1 failed to bind chitin.

The presence of the same 8-Cys CBD in at least four differ-ent proteins (chitinase and three Jessie lectins) suggests thegene encoding this unique motif has been duplicated and re-inserted into the E. histolytica genome at least three times.Similarly, the 8-Cys CBDs of plants are present in chitinasesand lectins (3, 19, 29) and the 6-Cys CBDs of insects arepresent in chitinases and peritrophins (24), while a Cys-richsialic acid-binding domain is conserved in the P. falciparum175-kDa erythrocyte binding protein and other homologousproteins in Plasmodia (15).

Summary. We have identified Entamoeba genes encodingproteins that contain unique 6-Cys or 8-Cys CBDs. A modelshowing the 6-Cys CBDs of Entamoeba Jacob lectins and the8-Cys CBDs of chitinase and Jessie lectins is shown in Fig. 3.The CBDs of Entamoeba are similar to other known CBDs inthat they have conserved Cys and aromatic residues, but theyare not related by a common ancestry (convergent evolution)

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(3, 9, 11, 24, 29). The 6-Cys and 8-Cys CBDs are much easierto define than are the carbohydrate recognition domains of thelarge and intermediate subunits of the E. histolytica Gal/Gal-NAc lectin (4, 8, 20, 21). Future studies beyond the scope of

the present experiments will attempt to determine whetherJacob and Jessie lectins are located in the E. histolytica cyst wall.

Nucleotide sequence accession numbers. The sequences ofE. histolytica and E. dispar Jacob lectins have been deposited in

FIG. 2. (A) Primary structures in single letter code of the N-terminal regions of E. histolytica (Eh) and E. invadens (Ei) chitinases, as well asthree E. histolytica hypothetical lectins (Jessie 1 to Jessie 3). The complete sequences of Jessie 1 and Jessie 2 are marked by asterisks, whileC-terminal sequences of chitinases and Jessie 3 have been omitted. Signal sequences, identified with SignalP (22), are shown in gray, whileconserved Cys residues in CBDs are shown in red. Other amino acids identical in all five CBDs are shown in blue, while residues identical in atleast three of five CBDs are shown in dark red. Hydrophilic spacers are shown in green, while potential N-linked glycosylation sites in Jessie 1 andJessie 2 are shown in light orange. (B) Confocal micrograph of the E. histolytica chitinase CBD expressed in transfected E. histolytica trophozoitesand identified with antichitinase antibodies and Texas Red-labeled anti-rabbit IgG antibodies, using methods described in reference 14. (C to E)Confocal micrographs of the myc-tagged Jessie 1 (C), Jessie 2 (D), and Jessie 3 CBD (E) expressed in transfected E. histolytica trophozoites andidentified with anti-myc antibodies and Alexa Fluor 488-labeled anti-mouse IgG antibodies. (F) Western blot analysis of chitin binding by thefull-length E. histolytica chitinase, the E. histolytica chitinase CBD, Jessie 1, Jessie 2, and the Jessie 3 CBD, detected with antichitinase or anti-mycantibodies and chemiluminescence.

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GenBank under accession numbers AF401984 and AF401985,respectively. The sequences of the E. histolytica Jessie lectinshave been deposited in GenBank under accession numbersAF401986 to AF401988.

This work was supported in part by National Institutes of Healthgrants AI33492 (to J.S.), GM31318 (to P.R.), and AI46516 (to B.L.).

We acknowledge the expert technical support of Jean Lai of theHarvard School of Public Health for confocal microscopy.

REFERENCES1. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller,

and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generationof protein database search programs. Nucleic Acids Res. 25:3389–3402.

2. Arroyo-Begovich, A., and A. Carabez-Trejo. 1982. Location of chitin in thecyst wall of Entamoeba invadens with the colloidal gold tracers. J. Parasitol.68:253–258.

3. Beintema, J. J. 1994. Structural features of plant chitinases and chitin-binding proteins. FEBS Lett. 350:159–163.

4. Cheng, X. J., M. A. Hughes, C. D. Huston, B. Loftus, C. A. Gilchrist, L. A.Lockhart, S. Ghosh, V. Miller-Sims, B. J. Mann, W. A. Petri, Jr., and H.Tachibana. 2001. Intermediate subunit of the Gal/GalNAc lectin of Entam-oeba histolytica is a member of a gene family containing multiple CXXCsequence motifs. Infect. Immun. 69:5892–5898.

5. Das, S., and F. D. Gillin. 1991. Chitin synthase in encysting Entamoebainvadens. Biochem. J. 280:641–647.

6. de la Vega, H., C. A. Specht, C. E. Semino, P. W. Robbins, D. Eichinger, D.Caplivski, S. Ghosh, and J. Samuelson. 1997. Cloning and expression ofEntamoeba chitinases. Mol. Biochem. Parasitol. 85:139–147.

7. Diamond, L. S., and C. G. Clark. 1993. A redescription of Entamoebahistolytica Schaudinn, 1903 (emended Walker, 1911) separating it from En-tamoeba dispar Brumpt, 1925. J. Eukaryot. Microbiol. 40:340–344.

8. Dodson, J. M., P. W. Lenkowski, Jr., A. C. Eubanks, T. F. Jackson, J.

Napodano, D. M. Lyerly, L. A. Lockhart, B. J. Mann, and W. A. Petri, Jr.1999. Infection and immunity mediated by the carbohydrate recognitiondomain of the Entamoeba histolytica Gal/GalNAc lectin. J. Infect. Dis. 179:460–466.

9. Doolittle, R. F. 1994. Convergent evolution: the need to be explicit. TrendsBiochem. Sci. 19:15–18.

10. Eichinger, D. 1997. Encystation of entamoeba parasites. Bioessays 19:633–639.

11. Frisardi, M., S. K. Ghosh, J. Field, K. Van Dellen, R. Rogers, P. Robbins,and J. Samuelson. 2000. The most abundant glycoprotein of amebic cystwalls (Jacob) is a lectin with five Cys-rich, chitin-binding domains. Infect.Immun. 68:4217–4224.

12. Ghosh, S., J. Field, R. Rogers, M. Hickman, and J. Samuelson. 2000. TheEntamoeba histolytica mitochondrion-derived organelle (crypton) containsdouble-stranded DNA and appears to be bound by a double membrane.Infect. Immun. 68:4319–4322.

13. Ghosh, S., M. Frisardi, L. Ramirez-Avila, S. Descoteaux, K. Sturm-Ramirez,O. A. Newton-Sanchez, J. I. Santos-Preciado, C. Ganguly, A. Lohia, S. Reed,and J. Samuelson. 2000. Molecular epidemiology of Entamoeba spp.: evi-dence of a bottleneck (demographic sweep) and transcontinental spread ofdiploid parasites. J. Clin. Microbiol. 38:3815–3821.

14. Ghosh, S. K., J. Field, M. Frisardi, B. Rosenthal, Z. Mai, R. Rogers, and J.Samuelson. 1999. Chitinase secretion by encysting Entamoeba invadens andtransfected Entamoeba histolytica trophozoites: localization of secretory ves-icles, endoplasmic reticulum, and Golgi apparatus. Infect. Immun. 67:3073–3081.

15. Gupta, S., K. Treenholme, R. M. Anderson, and K. P. Day. 1994. Antigenicdiversity and the transmission dynamics of Plasmodium falciparum. Science263:961–963.

16. Henrissat, B., and A. Bairoch. 1993. New families in the classification ofglycosyl hydrolases based on amino acid sequence similarities. Biochem. J.293:781–788.

17. Kornfeld, R., and S. Kornfeld. 1985. Assembly of asparagine-linked oligo-saccharides. Annu. Rev. Biochem. 54:631–664.

18. Kuranda, M. J., and P. W. Robbins. 1991. Chitinase is required for cellseparation during growth of Saccharomyces cerevisiae. J. Biol. Chem. 266:19758–19767.

19. Lerner, D. R., and N. V. Raikhel. 1992. The gene for stinging nettle lectin(Urtica dioica agglutinin) encodes both a lectin and a chitinase. J. Biol.Chem. 267:11085–11091.

20. Lotter, H., T. Zhang, K. B. Seydel, S. L. Stanley, Jr., and E. Tannich. 1997.Identification of an epitope on the Entamoeba histolytica 170-kD lectinconferring antibody-mediated protection against invasive amebiasis. J. Exp.Med. 185:1793–1801.

21. Mann, B. J., B. E. Torian, T. S. Vedvick, and W. A. Petri, Jr. 1991. Sequenceof a cysteine-rich galactose-specific lectin of Entamoeba histolytica. Proc.Natl. Acad. Sci. USA 88:3248–3252.

22. Nielsen, H., S. Brunak, and G. von Heijne. 1999. Machine learning ap-proaches for the prediction of signal peptides and other protein sortingsignals. Protein Eng. 12:3–9.

23. Ortner, S., C. G. Clark, M. Binder, O. Scheiner, G. Wiedermann, and M.Duchene. 1997. Molecular biology of the hexokinase isoenzyme pattern thatdistinguishes pathogenic Entamoeba histolytica from nonpathogenic Entam-oeba dispar. Mol. Biochem. Parasitol. 86:85–94.

24. Shen, Z., and M. Jacobs-Lorena. 1999. Evolution of chitin-binding proteinsin invertebrates. J. Mol. Evol. 48:341–347.

25. Sonnhammer, E. L. L., G. von Heijne, and A. Krogh. 1998. A hidden Markovmodel for predicting transmembrane helices in protein sequences, p. 175–182. In J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C.Sensen (ed.), Proceedings of the Sixth International Conference on Intelli-gent Systems for Molecular Biology. AAAI Press, Menlo Park, Calif.

26. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W:improving the sensitivity of progressive multiple sequence alignment throughsequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Res. 22:4673–4680.

27. Venegas, A., J. C. Goldstein, K. Beauregard, A. Oles, N. Abdulhayoglu, andJ. A. Fuhrman. 1996. Expression of recombinant microfilarial chitinase andanalysis of domain function. Mol. Biochem. Parasitol. 78:149–159.

28. Villagomez-Castro, J. C., C. Calvo-Mendez, and E. Lopez-Romero. 1992.Chitinase activity in encysting Entamoeba invadens and its inhibition byallosamidin. Mol. Biochem. Parasitol. 52:53–62.

29. Wright, C. S. 1989. Comparison of the refined crystal structures of two wheatgerm isolectins. J. Mol. Biol. 209:475–487.

30. Yamagami, T., and G. Funatsu. 1996. Limited proteolysis and reduction-carboxymethylation of rye seed chitinase-�—role of the chitin-binding do-main in its chitinase action. Biosci. Biotechnol. Biochem. 60:1081–1086.

Editor: J. M. Mansfield

FIG. 3. Cartoons of Entamoeba lectins with unique 6-Cys (Jacob)and 8-Cys (chitinase and Jessie) CBDs.

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