O-acetylated Sialic Acids Research Articles

Abstract 6844: Discovery of a novel sialic acid O-acetyltransferase in colon tissue

Abstract Sialic acids have critical functions relating to host-pathogenic interactions and the immune microenvironment. Subsequent post-translational O-acetyl modifications add an extra layer of control by modulating many of these interactions in both normal and transformed colorectal cells. The processes through which this modification is added to sialic acids are not completely understood. To date only one gene, CASD1, has been identified to encode a sialic acid O-acetyltransferase in human cells. We used whole-genome sequencing on normal human colon tissue to identify a haplotype strongly associated with colorectal sialic acid O-acetylation, as determined by histopathological mPAS staining. Only SNP’s of a single candidate gene within the haplotype correlated perfectly in a 91-sample validation set. CRISPR-Cas9 editing of this gene in 2D cell line and 3D normal colon organoid models yielded changes to modified sialic acid levels. These findings suggest that this candidate gene functions as a novel sialic acid O-acetyltransferase in colorectal tissue and could have implications as a diagnostic tool for patients with inflammatory diseases of the bowel or as an indicator for therapeutic targets in cancer. Citation Format: Bum Seok Lee, Ashley Cook, Maged Zeineldin, Surojit Sur, Laura Dobbyn, Maria Popoli, Sana Khalili, Bert Vogelstein, Nickolas Papadopoulos, Shibin Zhou, Chetan Bettegowda, Phillip Buckhaults, Tatianna Larman, Kathy Gabrielson, Kenneth W. Kinzler, Nicolas Wyhs. Discovery of a novel sialic acid O-acetyltransferase in colon tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6844.

Analysis of O-acetylated sialic acids by 3-nitrophenylhydrazine derivatization combined with LC-MS/MS.

Sialic acids are a family of monosaccharides that share a nine-carbon backbone and a carboxyl group. A recent derivatization method based on 3-nitrophenylhydrazine (3-NPH) provides a mild chemical labeling technique for biomolecules containing carbonyl or carboxyl groups. In this study, we utilized 3-NPH to label sialic acids via a two-step derivatization process. The derivatized species can produce a common reporter ion corresponding to C1-C3 with two labels, and a fragment differentiating between Neu5Ac, Neu5Gc, and KDN. This method is compatible with O-acetylated sialic acids and provides high sensitivity to Neu5Gc and KDN, and since the utilization of dual labeling significantly enhances the hydrophobicity of derivatives, it can effectively mitigate matrix effects when combined with parallel reaction monitoring technology. Negative-ion tandem mass spectrometry (MS/MS) analysis reveals a distinctive fragmentation profile for the 4-O-acetylated species, while the other sialic acids yield similar MS/MS spectra with a high abundance of reporter ions. Using the reporter ion as a transition, this analytical strategy is effective for analyzing complex biological samples. For example, it was successfully employed to quantify sialic acids in the intestinal tissues of several carp species, demonstrating its potential in sialylation research.

Sialic acid O-acetylation patterns and glycosidic linkage type determination by ion mobility-mass spectrometry

O-acetylation is a common modification of sialic acids that has been implicated in a multitude of biological and disease processes. A lack of analytical methods that can determine exact structures of sialic acid variants is a hurdle to determine roles of distinct O-acetylated sialosides. Here, we describe a drift tube ion mobility-mass spectrometry approach that can elucidate exact O-acetylation patterns as well as glycosidic linkage types of sialosides isolated from complex biological samples. It is based on the use of a library of synthetic O-acetylated sialosides to establish intrinsic collision cross section (CCS) values of diagnostic fragment ions. The CCS values were used to characterize O-acetylated sialosides from mucins and N-linked glycans from biologicals as well as equine tracheal and nasal tissues. It uncovered contrasting sialic acid linkage types of acetylated and non-acetylated sialic acids and provided a rationale for sialic acid binding preferences of equine H7 influenza A viruses.

Antimicrobial, anti-inflammatory and anti-arthritic activity of hemolymph lectin (NagLec) isolated from the freshwater crab, Oziotelphusanaga

Lectins are non-immune glycoproteins or proteins having a unique capacity to interact with carbohydrate ligands found on the surface of their host cells. In the present investigation, the lectin was purified from the hemolymph of freshwater crab, Oziotelphusa naga and its antimicrobial, anti-inflammatory and anti-arthritic activity was analysed. The preliminary characterization of the hemagglutinin was carried out to identify the erythrocyte and sugar specificity, optimum pH and temperature and cation dependency. The agglutinin was found to be highly specific to rabbit erythrocyte and inhibited by fetuin and α-lactose. Maximum hemagglutination activity was noted at pH 7.5–8 and temperature 20–40 °C. An O-acetyl sialic acid specific 75 kDa hemolymph lectin, designated as NagLec was isolated from the freshwater crab, Oziotelphusa naga by affinity chromatography on fetuin coupled Sepharose 4 B, with a purification fold of 185. The bacteria Staphylococcus aureus, Proteus mirabilis and fungus Candida albicans had the greatest zone of inhibition when treated with NagLec. The results of the Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) assays showed that the purified lectin inhibited the growth of Staphylococcus aureus at 0.031 and 0.065 μg/ml, which proved the bactericidal property of NagLec. NagLec generated alterations on the bacterial cells and led to protein leakage, which was dosage (24 and 48 μg/ml) and time dependent (10–40 min). COX and LOX enzyme was inhibited to 49.43% and 61.81% with 100 μg/ml concentration of NagLec respectively, demonstrating NagLec's ability to reduce inflammation. Furthermore, NagLec (500 μg) suppressed protein denaturation up to 77.12% whereas diclofenac sodium (a standard drug) was inhibited by 89.36%. The results indicate that NagLec, a sialic acid specific lectin isolated from the freshwater crab O. naga could be formulated as a nano drug in future owing to its antimicrobial, anti-inflammatory and anti-arthritic potential that could be targeted to specific pathogenic microbes and treat arthritis.

Impact of Asialoglycoprotein Receptor and Mannose Receptor Deficiency on Murine Plasma N-glycome Profiles

The asialoglycoprotein receptor (ASGPR) and the mannose receptor C-type 1 (MRC1) are well known for their selective recognition and clearance of circulating glycoproteins. Terminal galactose and N-Acetylgalactosamine are recognized by ASGPR, while terminal mannose, fucose, and N-Acetylglucosamine are recognized by MRC1. The effects of ASGPR and MRC1 deficiency on the N-glycosylation of individual circulating proteins have been studied. However, the impact on the homeostasis of the major plasma glycoproteins is debated and their glycosylation has not been mapped with high molecular resolution in this context. Therefore, we evaluated the total plasma N-glycome and plasma proteome of ASGR1 and MRC1 deficient mice. ASGPR deficiency resulted in an increase in O-acetylation of sialic acids accompanied by higher levels of apolipoprotein D, haptoglobin, and vitronectin. MRC1 deficiency decreased fucosylation without affecting the abundance of the major circulating glycoproteins. Our findings confirm that concentrations and N-glycosylation of the major plasma proteins are tightly controlled and further suggest that glycan-binding receptors have redundancy, allowing compensation for the loss of one major clearance receptor.

Generalized low levels of serum N-glycans associate with better health status.

Caloric restriction (CR) can prolong life and ameliorate age-related diseases; thus, its molecular basis might provide new insights for finding biomarker and intervention for aging and age-related disease. Glycosylation is an important post-translational modification, which can timely reflect the changes of intracellular state. Serum N-glycosylation was found changed with aging in humans and mice. CR is widely accepted as an effective anti-aging intervention in mice and could affect mouse serum fucosylated N-glycans. However, the effect of CR on the level of global N-glycans remains unknown. In order to explore whether CR affect the level of global N-glycans, we performed a comprehensive serum glycome profiling in mice of 30% calorie restriction group and ad libitum group at 7 time points across 60 weeks by MALDI-TOF-MS. At each time point, the majority of glycans, including galactosylated and high mannose glycans, showed a consistent low level in CR group. Interestingly, O-acetylated sialoglycans presented an upward change different from other derived traits, which is mainly reflected in two biantennary α2,6-linked sialoglycans (H5N4Ge2Ac1, H5N4Ge2Ac2). Liver transcriptome analysis further revealed a decreased transcriptional level of genes involved in N-glycan biosynthesis while increased level of acetyl-CoA production. This finding is consistent with changes in serum N-glycans and O-acetylated sialic acids. Therefore, we provided one possible molecular basis for the beneficial effect of CR from N-glycosylation perspective.

Precision Characterization of Site-Specific O-Acetylated Sialic Acids on N-Glycoproteins.

O-Acetylation is a common modification of sialic acid, playing a significant role in glycoprotein stability, immune response, and cell development. Due to the lack of efficient methods for direct analysis of O-acetylated sialoglycopeptides (O-AcSGPs), the majority of identified O-acetylated sialic acids (O-AcSia) until now had no glycosite/glycoprotein information. Herein, we introduced a new workflow for precise interpretation of O-AcSGPs with probability estimation by recognizing the characteristic B and Y ions of O-AcSias. With further optimization of mass spectrometry parameters, the method allowed us to identify a total of 171 unique O-AcSGPs in mouse serum. Although the majority of these O-AcSGPs were at a relatively low abundance compared with their non-O-acetylated states, they were mainly involved in peptidase/endopeptidase inhibitor activities. The method paves the way for large-scale structural and functional analyses of site-specific O-AcSias in various complex samples as well as further identification of many other similar chemical modifications on glycoproteins.

O-Acetyl Migration within the Sialic Acid Side Chain: A Mechanistic Study Using the Ab Initio Nanoreactor.

Many disease-causing viruses target sialic acids on the surface of host cells. Some viruses bind preferentially to sialic acids with O-acetyl modification at the hydroxyl group of C7, C8, or C9 on the glycerol-like side chain. Studies of proteins binding to sialosides containing O-acetylated sialic acids are crucial in understanding the related diseases but experimentally difficult due to the lability of the ester group. We recently showed that O-acetyl migration among hydroxyl groups of C7, C8, and C9 in sialic acids occurs in all directions in a pH-dependent manner. In the current study, we elucidate a full mechanistic pathway for the migration of O-acetyl among C7, C8, and C9. We used an ab initio nanoreactor to explore potential reaction pathways and density functional theory, pKa calculations, and umbrella sampling to investigate elementary steps of interest. We found that when a base is present, migration is easy in any direction and involves three key steps: deprotonation of the hydroxyl group, cyclization between the two carbons, and the migration of the O-acetyl group. This dynamic equilibrium may play a defensive role against pathogens that evolve to gain entry to the cell by binding selectively to one acetylation state.

Differences in Proportion of N-acetyllactosamine and O-acetylated Sialic Acid Have No Significant Effect on the Pharmacokinetics and Biological Activity of Darbepoetin Alfa

Differences in Proportion of N-acetyllactosamine and O-acetylated Sialic Acid Have No Significant Effect on the Pharmacokinetics and Biological Activity of Darbepoetin Alfa

Excavating phytochemicals from plants possessing antiviral activities for identifying SARS-CoV hemagglutinin-esterase inhibitors by diligent computational workflow

Coronaviruses (CoVs) belong to a group of RNA viruses that cause diseases in vertebrates including. Newer and deadlier than SARS CoV-2 are sought to appear in future for which the scientific community must be prepared with the strategies for their control. Spike protein (S-protein) of all the CoVs require angiotensin-converting enzyme2 (ACE2), while CoVs also require hemagglutinin-acetylesterase (HE) glycoprotein receptor to simultaneously interact with O-acetylated sialic acids on host cells, both these interactions enable viral particle to enter host cell leading to its infection. Target inhibition of viral S-protein and HE glycoprotein receptor can lead to a development of therapy against the SARS CoV-2. The proposition is to recognize molecules from the bundle of phytochemicals of medicinal plants known to possess antiviral potentials as a lead that could interact and mask the active site of, HE glycoprotein which would ideally bind to O-acetylated sialic acids on human host cells. Such molecules can be addressed as ‘HE glycoprotein blockers’. A library of 110 phytochemicals from Withania somnifera, Asparagus racemosus, Zinziber officinalis, Allium sativum, Curcuma longa and Adhatoda vasica was constructed and was used under present study. In silico analysis was employed with plant-derived phytochemicals. The molecular docking, molecular dynamics simulations over the scale of 1000 ns (1 μs) and ADMET prediction revealed that the Withania somnifera (ashwagandha) and Asparagus racemosus (shatavari) plants possessed various steroidal saponins and alkaloids which could potentially inhibit the COVID-19 virus and even other CoVs targeted HE glycoprotein receptor. Communicated by Ramaswamy H. Sarma

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.

Tissue Microarrays to Visualize Influenza D Attachment to Host Receptors in the Respiratory Tract of Farm Animals.

The trimeric hemagglutinin-esterase fusion protein (HEF) of influenza D virus (IDV) binds 9-O-acetylated sialic acid receptors, which are expressed in various host species. While cattle are the main reservoir for IDV, the viral genome has also been detected in domestic pigs. In addition, antibodies against IDV have been detected in other farm animals such as sheep, goats, and horses, and even in farmers working with IDV positive animals. Viruses belonging to various IDV clades circulate, but little is known about their differences in host and tissue tropism. Here we used recombinantly produced HEF proteins (HEF S57A) from the major clades D/Oklahoma (D/OK) and D/Oklahoma/660 (D/660) to study their host and tissue tropism and receptor interactions. To this end, we developed tissue microarrays (TMA) composed of respiratory tissues from various farm animals including cattle, domestic pigs, sheep, goats, and horses. Protein histochemical staining of farm animal respiratory tissue-microarrays with HEF proteins showed that cattle have receptors present over the entire respiratory tract while receptors are only present in the nasal and pharyngeal epithelium of pigs, sheep, goats, and horses. No differences in tropism for tissues and animals were observed between clades, while hemagglutination assays showed that D/OK has a 2-fold higher binding affinity than D/660 for receptors on red blood cells. The removal of O-acetylation from receptors via saponification treatment confirmed that receptor-binding of both clades was dependent on O-acetylated sialic acids.

Reversible O-Acetyl Migration within the Sialic Acid Side Chain and Its Influence on Protein Recognition.

O-Acetylation is a common naturally occurring modification of carbohydrates and is especially widespread in sialic acids, a family of nine-carbon acidic monosaccharides. O-Acetyl migration within the exocyclic glycerol-like side chain of mono-O-acetylated sialic acid reported previously was from the C7- to C9-hydroxyl group with or without an 8-O-acetyl intermediate, which resulted in an equilibrium that favors the formation of the 9-O-acetyl sialic acid. Herein, we provide direct experimental evidence demonstrating that O-acetyl migration is bidirectional, and the rate of equilibration is influenced predominantly by the pH of the sample. While the O-acetyl group on sialic acids and sialoglycans is stable under mildly acidic conditions (pH < 5, the rate of O-acetyl migration is extremely low), reversible O-acetyl migration is observed readily at neutral pH and becomes more significant when the pH increases to slightly basic. Sialoglycan microarray studies showed that esterase-inactivated porcine torovirus hemagglutinin-esterase bound strongly to sialoglycans containing a more stable 9-N-acetylated sialic acid analog, but these compounds were less resistant to periodate oxidation treatment compared to their 9-O-acetyl counterparts. Together with prior studies, the results support the possible influence of sialic acid O-acetylation and O-acetyl migration to host-microbe interactions and potential application of the more stable synthetic N-acetyl mimics.

Synthetic O-acetylated sialosides facilitate functional receptor identification for human respiratory viruses.

The transmission of viruses from animal reservoirs to humans poses major threats to public health. Preparedness for future zoonotic outbreaks requires a fundamental understanding of how viruses of animal origin have adapted to binding to a cell surface component and/or receptor of the new host. Here we report on the specificities of human and animal viruses that engage with O-acetylated sialic acid, which include betacoronaviruses, toroviruses and influenza C and D viruses. Key to these studies was the development of a chemoenzymatic methodology that can provide almost any sialate-acetylation pattern. A collection of O-acetylated sialoglycans was printed as a microarray for the determination of receptor specificity. These studies showed host-specific patterns of receptor recognition and revealed that three distinct human respiratory viruses uniquely bind 9-O-acetylated α2,8-linked disialoside. Immunofluorescence and cell entry studies support that such a glycotope as part of a ganglioside is a functional receptor for human coronaviruses.

A Chemoenzymatic Synthon Strategy for Synthesizing N-Acetyl Analogues of O-Acetylated N. meningitidis W Capsular Polysaccharide Oligosaccharides.

O-Acetylated sialic acid has been found in the Neisseria meningitidis serogroup W (NmW) capsular polysaccharide (CPS) and is a required structural component of clinically used NmW CPS-based polysaccharide and polysaccharide-conjugate vaccines. The role of sialic acid O-acetylation in NmW CPS, however, is not clearly understood. This is partially due to the lack of a precise control of the percentage and the location of O-acetylation which is labile and susceptible to migration. We explore chemoenzymatic synthetic strategies for preparing N-acetylated analogues of O-acetylated NmW CPS oligosaccharides which can serve as structurally stable probe mimics. Substrate specificity studies of NmW CPS polymerase (NmSiaDW) identified 4-azido-4-deoxy-N-acetylmannosamine (ManNAc4N3) and 6-azido-6-deoxy-N-acetylmannosamine (ManNAc6N3) as suitable chemoenzymatic synthons for synthesizing N-acetyl analogues of NmW CPS oligosaccharides containing 7-O-acetyl-N-acetylneuraminic acid (Neu5,7Ac2) and/or 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2). The synthesis was achieved by NmSiaDW-dependent sequential one-pot multienzyme (OPME) strategy with in situ generation of the corresponding sugar nucleotides from simple monosaccharides or derivatives to form N3-oligosaccharides which were converted to the desired NAc-oligosaccharides by an efficient one-step chemical transformation.

Characterization of a novel O-acetyl sialic acid specific lectin from the hemolymph of the marine crab, Atergatis integerrimus (Lamarck, 1818)

An O-acetyl sialic acid specific lectin was purified from the hemolymph of the marine crab Atergatis integerrimus by affinity chromatography using BSM (Bovine Submaxillary Mucin) coupled to cyanogen bromide activated Sepharose 4B and biospecific adsorption using formalinized buffalo erythrocytes. The purified AiL (Atergatis integerrimus lectin) showed an 1218 fold increase in specific activity when compared to the crude hemolymph agglutinin. The lectin, on non - denaturing PAGE showed a single band of 216 kDa and when subjected to SDS - PAGE, the lectin resolved into three subunits of molecular weight 70, 72 and 74 kDa. Physico chemical characterization revealed the lectin as pH and temperature sensitive, calcium dependent and sensitive to calcium chelators. Based on the calcium dependency of the lectin, AiL could be classified as a C-type lectin. The purified lectin agglutinated buffalo erythrocytes with greater avidity and was inhibited by the glycoproteins BSM, thyroglobulin, fetuin, PSM, and sugars raffinose, trehalose, l - fucose, α - Lactose, melibiose and GluNAc suggesting the affinity of the lectin to sialic acid. Reduction in HA with asialo buffalo erythrocytes and HAI titer with desialylated BSM, confirms the sialic acid specificity of the lectin. The reduction in HAI following de - O - acetylation confirms the specificity of the lectin for O - acetyl sialic acid. FTIR analysis confirms the purified lectin as a glycoprotein with spectral bands corresponding to amide bands and saccharides. Thus this study paves way to assess the therapeutic application of this lectin that could be targeted to modified sialic acid moieties that are expressed on the malignant cells and pathogenic microbes and also deduce the crystal structure of the lectin.

Age-Related Changes in O-Acetylation of Sialic Acids Bound to N-Glycans of Male Rat Serum Glycoproteins and Influence of Dietary Intake on Their Changes

O-Acetylation of sialic acids has been widely found in eukaryotic cells. Such modifications of sialic acids are tissue-specific and seem to be developmentally regulated. In this study, we performed comprehensive analysis of age-related changes in the serum N-glycans of male rats using capillary electrophoresis (CE) and investigated the changes in the O-acetylation of sialic acids bound to N-glycans with aging and different diets. The present method offered sufficient resolution to assess the degree of O-acetylation of the N-glycans and allowed for the determination of the age-related changes in O-acetylation of sialic acids. Using the CE-based method, we found that the relative abundance of disialo-biantennary N-glycans modified with 9-O-acetylated N-acetylneuraminic acid (Neu5,9Ac) significantly increased with aging. In addition, the relative abundances of N-glycans with two Neu5,9Ac reversed to those of N-glycans with only Neu5Ac during 12 weeks. Next, we evaluated the influence of high-fat diet and food restriction on age-related changes in O-acetylation. Although the total amount of disialo-biantennary N-glycans increased with aging, age-related O-acetylation of sialic acids was suppressed by a high-fat diet. On the other hand, food restriction enhanced the O-acetylation of sialic acids, and the relative abundance of N-glycans with two Neu5,9Ac residues at 15 weeks of age was higher than that observed in the standard diet group. These findings suggest that the O-acetylation of sialic acids is closely related to changes in energy metabolisms such as glycolysis or fatty acid metabolism.

Human Sialic acid O-acetyl esterase (SIAE) \u2013 mediated changes in sensitivity to etoposide in a medulloblastoma cell line

Medulloblastoma (MB), the most common malignant paediatric brain tumour occurs in the cerebellum. Advances in molecular genomics have led to the identification of defined subgroups which are associated with distinct clinical prognoses. Despite this classification, standard therapies for all subgroups often leave children with life-long neurological deficits. New therapeutic approaches are therefore urgently needed to reduce current treatment toxicity and increase survival for patients. GD3 is a well-studied ganglioside which is known to have roles in the development of the cerebellum. Post-partum GD3 is not highly expressed in the brain. In some cancers however GD3 is highly expressed. In MB cells GD3 is largely acetylated to GD3A. GD3 is pro-apoptotic but GD3A can protect cells from apoptosis. Presence of these gangliosides has previously been shown to correlate with resistance to chemotherapy. Here we show that the GD3 acetylation pathway is dysregulated in MB and as a proof-of-principle we show that increased GD3 expression sensitises an MB cell line to etoposide.

Seventeen O-acetylated N-glycans and six O-acetylation sites of Myozyme identified using liquid chromatography-tandem mass spectrometry

O-acetylated sialic acid (SA) attached to the N-glycans of therapeutic glycoproteins reportedly inhibit sialidase activity, increase protein half-life, decrease protein antigenicity, and stabilize protein conformation. Recombinant human acid α-glucosidase (Myozyme) is the only drug approved by the United States Food and Drug Administration for the treatment of Pompe disease. In this study, unreported N-glycans containing O-acetylated SA in Myozyme and the relative quantities of total glycans were investigated using liquid chromatography (LC)-electrospray ionization (ESI)-high-energy collisional dissociation (HCD) tandem mass spectrometry (MS/MS). The 17 N-glycans (6.4% of total glycans) containing mono-, di-, mono/di-, and di/di-O-acetylated N-acetylneuraminic acid (Neu5Ac) were identified with mass accuracy, glycan-generated fragment ions, and the retention time on an LC column. The analysis of peptides containing mono- and/or di-O-acetylated Neu5Ac ions sorted from all peptides using nano-LC-ESI-HCD-MS/MS confirmed six O-acetylation sites (Asn 140, Asn 233, Asn 390, Asn 470, Asn 652, and Asn 882), at least five of which (Asn 140, Asn 233, Asn 390, Asn 470, and Asn 652) could contribute to the drug efficacy or cellular uptake of Myozyme. This is the first study to identify N-glycans containing O-acetylated Neu5Ac and O-acetylation sites in Myozyme.

New Rabbit Erythrocyte Specific Mycelial Lectins from Fusarium sp. with Complex Saccharide Specificity

Eight Fusarium sp. namely, F. acutatum, F. globosum, F. graminearum, F. lactis, F. nivale, F. proliferatum, F. pseudoanthophilum and F. robustum were screened for the presence of lectins by hemagglutination activity using human ABO, porcine, ovine, goat and rabbit erythrocytes. Mycelial extracts of all the fungal cultures except F. graminearum displayed unique lectin activity with only rabbit erythrocytes. Enzymatic treatment of rabbit erythrocytes with neuraminidase has significantly enhanced the titre of all the lectin positive extracts of fungal cultures. In contrast, most of the lectins showed a decline in lectin activity with protease treated rabbit erythrocytes. Saccharide specificity studies have shown that majority of the lectins were inhibitory towards O-acetyl sialic acids. None of the lectins from Fusarium sp. were inhibited by dextran, meso-inositol and N-acetyl-D-glucosamine. Most of the fungal cultures displayed highest hemagglutination activity during 10th day of growth in broth cultures. The unique saccharide specificity of Fusarium sp. lectins can be used for elucidating their clinical role in glycobiology research.