Identification of proteins interacting with purified brush
border from human enterocytes

The first peptide, LYLPYYFSVTK, corresponded to the E. histolytica Gal/GalNAc lectin Igl1 and Igl2 proteins (amino acids 457­467 of locus 53.m00171/119.m00118 respectively). Igl is a 150 kDa protein family that interacts with the Gal/GalNAc lectin complex (Cheng et al., 1998). The protein must have been proteolysed on the amoeba surface or during the experiment leading to a molecular weight of 75 kDa instead of 150 kDa.

from "A lysine- and glutamic acid-rich protein, KERP1, from Entamoeba histolytica binds to human entero"
by Marie Seigneur, Joelle Mounier, Marie-Christine Prevost and Nancy Guillén
in Cellular Microbiology (2005) 7, (4), 56

Isolation of BB:

Isolation of BB from Caco2 cells was based on standard protocols used to obtain BB from intestinal mucosa (Keller and Mooseker, 1982).

cell cuture:
Briefly, 3 ¥ 107 cells grown for 14 days were washed in PBS, washed in buffer A (Imidazole 10 mM pH 7.4, EDTA 5 mM, EGTA 1 mM, DTT 0.2 mM and protease inhibitor from Sigma), incubated for 20 min in buffer A and scraped.

homoenisation:
After centrifugation during 5 min at 300 g the cell pellet was resuspended in 0.5 ml of buffer A and passed once through the cell cracker (EMBL, Precision Engineering, Heidelberg, Germany).
Cell homogenate was adjusted to 4 ml with buffer A and centrifuged for 10 min at 1000 g. The pellet was washed three times in buffer A, and twice in buffer B.

BBisolation
Brush borders were isolated from contaminated nuclei by resuspending the final pellet in 42% sucrose in buffer B and layered on a cushion of 50% sucrose before centrifugation at 45 000 g. Brush borders were collected at the interface of the two sucrose solutions. The quality of the preparation was monitored by light microscopy.

E. histolytica crude protein extracts
Total E. histolytica crude protein extracts were obtained from 106 trophozoites. These were washed in PBS and lysed in 50 ml of 10 mM Tris (pH 7.5), protease inhibitor mixture [10 mM leupeptine (Sigma), 1 mM N-ethylmaleimide (Sigma), 2 mM p-chloromercuribenzoate (Sigma), 2 mM 4-(2-aminoethyl) benzenesulphonyl fluoride (Uptima), complete mini EDTA-free (protease inhibitor cocktail, Roche)] and 1% SDS at 100C for 10 min. Entamoeba histolytica cytoplasm, vesicles and internal membranes and plasma membranes were isolated using the amoebaspecific sugar gradient separation technique (Aley et al., 1980). Biotinylation coupled to streptavidin revelation of trophozoite surface proteins were made as described by Andrews and Bjorvatn (1991).

Brush border from differentiated Caco2 cells was purified and coated on affigel beads. Purified biotinylated parasite plasma membrane proteins were submitted to interaction with these beads. After elution, we recovered a fraction enriched in biotinylated proteins interacting with the BB, indicating that the conditions used for this affinity chromatography were useful. Nevertheless, abundant parasite proteins were still bound to the beads especially the Gal/GalNAc lectin. In preparative experiments using non-biotinylated proteins, we succeeded in recovering proteins with molecular masses of roughly 75, 37 and 20 kDa

The three bands recovered from acrylamide gel were endolysine digested, the peptides were separated by HPLC and the amino acid sequence of some peptides was successfully determined. Computer search for protein identity by using the peptide sequences allowed us to determine the nature of some proteins revealed in this experiment.

LYLPYYFSVTK
Leu-Tyr-Leu-ProTyr-Tyr-Phe-Ser-Val-Thr-Lys
http://www.tigr.org/tdb/e2k1/eha1/
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 53.m00171
119.m00118
Gal/GalNAc lectin complex

http://tigrblast.tigr.org/er-blast/index.cgi?project=eha1