Entamoeba histolytica, which causes
amebic dysentery and
liver abscesses, is spread via
chitin-walled
cysts. The most abundant
protein in the
cyst wall of Entamoeba invadens, a model for amebic encystation, is a
lectin called EiJacob1. EiJacob1 has five tandemly arrayed, six-Cys chitin-binding domains separated by low-complexity Ser- and Thr-rich spacers. E. histolytica also has numerous predicted Jessie
lectins and
chitinases, which contain a single, N-terminal eight-Cys chitin-binding domain. We hypothesized that E. invadens
cyst walls are composed entirely of
proteins with six-Cys or eight-Cys chitin-binding domains and that some of these
proteins contain
sugars. E. invadens genomic sequences predicted seven Jacob
lectins, five Jessie
lectins, and three
chitinases. Reverse transcription-PCR analysis showed that mRNAs encoding Jacobs, Jessies, and
chitinases are increased during E. invadens encystation, while mass spectrometry showed that the
cyst wall is composed of an approximately 30:70 mix of Jacob
lectins (cross-linking
proteins) and Jessie and
chitinase lectins (possible
enzymes). Three Jacob
lectins were cleaved prior to Lys at conserved sites (e.g., TPSVDK) in the Ser- and Thr-rich spacers between chitin-binding domains. A model
peptide was cleaved at the same site by
papain and E. invadens Cys
proteases, suggesting that the latter cleave Jacob
lectins in vivo. Some Jacob
lectins had O-phosphodiester-linked
carbohydrates, which were one to seven
hexoses long and had deoxysugars at reducing ends. We concluded that the major
protein components of the E. invadens
cyst wall all contain chitin-binding domains (
chitinases, Jessie
lectins, and Jacob
lectins) and that the Jacob
lectins are differentially modified by site-specific Cys
proteases and O-phosphodiester-linked
glycans.