An acetylation site in lectin domain modulates the biological activity of polypeptide GalNAc-transferase-2

Abstract

Polypeptide GalNAc-transferases (ppGalNAc-Ts) are a family of enzymes that catalyze the initiation of mucin-type O-glycosylation. All ppGalNAc-T family members contain a common (QXW)3 motif, which is present in the R-type lectin group. The acetylation site K521 is part of the QKW motif of β-trefoil in the lectin domain of ppGalNAc-T2. We used a combination of acetylation and site-directed mutagenesis approaches to examine the functional role of K521 in ppGalNAc-T2. Binding assays of non-acetylated and acetylated forms of the mutant ppGalNAc-T2K521Q to various naked and αGalNAc-glycosylated mucin peptides indicated that the degree of interaction of lectin domain with αGalNAc depends on the peptide sequence of mucin. Studies of the inhibitory effect of various carbohydrates on the interactions of ppGalNAc-T2 with MUC1αGalNAc indicate that point K521Q mutation enhance the carbohydrate specificity of lectin domain for αGalNAc. K521Q mutation resulted in an enzyme activity lower than that of the wild-type ppGalNAc-T2, similar to the acetylation of ppGalNAc-T2. We conclude that an acetylation site in the QKW motif of the lectin domain modulates carbohydrate recognition specificity and catalytic activity of ppGalNAc-T2 for partially preglycosylated acceptors and a certain naked peptide. Posttranslational modifications of ppGalNAc-Ts, such as acetylation, may play key roles in modulating the functions of the R-type lectin domains in cellular homeostasis.

d‐Amino acid substitutions and dimerization increase the biological activity and stability of an IL‐15 antagonist peptide

“…However, the N‐terminal acetylation of the [A4a]P8 peptide and its dimer decreases the IC 50 of these peptides at least three‐fold with respect to the values calculated for the nonacetylated variants. Other studies show that N‐terminal acetylation of certain peptides module the biological activity of the modified molecule with respect to the native peptide 41–43 . Demonstrating that the N‐acetylation of the monomer and dimer of the [A4a]P8 peptide affects its biological activity represents a drawback in the development of an IL‐15 antagonist peptide capable of displacing the high‐affinity interaction between the human IL‐15 and its private alpha chain 44…”

Extrinsic Functions of Lectin Domains in O-N-Acetylgalactosamine Glycan Biosynthesis

“…Color was developed with 0.5 mg/ml o-phenylenediamine and 0.02% H 2 O 2 in sodium citrate (pH 5.0) at room temperature, and the reaction was stopped by addition of 0.5 N H 2 SO 4 (50 l/well). Absorbance was read by microplate reader at 490 nm (33). Non-linear fitting of curves and enzyme kinetic parameters were obtained using the GraphPad Prism 5 software program.…”