Dataset Information

Expression and characterization of the first snail-derived UDP-N-acetyl-?-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.

SUBMITTER: Taus C

PROVIDER: S-EPMC3825155 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Publications

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

Taus Christopher C Lucini Chantal C Sato Takeshi T Furukawa Kiyoshi K Grabherr Reingard R Staudacher Erika E

Glycoconjugate journal 20130723 9

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-b ...[more]

PMID: 23877648

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Project description:O-Glycan biosynthesis is initiated by the transfer of N-acetylgalactosamine (GalNAc) from a nucleotide sugar donor (UDP-GalNAc) to Ser/Thr residues of an acceptor substrate. The detailed transfer mechanism, catalyzed by the UDP-GalNAc polypeptide:N-acetyl-alpha-galactosaminyltransferases (ppGalNAcTs), remains unclear despite structural information available for several isoforms in complex with substrates at various stages along the catalytic pathway. We used all-atom molecular dynamics simulations with explicit solvent and counterions to study the conformational dynamics of ppGalNAcT-2 in several enzymatic states along the catalytic pathway. ppGalNAcT-2 is simulated both in the presence and in the absence of substrates and reaction products to examine the role of conformational changes in ligand binding. In multiple 40-ns-long simulations of more than 600 ns total run time, we studied systems ranging from 45,000 to 95,000 atoms. Our simulations accurately identified dynamically active regions of the protein, as previously revealed by the X-ray structures, and permitted a detailed, atomistic description of the conformational changes of loops near the active site and the characterization of the ensemble of structures adopted by the transferase complex on the transition pathway between the ligand-bound and ligand-free states. In particular, the conformational transition of a functional loop adjacent to the active site from closed (active) to open (inactive) is correlated with the rotameric state of the conserved residue W331. Analysis of water dynamics in the active site revealed that internal water molecules have an important role in enhancing the enzyme flexibility. We also found evidence that charged side chains in the active site rearrange during site opening to facilitate ligand binding. Our results are consistent with the single-displacement transfer mechanism previously proposed for ppGalNAcTs based on X-ray structures and mutagenesis data and provide new evidence for possible functional roles of certain amino acids conserved across several isoforms.

Dataset Information

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

Publications

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

Similar Datasets

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