The sialyl-O-acetylesterase NanS of Tannerella forsythia encompasses two catalytic modules with different regiospecificity for O7 and O9 of sialic acid.

The periodontal pathogen Tannerella forsythia utilizes host sialic acids as a nutrient source. To also make O-acetylated sialyl residues susceptible to the action of its sialidase and sialic acid uptake system, Tannerella produces NanS, an O-acetylesterase with two putative catalytic domains. Here, we analyzed NanS by homology modeling, predicted a catalytic serine-histidine-aspartate triad for each catalytic domain and performed individual domain inactivation by single alanine exchanges of the triad nucleophiles S32 and S311. Subsequent functional analyses revealed that both domains possess sialyl-O-acetylesterase activity, but differ in their regioselectivity with respect to position O9 and O7 of sialic acid. The 7-O-acetylesterase activity inherent to the C-terminal domain of NanS is unique among sialyl-O-acetylesterases and fills the current gap in tools targeting 7-O-acetylation. Application of the O7-specific variant NanS-S32A allowed us to evidence the presence of cellular 7,9-di-O-acetylated sialoglycans by monitoring the gain in 9-O-acetylation upon selective removal of acetyl groups from O7. Moreover, we established de-7,9-O-acetylation by wild-type NanS as an easy and efficient method to validate the specific binding of three viral lectins commonly used for the recognition of (7),9-O-acetylated sialoglycans. Their binding critically depends on an acetyl group in O9, yet de-7,9-O-acetylation proved advantageous over de-9-O-acetylation as the additional removal of the 7-O-acetyl group eliminated ligand formation by 7,9-ester migration. Together, our data show that NanS gained dual functionality through recruitment of two esterase modules with complementary activities. This enables Tannerella to scavenge 7,9-di-O-acetylated sialyl residues and provides a novel, O7-specific tool for studying sialic acid O-acetylation.

Low galactosylation of IgG associates with higher risk for future diagnosis of rheumatoid arthritis during 10 years of follow-up.

Antibodies are known to have an important role in the development of rheumatoid arthritis (RA), one of the most prevalent chronic inflammatory diseases which primarily involves the joints. Most RA patients develop autoantibodies against immunoglobulin G (IgG) and changes in IgG glycosylation have been associated with RA. We undertook this study to determine whether altered IgG glycosylation precedes the disease diagnosis. We studied IgG glycosylation in RA in two prospective cohorts (N = 14,749) by measuring 28 IgG glycan traits in 179 subjects who developed RA within 10-years follow-up and 358 matched controls. Ultra-performance liquid chromatography method based on hydrophilic interactions (HILIC-UPLC) was used to analyse IgG glycans. Future RA diagnosis associated with traits related to lower galactosylation and sialylation of IgG when comparing the cases to the matched controls. In RA cases, these traits did not correlate with the time between being recruited to the study and being diagnosed with RA (median time 4.31 years). The difference in IgG glycosylation was relatively stable and present years before diagnosis. This indicates that long-acting factors affecting IgG glycome composition are among the underlying mechanisms of RA and that decreased galactosylation is a pre-existing risk factor involved in the disease development.

Semi-high-throughput isolation and N-glycan analysis of human fibrinogen using monolithic supports bearing monoclonal anti-human fibrinogen antibodies.

Fibrinogen (FIB) is a secretory glycoprotein synthesized by hepatocytes that has a key role in blood clotting. Its glycosylation has not been studied in detail and little is known about the biological variability of FIB N-glycosylation, mainly due to the lack of fast, simple, and robust approaches to purify FIB from blood plasma samples. In recent years, customised chromatographic monoliths have been used for a variety of biological applications due to their unique characteristics. Here we describe development and optimisation of monolithic supports bearing monoclonal anti-human fibrinogen antibodies in a single column as well as in multi-well plate formats with high FIB specificity and binding capacity for fast immunoaffinity purification of FIB from human blood samples. The developed semi-high-throughput workflow has been successfully applied for FIB immunoaffinity isolation and subsequent ultra performance liquid chromatography N-glycosylation analysis in ten healthy human individuals, demonstrating the potential of monolithic supports in glycomics studies.

The Resveratrol Tetramer r-Viniferin Induces a Cell Cycle Arrest Followed by Apoptosis in the Prostate Cancer Cell Line LNCaP.

Polyphenols are secondary plant metabolites that possess potentially health-promoting properties and which occur in various edible plants and plant products. Especially the stilbenoid resveratrol has been extensively studied regarding its anticarcinogenic and chemopreventive activities. However, research has recently focused on the investigation of other natural or synthetic compounds in order to find substances that show a higher bioactivity and/or bioavailability than resveratrol. In this context, we exemplarily investigated the cytotoxic/growth-inhibiting properties of the resveratrol tetramer r-viniferin on the prostate cancer cell line LNCaP and compared them with those of resveratrol. By using the sulforhodamine B assay followed by cell cycle analysis via flow cytometry and commercially available apoptosis/necrosis assay kits, we show that both compounds were able to inhibit the growth of LNCaP cells and to induce a cell cycle arrest in the G1 phase. However, r-viniferin was significantly more potent in inhibiting cellular growth than resveratrol and the only compound that increased the apoptotic cellular fraction as well as the activity of apoptosis-associated enzymes. In conclusion, r-viniferin leads to cytotoxicity in LNCaP cells at fairly low concentrations, and it is therefore conceivable that it might be used as a chemopreventive agent or as an adjuvant in prostate cancer therapy.

Role of Sialyl-O-Acetyltransferase CASD1 on GD2 Ganglioside O-Acetylation in Breast Cancer Cells.

The O-acetylated form of GD2, almost exclusively expressed in cancerous tissues, is considered to be a promising therapeutic target for neuroectoderm-derived tumors, especially for breast cancer. Our recent data have shown that 9-O-acetylated GD2 (9-OAcGD2) is the major O-acetylated ganglioside species in breast cancer cells. In 2015, Baumann et al. proposed that Cas 1 domain containing 1 (CASD1), which is the only known human sialyl-O-acetyltransferase, plays a role in GD3 O-acetylation. However, the mechanisms of ganglioside O-acetylation remain poorly understood. The aim of this study was to determine the involvement of CASD1 in GD2 O-acetylation in breast cancer. The role of CASD1 in OAcGD2 synthesis was first demonstrated using wild type CHO and CHOΔCasd1 cells as cellular models. Overexpression using plasmid transfection and siRNA strategies was used to modulate CASD1 expression in SUM159PT breast cancer cell line. Our results showed that OAcGD2 expression was reduced in SUM159PT that was transiently depleted for CASD1 expression. Additionally, OAcGD2 expression was increased in SUM159PT cells transiently overexpressing CASD1. The modulation of CASD1 expression using transient transfection strategies provided interesting insights into the role of CASD1 in OAcGD2 and OAcGD3 biosynthesis, and it highlights the importance of further studies on O-acetylation mechanisms.