Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Sep 1;382(Pt 2):501-10.
doi: 10.1042/BJ20031877.

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

Teresa Freire  1 , Cecilia Fernández, Cora Chalar, Rick M Maizels, Pedro Alzari, Eduardo Osinaga, Carlos Robello
Affiliations

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

Teresa Freire et al. Biochem J. .

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Sequence analysis of Eg-ppGalNAc-T1
(A) Multiple sequence alignment of the predicted amino acid sequence from Eg-ppGalNAc-T1 cDNA with the C. elegans ppGalNAc-T family. Protein sequences were aligned for maximum identity by introducing gaps using ClustalW (1.82) as a progressive tool. Amino acids are shaded black if identical, dark grey if strongly similar and light grey if weakly similar. Conserved structural domains are highlighted with arrows and the localization of the catalytic domain is indicated by dotted lines. Boxed amino acids represent conserved residues from the lectin domain. The putative CYTH domain of Eg-ppGalNAc-T1 is underlined. The GenBank® accession numbers of the aligned sequences are AY353720 (Eg-ppGalNAc-T1), AF031833 (gly-3), AF031834 (gly-4), AF031835 (gly-5a), AF031836 (gly-5b), AF031837 (gly-5c), AF031838 (gly-6a), AF031841 (gly-7), AF031842 (gly-8) and AF031843 (gly-9). (B) Alignment of the C-terminal region from Eg-ppGalNAc-T1 and the CYTH consensus. Protein sequences were aligned for maximum identity by introducing gaps using ClustalW (1.82) as a progressive tool. Amino acids are shaded in black if identical and in grey if similar.
Figure 2
Figure 2. Expression of Eg-ppGalNAc-T1 in COS-7-transfected cells and purification of the recombinant enzyme
(A) A partial sequence of Eg-ppGalNAc-T1 lacking the predicted transmembrane domain was expressed in COS-7 cells. The expression of Eg-ppGalNAc-T1 was confirmed by Western blot using a rabbit antiserum raised against a fragment of Eg-ppGalNAc-T1 catalytic domain. No reactivity was observed when the antiserum was used to probe an extract from non-transfected cells. (B) The recombinant protein was affinity-purified from the cell extracts over a column prepared with the purified IgG fraction from the antiserum used in (A), immobilized on to CNBr-activated Sepharose, and analysed by SDS/PAGE (10% gel) followed by silver staining.
Figure 3
Figure 3. pH dependence of Eg-ppGalNAc-T1 activity
The transferase activity was evaluated with dOSM and UDP-(3H)-GalNAc at various pH values. Results are expressed in pmoles of transferred 3H-GalNAc·(mg of protein)−1·h−1. Bars correspond to the S.D. of three determinations.
Figure 4
Figure 4. Expression of Eg-ppGalNAc-T1 in E. granulosus larval stages: in situ hybridization analysis
Protoscolex and cyst wall sections were hybridized with digoxigenin-labelled anti-sense RNA probes directed against the catalytic domain (A–C) or the C-terminus (D–F) of the enzyme; the reaction was developed using an alkaline phosphatase-conjugated anti-digoxigenin antibody. A protoscolex section assayed in parallel with a sense probe (inset to A) showed no reactivity. Arrowheads indicate the protoscolex suckers and asterisks the germinal layer. Localization of other structures mentioned in the text is marked in the evaginated larval worm shown in F (SU, suckers; P, parenchyma; ST, subtegument). Scale bars: (A, D), 100 μm; (B, C, E, F), 50 μm.
Figure 5
Figure 5. Expression of Eg-ppGalNAc-T1 in E. granulosus larval stages: immunohistochemical analysis
Protoscolex and cyst wall sections were incubated with polyclonal antibodies raised against recombinant fragments corresponding to the catalytic domain (A, B) or the C-terminus (C, D) of the enzyme; the reaction was developed using a goat anti-rabbit conjugated to Alexa-Fluor 488. A protoscolex section, assayed in parallel with PBS (negative control), showed no reactivity (E). Arrowheads indicate the protoscolex suckers and asterisks the germinal layer. See Figure 4(F) for the localization of other structures mentioned in the text. Scale bars: (A, C, E), 100 μm; (B, D), 50 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Thompson R. C. A. Biology and systematics of Echinococcus. In: Lymbery A. J., Thompson R. C. A., editors. Biology and Systematics of Echinococcus. Wallingford: CAB International; 1995. pp. 1–50.
    1. Theodoropoulos G., Hicks S., Corfield A., Miller B., Carrington S. The role of mucins in host–parasite interactions: part II – helminth parasites. Trends Parasitol. 2001;17:130–135. - PubMed
    1. Loukas A., Hintz M., Linder D., Mullin N., Parkinson J., Tetteh K., Maizels R. A family of secreted mucins from the parasitic nematode Toxocara canis bears diverse mucin domains but shares similar flanking six-cysteine repeat motifs. J. Biol. Chem. 2000;275:39600–39607. - PubMed
    1. Hülsmeier A., Gehrig P., Geyer R., Sack R., Gottstein B., Deplazes P., Köhler P. A major Echinococcus multilocularis antigen is a mucin-type glycoprotein. J. Biol. Chem. 2002;277:5742–5748. - PubMed
    1. Hakomori S. Aberrant glycosylation in tumors and tumor-associated carbohydrate antigens. Adv. Cancer Res. 1989;52:257–331. - PubMed

Publication types

MeSH terms