The prevalence of gentamicin 2'-N-acetyltransferase in the Proteeae and its role in the O-acetylation of peptidoglycan

Abstract The prevalence of aac(2')-Ia , a gene coding for gentamicin 2'-JV-acetyltransferase in Providenda stuartii , among species of the Proteeae was investigated to determine if it is a common resistance factor and whether the correlation observed in P. stuartii between its expression and the levels of peptidoglycan O -acetylation represents a general feature of bacteria producing this form of modified peptidoglycan. An evaluation of the MICs of gentamicin for each of the species of the Proteeae did not reveal any apparent relationship between resistance and the degree of O-acetylation of peptidoglycan. The entire aac(2')-Ia gene was used as a probe in Southern hybridization experiments against genomic DNA from each species of the Proteeae. A sequence with strong homology to aac(2')-Ia was present only in Proteus penneri while weak hybridization was also observed to the restriction digested DNA from Providenda rettgeri . Other bacteria that O -acetylate peptidoglycan were also screened with this probe and a homologous DNA sequence was only found in Neisseria subflava . These data suggest that AAC(2')-Ia may contribute to the rO -acetylation of peptidoglycan in P. stuartii , but a more specific enzyme must also be produced for this function.     

Reduction of sialic acid O-acetylation in human colonic mucins in the adenoma-carcinoma sequence

The oligo-O-acetylation of sialic acids found in normal colonic mucins is greatly reduced in colorectal cancer. Mucins prepared from cancer tissue in adenocarcinoma showed this reduction, while normal O-acetylation was detected in resection margin and control cases and total mucin sialic acid content was significantly decreased in cancer vs control samples. A reduction of the O-acetyl transferase activity catalysing the O-acetylation reaction was also found. A series of cultured human colorectal cell lines derived from the same premalignant adenomatous line, and representative of the adenoma-carcinoma sequence were examined and revealed a depletion of oligo-O-acetylation in the original diploid premalignant line, re-expression in a further premalignant line and reduction in malignant mucinous and adenocarcinoma cell lines. Reduction of sialic acid O-acetylation appears as an early event in the process of malignant transformation in human colorectal cancer.     

Structure of hardwood glucuronoxylans: modifications and impact on pulp retention during wood kraft pulping

The behaviour of different hardwood glucuronoxylans during the kraft pulping process was investigated. Woods and pulps xylans were isolated and characterized by size exclusion chromatography, methylation (linkage) analysis and ¹H NMR. Eucalyptus globulus and Eucalyptus urograndis showed xylan retention significantly higher than that of Betula pendula. The higher retention of Eucalyptus xylans was assigned to (i) their higher average molecular weight (31 against 24 KDa in B. pendula) and to (ii) the presence of O-2 substituted 4-O-methyl--d-glucuronic acid groups ([→2)-GlcpA-(1→]) with galactopyranosyl/glucopyranosyl residues belonging to fragments of galactan/glucan chains that were absent in B. pendula xylans. A significant part of uronic acids, particularly [→2)-GlcpA-(1→] units, remain in fibres until the end of pulping. The acetylation degree and distribution of acetyl groups between Xylp units, in general terms, was similar in the three types of xylans. Unexpectedly, about 20% of the acetyl groups persisted in pulps xylans till the end of pulping.     

Structure of the lipopolysaccharide of Pseudomonas aeruginosa O-12 with a randomly O-acetylated core region

The lipopolysaccharide of Pseudomonas aeruginosa O-12 was studied by strong alkaline and mild acid degradations and dephosphorylation followed by fractionation of the products by GPC and high-performance anion-exchange chromatography and analyses by ESI FT-MS and NMR spectroscopy. The structures of the lipopolysaccharide core and the O-polysaccharide repeating unit were elucidated and the site and the configuration of the linkage between the O-polysaccharide and the core established. The core was found to be randomly O-acetylated, most O-acetyl groups being located on the terminal rhamnose residue of the outer core region.     

Different modes of sialyl-Tn expression during malignant transformation of human colonic mucosa

Monoclonal antibodies TKH2 and B72.3, which react with the mucin-associated sialyl-Tn(STn) antigen, preferentially bind to cancerous but not normal colonic tissues. If O-acetyl groups are removed by saponification of tissues, MAb TKH2 will react with normal colonocytes, whereas MAb B72.3 remains non-reactive. To explain this difference in binding specificity, we tested both MAbs against synthetic constructs of single (monomeric) or clustered (trimeric) STn epitopes by enzyme immunoassay. Both MAb TKH2 and MAb B72.3 reacted with trimeric STn, but MAb TKH2 demonstrated greater binding than MAb B72.3 to monomeric STn. This suggests that normal colonic mucosa expresses monomeric STn epitopes, but that with transformation to malignancy, clustered STn epitopes appear. The appearance of clustered STn epitopes during colonic carcinogenesis represents a novel pattern of carbohydrate antigen expression and implicates alterations at the level of apomucins and/or glycosyltransferases responsible for cluster epitope formation.     

Structural variation in the glycan strands of bacterial peptidoglycan

The normal, unmodified glycan strands of bacterial peptidoglycan consist of alternating residues of beta-1,4-linked N-acetylmuramic acid and N-acetylglucosamine. In many species the glycan strands become modified after their insertion into the cell wall. This review describes the structure of secondary modifications and of attachment sites of surface polymers in the glycan strands of peptidoglycan. It also provides an overview of the occurrence of these modifications in various bacterial species. Recently, enzymes responsible for the N-deacetylation, N-glycolylation and O-acetylation of the glycan strands were identified. The presence of these modifications affects the hydrolysis of peptidoglycan and its enlargement during cell growth. Glycan strands are frequently deacetylated and/or O-acetylated in pathogenic species. These alterations affect the recognition of bacteria by host factors, and contribute to the resistance of bacteria to host defence factors such as lysozyme.     

The O-acetylation patterns in the O-antigens of Hafnia alvei strains PCM 1200 and 1203, serologically closely related to PCM 1205

Serological tests revealed immunochemical similarities between the lipopolysaccharides of Hafnia alvei strains PCM 1200, 1203 and 1205. Immunoblotting and ELISA showed cross-reactions between the strains. NMR spectroscopy showed that the O-deacetylated O-specific polysaccharides isolated from lipopolysaccharides of H. alvei strains PCM 1200 and 1203 possessed the same composition and sequence as the O-deacetylated O-specific polysaccharide of H. alvei strain PCM 1205, that is a glycerol teichoic-acid-like polymer with a repeating unit of the following structure: [carbohydrate structure: see text] NMR spectroscopic studies of the polysaccharides concluded that O-3 of the side chain beta-D-GlcpNAc is partially O-acetylated (50-80%) in both investigated strains. In strain PCM 1203 an additional O-acetyl group (50-80%) is linked to O-6 of the chain -->3)-alpha-D-GlcpNAc-(1--> residue. The structural features of the isolated O-specific polysaccharides were also the same as those of the O-specific polysaccharides on the bacterial cells directly observed by the HR-MAS NMR technique.     

Victor Ginsburg's influence on my research of the role of sialic acids in biological recognition

Sialic acids are monosaccharides with relatively strong acidity which belong to the most important molecules of higher animals and also occur in some microorganisms. They are bound to complex carbohydrates and occupy prominent positions, especially in cell membranes. Their structural diversity is high and, correspondingly, the mechanisms for their biosynthesis complex. Sialic acids are involved in a great number of cell functions. Due to their cell surface location these acidic molecules shield macromolecules and cells from enzymatic and immunological attacks and thus contribute to innate immunity. In contrast to this masking role, enabling, for example, blood cells and serum glycoproteins a longer life-time, sialic acids also represent recognition sites for various physiological receptors, such as the selectins and siglecs, as well as for toxins and microorganisms and thus allow their colonization. The recognition function of sialic acids can again be masked by O-acetylation, which modifies the interaction with receptors. Many viruses use sialic acids for the infection of cells. As sialic acids play also a decisive role in tumor biology, they prove to be rather versatile molecules that modulate biological and pathological cellular events in a sensitive way. Thus, they are most prominent representatives of mediators of molecular and cellular recognition.     

Inhibition of acetyl-coenzyme A dependent activation of N-hydroxyarylamines by phenolic compounds, pentachlorophenol and 1-nitro-2-naphthol

Pentachlorophenol (PCP) and 1-nitro-2-naphthol were found to be potent inhibitors of enzymatic acetyl-CoA dependent activation, which is suggested as proceeding through direct O-acetylation, of N-hydroxyarylamines to tRNA binding by liver cytosolic enzymes from hamsters and rats. IC₅₀ values of PCP for the activation of 2-hydroxyamino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole (N-OH-Glu-P-1), 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-OH-Trp-P-2) and N-hydroxy-2-aminofluorene (N-OH-AF) were 20, 25 and 17 μM, respectively, in hamster cytosol system. Similar inhibition was observed with rat liver cytosol (IC₅₀ values of PCP and 1-nitro-2-naphthol were 13 and 12 μM, respectively, for the binding of N-OH-Glu-P-1). PCP is known as an inhibitor of sulfotransferase; however, another potent inhibitor of sulfotransferase, 2,6-dichloro-4-nitrophenol, did not inhibit the acetyl-CoA dependent binding. Antibiotic thiolactomycin, which inhibits bacterial O-acetyltransferase, did not affect the activation by hamster and rat cytosol, indicating the difference in property between bacterial and mammalian enzymes. The kinetic data obtained with hamster cytosol suggested the competitive inhibition of PCP with substrate, N-OH-Glu-P-1, and non-competitive inhibition with acetyl-CoA. In addition to the O-acetylation, PCP and 1-nitro-2-naphthol also inhibited N-acetylation of arylamines and N, O-acetyltransfer reaction of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) by hamster cytosol. IC₅₀ values for these two types of acetyltransfer reactions, however, were slightly higher than those observed for acetyl-CoA dependent activations of N-hydroxyarylamines.     

Sialic acid O-acetylation: From biosynthesis to roles in health and disease

Sialic acids are nine-carbon sugars that frequently cap glycans at the cell surface in cells of vertebrates as well as cells of certain types of invertebrates and bacteria. The nine-carbon backbone of sialic acids can undergo extensive enzymatic modification in nature and O-acetylation at the C-4/7/8/9 position in particular is widely observed. In recent years, the detection and analysis of O-acetylated sialic acids have advanced, and sialic acid-specific O-acetyltransferases (SOATs) and O-acetylesterases (SIAEs) that add and remove O-acetyl groups, respectively, have been identified and characterized in mammalian cells, invertebrates, bacteria, and viruses. These advances now allow us to draw a more complete picture of the biosynthetic pathway of the diverse O-acetylated sialic acids to drive the generation of genetically and biochemically engineered model cell lines and organisms with altered expression of O-acetylated sialic acids for dissection of their roles in glycoprotein stability, development, and immune recognition, as well as discovery of novel functions. Furthermore, a growing number of studies associate sialic acid O-acetylation with cancer, autoimmunity, and infection, providing rationale for the development of selective probes and inhibitors of SOATs and SIAEs. Here, we discuss the current insights into the biosynthesis and biological functions of O-acetylated sialic acids and review the evidence linking this modification to disease. Furthermore, we discuss emerging strategies for the design, synthesis, and potential application of unnatural O-acetylated sialic acids and inhibitors of SOATs and SIAEs that may enable therapeutic targeting of this versatile sialic acid modification.