The beta-N-acetylhexosaminidase of Entamoeba histolytica is composed of two homologous chains and has been localized to cytoplasmic granules.
We have purified a beta-N-acetylhexosaminidase from trophozoites of Entamoeba histolytica to homogeneity. In SDS-PAGE, the enzyme yielded a single protein band at an apparent M(r) of 64,000. The elution behaviour of the native enzyme upon molecular sieve chromatography corresponded to a molecular mass of approximately 132,000 suggesting that the enzyme is a dimer. Upon sedimentation velocity centrifugation, hexosaminidase activity sedimented at 12S, implying aggregation to a higher molecular mass complex with an apparent M(r) of approximately 400,000. Based on the N-terminal sequence of the purified enzyme and on data extracted from the E. histolytica genomic data base, we amplified and cloned two genes (EhHEXA and EhHEXB) coding for two presumptive, highly similar hexosaminidase chains which we designated as Ehhexalpha and Ehhexbeta. Northern blot analysis indicated that the two genes were expressed to a similar level, and Western blotting with chain-specific antisera showed that the trophozoites synthesize both proteins. By cell fractionation, the hexosaminidase was found to be a major component of cytoplasmic granules; these contain tissue-destructive factors and are released after collagen-induced exocytosis to the cell surface. In agreement with this observation, immunocytochemistry with an antiserum cross-reacting with both hexosaminidase chains revealed strong fluorescence in surface patches, which we interpret as released granules, and in vesicles throughout the cell. Its localization in cytoplasmic granules strengthens the notion that the hexosaminidase complex may contribute to amoebic pathogenicity.
Riekenberg
Sabine
S
Department of Biochemistry, University of Osnabrueck, 49069 Osnabrueck, Germany.
Flockenhaus
Bettina
B
Vahrmann
Anke
A
Müller
Monika C M
MC
Leippe
Matthias
M
Kiess
Michael
M
Scholze
Henning
H
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002458:Cell Fractionation; D002850:Chromatography, Gel; D003594:Cytoplasmic Granules / Q000201:enzymology*; D016054:DNA, Protozoan / Q000737:chemistry / Q000302:isolation & purification; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D015870:Gene Expression; D017125:Genes, Protozoan; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015800:Protozoan Proteins / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis; D016053:RNA, Protozoan / Q000032:analysis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D001619:beta-N-Acetylhexosaminidases / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D016054:DNA, Protozoan; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D001619:beta-N-Acetylhexosaminidases