Characterization of a UDP-N-acetyl-D-galactosamine:polypeptide
N-acetylgalactosaminyltransferase with an unusual lectin domain from the
platyhelminth parasite Echinococcus granulosus.


Freire T, Fernandez C, Chalar C, Maizels RM, Alzari P, Osinaga E, Robello C.

Departamento de Bioquimica, Facultad de Medicina, Universidad de la Republica,
Av. Gral. Flores 2125, Montevideo CP 11800, Uruguay.

As part of a general project aimed at elucidating the initiation of mucin-type
O-glycosylation in helminth parasites, we have characterized a novel ppGalNAc-T
(UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase)
from the cestode Echinococcus granulosus (Eg-ppGalNAc-T1). A full-length cDNA
was isolated from a library of the tissue-dwelling larval stage of the parasite,
and found to code for a 654-amino-acid protein containing all the structural
features of ppGalNAc-Ts. Functional characterization of a recombinant protein
lacking the transmembrane domain showed maximal activity at 28 degrees C, in the
range 6.5-7.5 pH units and in the presence of Cu2+. In addition, it transferred
GalNAc to a broad range of substrate peptides, derived from human mucins and
O-glycosylated parasite proteins, including acceptors containing only serine or
only threonine residues. Interestingly, the C-terminal region of Eg-ppGalNAc-T1
bears a highly unusual lectin domain, considerably longer than the one from
other members of the family, and including only one of the three ricin B repeats
generally present in ppGalNAc-Ts. Furthermore, a search for conserved domains
within the protein C-terminus identified a fragment showing similarity to a
recently defined domain, specialized in the binding of organic phosphates
(CYTH). The role of the lectin domain in the determination of the substrate
specificity of these enzymes suggests that Eg-ppGalNAc-T1 would be involved in
the glycosylation of a special type of substrate. Analysis of the tissue
distribution by in situ hybridization and immunohistochemistry revealed that
this transferase is expressed in the hydatid cyst wall and the subtegumental
region of larval worms. Therefore it could participate in the biosynthesis of
O-glycosylated parasite proteins exposed at the interface between E. granulosus
and its hosts.