Expression and characterization of the first snail-derived UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase

Abstract

UDP-GalNAc:polypeptide GalNAc transferase (ppGalNAcT; EC 2.4.1.41) catalyzes the first step in mucin-type O-glycosylation. To date, several members of this large enzyme family have been analyzed in detail. In this study we present cloning, expression and characterization of the first representative of this type of glycosyltransferase from mollusk origin, namely from Biomphalaria glabrata. The full length sequence of the respective gene was obtained by screening of a cDNA library using homology-based PCR. The entire gene codes for a protein consisting of 600 amino acids comprising the features of a typical type II membrane protein containing a cytoplasmic tail at the N-terminus, a transmembrane and a catalytic domain as well as a ricin-like motif at the C-terminus. Sequence comparison with ppGalNAcTs from various species revealed high similarities in terms of structural architecture. The enzyme is O-glycosylated but does not have any putative N-glycosylation sites. All four tested acceptor peptides were functional substrates, with Muc2 being the best one. Further biochemical parameters tested, confirmed a close relationship to the family of yet known ppGalNAcTs.

UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase from the snail Biomphalaria glabrata – structural reflections

“…Glycosyltransferases are known to require divalent cations for their activity. For Bge-ppGalNAcT we found no activity in the presence of the complexing agent EDTA and an order of increasing activity using Ca 2+ < Mg 2+ < Co 2+ < Mn 2+ , while Cu 2+ completely abolished the activity [17]. Trying to explain this order we found that the activity of Bge-ppGalNAcT in the presence of various metals notably follows the Irving-Williams series of relative stabilities of metal complexes.…”

“…A cDNA library was synthesized from embryonic cells from Biomphalaria glabrata (Bge cells, NR-40248, BEI Resources, NIAID, NIH). The enzyme, Bge-ppGalNAcT, without cytoplasmic tail and transmembrane domain (amino acid 1-26) was expressed and purified exactly as in [17]. A further truncated version, ΔppGalNAcT, omitting the complete lectin domain (lacking amino acid 477-600) was obtained in the same way.…”

“…In invertebrates homologous enzymes have been described in insects (Drosophila melanogaster, Bombyx mori), worms (Caenorhabditis elegans, Fasciola hepática, Echinococcus granulosus), parasites (Toxoplasma gondi, Trypanosoma cruzi, Cryptosporidium species) and the purple sea urchin (Stronglylocetrotus purpuratus) [6][7][8][9][10][11][12][13][14][15][16], but never in bacteria, plants or fungi. A ppGalNAcT from the freshwater snail Biomphalaria glabrata (GenBank: KC182513) is the only characterised member of this enzyme family from mollusc origin [17,18].…”

“…All of them display a GT-A fold but can be clustered in groups and subgroups by some differences in their primary structures [2]. The enzyme from Biomphalaria glabrata is a homologue of human ppGalNAcT2 and therefore belongs to group Ib [17].…”

Position of human blood group O(H) and phenotype‐determining enzymes in growth and infectious disease

“…), as early metazoan eukaryotic O ‐glycans, Tn, and T 24 antigens have been identified. These ancestral glycans arising from O ‐glycosylations are used (with similar peptide backbones) by lower metazoans such as mollusks and the fruit fly Drosophila melanogaster , and in the snail Helix pomatia , they are associated with the release of a hexamerically structured Tn‐complementary hemagglutinating defense protein. Hammarström showed that the binding patterns and the capacity of this molluscan protein to bind human blood group A RBCs are strikingly similar to those of the mammalian IgM molecule, giving rise to speculation regarding an evolutionary relationship with the mammalian nonimmune anti‐A‐reactive IgM molecule.…”