9 research outputs found
Characterization of the Sialate-7(9)-O-Acetyltransferase from the Microsomes of Human Colonic Mucosa
Genetic and biochemical modulation of sialic acid O-acetylation on group B Streptococcus: Phenotypic and functional impact
Group B Streptococcus (GBS) is an important human pathogen and a model system for studying the roles of bacterial glycosylation in host–microbe interactions. Sialic acid (Sia), expressed prominently in the GBS capsular polysaccharide (CPS), mimics mammalian cell surface Sia and can interact with host Sia-binding proteins to subvert immune clearance mechanisms. Our earlier work has shown that GBS partially O-acetylates CPS Sia residues and employs an intracellular O-acetylation/de-O-acetylation cycle to control the final level of this surface Sia modification. Here, we examine the effects of point mutations in the NeuD O-acetyltransferase and NeuA O-acetylesterase on specific glycosylation phenotypes of GBS, pinpointing an isogenic strain pair that differs dramatically in the degree of the O-acetyl modification (80% versus 5%) while still expressing comparable levels of overall sialylation. Using these strains, higher levels of O-acetylation were found to protect GBS CPS Sia against enzymatic removal by microbial sialidases and to impede engagement of human Siglec-9, but not to significantly alter the ability of GBS to restrict complement C3b deposition on its surface. Additional experiments demonstrated that pH-induced migration of the O-acetyl modification from the 7- to 9-carbon position had a substantial impact on GBS–Siglec-9 interactions, with 7-O-acetylation exhibiting the strongest interference. These studies show that both the degree and position of the GBS O-acetyl modification influence Sia-specific interactions relevant to the host–pathogen relationship. We conclude that native GBS likely expresses a phenotype of intermediate Sia O-acetylation to strike a balance between competing selective pressures present in the host environment
9-O-Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate
Sialic acids, terminal sugars of glycoproteins and glycolipids, play important roles in development, cellular recognition processes and host–pathogen interactions. A common modification of sialic acids is 9-O-acetylation, which has been implicated in sialoglycan recognition, ganglioside biology, and the survival and drug resistance of acute lymphoblastic leukaemia cells. Despite many functional implications, the molecular basis of 9-O-acetylation has remained elusive thus far. Following cellular approaches, including selective gene knockout by CRISPR/Cas genome editing, we here show that CASD1—a previously identified human candidate gene—is essential for sialic acid 9-O-acetylation. In vitro assays with the purified N-terminal luminal domain of CASD1 demonstrate transfer of acetyl groups from acetyl-coenzyme A to CMP-activated sialic acid and formation of a covalent acetyl-enzyme intermediate. Our study provides direct evidence that CASD1 is a sialate O-acetyltransferase and serves as key enzyme in the biosynthesis of 9-O-acetylated sialoglycans
High level of sialate-O-acetyltransferase activity in lymphoblasts of childhood acute lymphoblastic leukaemia (ALL): enzyme characterization and correlation with disease status
Previous studies had established an over-expression
of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs)
on lymphoblasts of childhood acute lymphoblastic leukaemia
(ALL). Here, we report the discovery and characterization
of sialate-O-acetyltransferase enzyme in ALL-cell
lines and lymphoblasts from bone marrow of children
diagnosed with B- and T-ALL. We observed a positive
correlation between the enhanced sialate-O-acetyltransferase
activity and the enhanced expression of Neu5,9Ac2-GPs
in these lymphoblasts. Sialate-O-acetyltransferase activity
in cell lysates or microsomal fractions of lymphoblasts of
patients was always higher than that in healthy donors
reaching up to 22-fold in microsomes. Additionally, the Vmax of this enzymatic reaction with AcCoA was over
threefold higher in microsomal fractions of lymphoblasts.
The enzyme bound to the microsomal fractions showed
high activity with CMP-N-acetylneuraminic acid, ganglioside
GD3 and endogenous sialic acid as substrates. Nacetyl-
7-O-acetylneuraminic acid was the main reaction
product, as detected by radio-thin-layer chromatography
and fluorimetrically coupled radio-high-performance liquid
chromatography. CMP and coenzyme A inhibited the
microsomal enzyme. Sialate-O-acetyltransferase activity
increased at the diagnosis of leukaemia, decreased with
clinical remission and sharply increased again in relapsed
patients as determined by radiometric-assay. A newlydeveloped
non-radioactive ELISA can quickly detect
sialate-O-acetyltransferase, and thus, may become a suitable
tool for ALL-monitoring in larger scale. This is the
first report on sialate-O-acetyltransferase in ALL being one
of the few descriptions of an enzyme of this type in human
Sialic acid metabolism and sialyltransferases: natural functions and applications
Sialic acids are a family of negatively charged monosaccharides which are commonly presented as the terminal residues in glycans of the glycoconjugates on eukaryotic cell surface or as components of capsular polysaccharides or lipooligosaccharides of some pathogenic bacteria. Due to their important biological and pathological functions, the biosynthesis, activation, transfer, breaking down, and recycle of sialic acids are attracting increasing attention. The understanding of the sialic acid metabolism in eukaryotes and bacteria leads to the development of metabolic engineering approaches for elucidating the important functions of sialic acid in mammalian systems and for large-scale production of sialosides using engineered bacterial cells. As the key enzymes in biosynthesis of sialylated structures, sialyltransferases have been continuously identified from various sources and characterized. Protein crystal structures of seven sialyltransferases have been reported. Wild-type sialyltransferases and their mutants have been applied with or without other sialoside biosynthetic enzymes for producing complex sialic acid-containing oligosaccharides and glycoconjugates. This mini-review focuses on current understanding and applications of sialic acid metabolism and sialyltransferases