title: Characterization of a UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase with an unusual lectin domain from the platyhelminth parasite Echinococcus granulosus.
abstract: 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.
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, Montevideo CP 11800, Uruguay.
authors.forename: Teresa
authors.initials: T
authors.lastname: Freire
authors.forename: Cecilia
authors.initials: C
authors.lastname: Fernández
authors.forename: Cora
authors.initials: C
authors.lastname: Chalar
authors.forename: Rick M
authors.initials: RM
authors.lastname: Maizels
authors.forename: Pedro
authors.initials: P
authors.lastname: Alzari
authors.forename: Eduardo
authors.initials: E
authors.lastname: Osinaga
authors.forename: Carlos
authors.initials: C
authors.lastname: Robello
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002418:Cattle Diseases / Q000201:enzymology / Q000469:parasitology; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003300:Copper / Q000502:physiology; D018076:DNA, Complementary / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics; D004443:Echinococcosis / Q000201:enzymology / Q000662:veterinary; D048209:Echinococcus granulosus / Q000201:enzymology*; D015801:Helminth Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D037102:Lectins / Q000737:chemistry* / Q000235:genetics; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D010455:Peptides / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity / Q000235:genetics; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D003300:Copper; D018076:DNA, Complementary; D018509:DNA, Helminth; D015801:Helminth Proteins; D037102:Lectins; D008345:Manganese; D017350:N-Acetylgalactosaminyltransferases; D010455:Peptides; D000097763:Polypeptide N-acetylgalactosaminyltransferase