Higher Levels Of Sialic Acid Research Articles

The glycosphingolipid repertoire affects HLA class I function

HLA class I (HLA-I) molecules drive immune responses by presenting antigen-derived peptides to T-lymphocytes. Loading of these peptides into the HLA-I binding groove occurs via a multistep process which can be hijacked by viruses and tumors for immune escape. Using iterative genome-wide haploid genetic screening we identified the protease SPPL3 to enhance HLA-I antigen presentation by inhibiting B3GNT5 activity, an enzyme of the glycosphingolipid (GSL) synthesis pathway. GSL profiling by LCMS demonstrated SPPL3-deficient cells to express high levels of sialic acid containing B3GNT5-synthesized GSLs compared to wild type cells. These GSLs localize in close proximity to HLA-I molecules -as revealed by LCMS analysis of the local membrane surrounding HLA-I-thereby shielding membrane proximal HLA-I epitopes from epitope specific antibody recognition. On a functional level, HLA-I shielding also resulted in loss of ligand (LIR-1) binding and a defect in CD8+ T cells activation. HLA-I shielding GSLs are overexpressed on several subtypes of cancer, suggesting a novel form of immune escape. Moreover, pharmacological inhibition of these GSLs on AML cells abolished HLA-I shielding. Our findings demonstrate that the composition of the GSL repertoire determines the efficacy of antigen presentation and represents a potential treatment target in infection and cancer.

Sialic Acid as a Biomarker Studied in Breast Cancer Cell Lines In Vitro Using Fluorescent Molecularly Imprinted Polymers

Sialylations are post-translational modifications of proteins and lipids that play important roles in many cellular events, including cell-cell interactions, proliferation, and migration. Tumor cells express high levels of sialic acid (SA), which are often associated with the increased invasive potential in clinical tumors, correlating with poor prognosis. To overcome the lack of natural SA-receptors, such as antibodies and lectins with high enough specificity and sensitivity, we have used molecularly imprinted polymers (MIPs), or “plastic antibodies”, as nanoprobes. Because high expression of epithelial cell adhesion molecule (EpCAM) in primary tumors is often associated with proliferation and a more aggressive phenotype, the expression of EpCAM and CD44 was initially analyzed. The SA-MIPs were used for the detection of SA on the cell surface of breast cancer cells. Lectins that specifically bind to the a-2,3 SA and a-2,6 SA variants were used for analysis of SA expression, with both flow cytometry and confocal microscopy. Here we show a correlation of EpCAM and SA expression when using the SA-MIPs for detection of SA. We also demonstrate the binding pattern of the SA-MIPs on the breast cancer cell lines using confocal microscopy. Pre-incubation of the SA-MIPs with SA-derivatives as inhibitors could reduce the binding of the SA-MIPs to the tumor cells, indicating the specificity of the SA-MIPs. In conclusion, the SA-MIPs may be a new powerful tool in the diagnostic analysis of breast cancer cells.

Enhancement of sialylation in rIgG in glyco-engineered Chinese hamster ovary cells

Since about 70% of commercial biopharmaceutical products have been produced in Chinese hamster ovary (CHO) cells, this cell line is undeniably a workhorse for biopharmaceuticals production. Meanwhile, sialic acid terminals were reported to affect anti-inflammatory activity, antibody-dependent cellular cytotoxicity efficacy of IgG antibodies. Taking these findings together, we aimed to establish CHO cell lines that highly produce sialic acid terminals by overexpressing two N-acetylneuraminic acid-based key enzymes, α(2,6)-sialyltransferase and UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase using dihydrofolate reductase/methotrexate gene amplification method. Indeed, the number of total sialic acid terminal glycan structures increased tremendously, by 12-fold compared to the wild type in total protein extracts. With the methotrexate supplementation, a targeted cell line, CHOmt17-100, showed up to 1.4 times more sialylated structures of glycoforms in total proteins. Interestingly, immunoglobulin G, used as the model protein in CHOmt17-100, showed about 53% sialylated structures in its glycoforms. These resultant sialylated glycans exhibited more than approximately 14.5 times increase as compared to that of the wild type. Moreover, the resultant glycan structures mostly had N-acetylneuraminic acid terminals, while N-glycolylneuraminic acid terminal composition remained less than 5% as compared to the wild type. Engineered antibodies derived from CHO cell lines that produce high levels of sialic acid will contribute to the examination of glycoforms' efficacy and usefulness toward bio-better products.

Bovine Milk Oligosaccharides with Sialyllactose Improves Cognition in Preterm Pigs.

Optimal nutrition is important after preterm birth to facilitate normal brain development. Human milk is rich in sialic acid and preterm infants may benefit from supplementing formula with sialyllactose to support neurodevelopment. Using pigs as models, we hypothesized that sialyllactose supplementation improves brain development after preterm birth. Pigs (of either sex) were delivered by cesarean section at 90% gestation and fed a milk diet supplemented with either an oligosaccharide-enriched whey with sialyllactose (n = 20) or lactose (n = 20) for 19 days. Cognitive performance was tested in a spatial T-maze. Brains were collected for ex vivo magnetic resonance imaging (MRI), gene expression, and sialic acid measurements. For reference, term piglets (n = 14) were artificially reared under identical conditions and compared with vaginally born piglets naturally reared by the sow (n = 12). A higher proportion of sialyllactose supplemented preterm pigs reached the T-maze learning criteria relative to control preterm pigs (p < 0.05), and approximated the cognition level of term reference pigs (p < 0.01). Furthermore, supplemented pigs had upregulated genes related to sialic acid metabolism, myelination, and ganglioside biosynthesis in hippocampus. Sialyllactose supplementation did not lead to higher levels of sialic acid in the hippocampus or change MRI endpoints. Contrary, these parameters were strongly influenced by postconceptional age and postnatal rearing conditions. In conclusion, oligosaccharide-enriched whey with sialyllactose improved spatial cognition, with effects on hippocampal genes related to sialic acid metabolism, myelination, and ganglioside biosynthesis in preterm pigs. Dietary sialic acid enrichment may improve brain development in infants.

Glycan modifications to the gp120 immunogens used in the RV144 vaccine trial improve binding to broadly neutralizing antibodies.

To date, the RV144 HIV vaccine trial has been the only study to show that immunization can confer protection from HIV infection. While encouraging, the modest 31.2% (P = 0.04) efficacy achieved in this study left significant room for improvement, and created an incentive to optimize the AIDSVAX B/E vaccine immunogens to increase the level of vaccine efficacy. Since the completion of the RV144 trial, our understanding of the antigenic structure of the HIV envelope protein, gp120, and of the specificity of broadly neutralizing monoclonal antibodies (bN-mAbs) that bind to it, has significantly improved. In particular, we have learned that multiple families of bN-mAbs require specific oligomannose glycans for binding. Both of the monomeric gp120 immunogens (MN- and A244-rgp120) in the AIDSVAX B/E vaccine used in the RV144 trial were enriched for glycans containing high levels of sialic acid, and lacked critical N-linked glycosylation sites required for binding by several families of bN-mAbs. The absence of these epitopes may have contributed to the low level of efficacy achieved in this study. In this report, we describe our efforts to improve the antigenic structure of the rgp120 immunogens used in the vaccine by optimizing glycan-dependent epitopes recognized by multiple bN-mAbs. Our results demonstrated that by shifting the location of one PNGS in A244-rgp120, and by adding two PNGS to MN-rgp120, in conjunction with the production of both proteins in a cell line that favors the incorporation of oligomannose glycans, we could significantly improve the binding by three major families of bN-mAbs. The immunogens described here represent a second generation of gp120-based vaccine immunogens that exhibit potential for use in RV144 follow-up studies.

Differential Glycosylation Between Recombinant Factor VIII Produced in Baby Hamster Kidney and Chinese Hamster Ovary Cells Confers Differences in Immunogenicity in a Humanized Hemophilia Α Mouse Model

IntroductionThe formation of factor VIII (FVIII)- neutralizing antibodies is the most critical complication in the treatment of hemophilia A (HA). Recent clinical evidence suggests that recombinant FVIII (rFVIII) produced in baby hamster kidney (BHK-rFVIII) cells is more immunogenic than that produced in Chinese hamster ovary (CHO-rFVIII) cells. This difference in FVIII immunogenicity may be attributed to differences in protein glycosylation, which can impact the removal of FVIII from circulation through mechanisms leading to clearance and antigen presentation. Here, we document significant differences among the 25 potential N-linked glycans between these products, and we provide in vivo animal model-based evidence that supports these clinical observations.MethodsFactor VIII lectin binding was assessed by ELISA to detect exposed glycans. Commercially-available rFVIII products were adsorbed at 1 ug/mL and specific glycan moieties were detected using a panel of biotinylated lectins and HRP-conjugated streptavidin. Confirmation of differences and determination of N-linked glycan structures was conducted by LC-MS/MS.Eight to 12 week old transgenic C57BL/6 HA mice were used in these studies. This model contains a murine f8 exon 16 KO and additionally harbors a human F8 transgene with a R593C point mutation. While these mice have undetectable plasma levels of human FVIII antigen and activity, they are tolerant to intravenously infused human rFVIII. Clearance was assessed following a 6 IU (~240 IU/kg) infusion of each rFVIII product, and FVIII activity was measured by chromogenic assay and normalized to a 5-minute time point. The number of interferon (IFN)-γ secreting splenocytes from rFVIII-primed naïve mice was determined by ELISPOT.FVIII immune responses were elicited by subcutaneous infusion (6 IU twice-weekly for 2 weeks) and adjuvant-coupled intravenous infusion (1 ug lipopolysacharride with the first infusion as described above) with either rFVIII product. Week 5 plasma samples were assessed for FVIII-specific IgG by ELISA, and FVIII inhibitors by one-stage clotting assay.ResultsLectin binding showed that rFVIII produced in BHK cell lines exhibit lower proportions of high-mannose glycans (p<0.01), and greater levels of sialic acid capping (p<0.01) and fucosylated glycans (p<0.01). Mass spectra confirmed higher levels of sialic acid and identified two additional N-linked sites bearing high mannose glycans on CHO-rFVIII. In this mouse model we observed that BHK-rFVIII had a circulating half-life of 6.06 hours compared to the 10.01 hour half-life of CHO-rFVIII (p<0.0001).The immunogenicity of the BHK- and CHO-rFVIII products was next evaluated in vivo. In mice primed with a single 6 IU dose of BHK-rFVIII, we identified a higher proportion of FVIII-specific IFN-γ secreting splenocytes after seven days. Furthermore, long-term studies showed that 100% of mice subcutaneously exposed to BHK-rFVIII developed anti-FVIII IgG compared to the 47% that received CHO-rFVIII (p<0.01). Coincidently, when FVIII inhibitors were measured, we observed an incidence of 100% vs 37% (p<0.01), respectively. While the titres of FVIII-specific IgG were higher in mice exposed to BHK-rFVIII (p<0.01), there were no significant differences in the inhibitor concentrations. Similarly, we observed increased titres of FVIII-specific IgG in mice exposed intravenously (1 ug LPS with the first infusion) to BHK-rFVIII compared to CHO-rFVIII. However, there were no differences in the incidence of FVIII-specific IgG, nor in the incidence and concentration of inhibitors between the intravenously-infused mice.ConclusionsOur results demonstrate that BHK-rFVIII exhibits altered pharmacokinetic and immunogenic properties compared to CHO-rFVIII in humanized hemophilia A mice. The observed early increase in the proportion FVIII-specific IFN-γ producing cells in the spleen suggests an intrinsic immunogenic element of BHK-rFVIII. Similarly, the substantially increased immunogenicity of BHK-rFVIII in mice when treated subcutaneously complements the previously-reported clinical evidence. These differences may be attributed to the significant disparities in N-linked glycosylation, most notably high mannose and sialic acid containing glycans. Additional studies are underway to directly address the role of the these specific glycans and their potential impact on immunogenicity of rFVIII. DisclosuresLillicrap:Octapharma: Research Funding; Baxalta: Research Funding; Biogen-Idec: Research Funding; Bayer: Research Funding.

Correlation between sialic acid levels in the synovial fluid and the radiographic severity of knee osteoarthritis.

Osteoarthritis (OA) is associated with the presence of inflammation. Sialic acid (SA), an acetylated derivative of neuraminic acid, is reported to be a useful biomarker of inflammation. The aim of the present study was to investigate the correlation between SA levels in the serum and synovial fluid (SF) and radiographic severity in patients with knee OA. A total of 234 patients with knee OA were recruited for the study, as well as 20 patients that had suffered a knee injury or fracture (without knee OA) and 160 healthy controls. Radiological grading of OA in the knee was conducted according to the Kellgren-Lawrence (KL) grading system. SA levels in the serum and SF were measured using Warren’s thiobarbituric acid assay. The results demonstrated that knee OA patients exhibited significantly elevated levels of serum SA when compared with the healthy controls, and also significantly elevated levels of SF SA when compared with the knee fracture patients. Higher SA levels in the SF were identified in knee OA patients with KL grade 4 as compared with patients with KL grade 2 or 3. In addition, OA patients of KL grade 3 had significantly higher SA levels in the SF as compared with patients with KL grade 2 (P<0.01). The SA levels in the SF of the knee OA patients positively correlated with the KL grades (r=0.353; P<0.01). However, there was no significant correlation identified between serum SA levels and KL grade. Therefore, SA levels in the SF positively correlated with the radiographic severity of OA, thus, SA levels in the SF may serve as a biomarker for the progression of OA.

P.3.7 A prodrug strategy to increase oral absorption and bioavailability of Sialic acid in an HIBM mouse model

Sialic acid (SA) is intended for use as a substrate replacement therapy for the treatment of hereditary inclusion body myopathy (HIBM or also DMRV, now known as GNE Myopathy), a severe, neuromuscular disease caused by a defect in the sialic acid biosynthetic pathway. Oral SA provided in the drinking water to an HIBM mouse model is effective in restoring biochemical sialylation and reducing pathology while also preventing the progressive decline in muscle function. However, the pharmaceutical properties of SA in terms of absorption and delivery are not optimal, and the potential exists to make a therapeutically more potent compound for substrate replacement therapy. A series of prodrugs of SA were synthesized and tested in order to achieve a higher degree of absorption, increase delivery to muscle and potentially improve the therapeutic efficacy in humans. Three prodrugs candidates were developed and selected based on their ability to increase uptake in vitro and in vivo. Uptake into induced C2C12 mouse myotube cultures indicated that the prodrugs achieved many fold higher SA levels in the myotubes compared to SA treatment at the same concentration. In HIBM mice after 14 days of treatment with oral prodrugs or SA (BID, 200 mg/kg/day), results showed that the prodrugs consistently had higher levels of free SA in the serum compared to SA and the prodrugs had a substantially prolonged half-life over SA. The high free SA levels achieved suggest that the prodrugs are accumulating in tissues, including muscle, and act as a reservoir to release SA into the serum. Additionally, levels of uncleaved prodrugs were relatively low in the serum suggesting that the prodrug candidates are being metabolized appropriately in the tissues. A 60 day study in HIBM mice is currently underway to confirm the previous results and to evaluate the efficacy and potential toxicity of the three prodrug candidates as compared to SA.

Sialic acid levels in the blood in pregnant women with impaired glucose tolerance test

Gestational diabetes (GDM) is a metabolic disease characterized by the impairment of glucose tolerance during pregnancy. Sialic acid (SA) is a component of glycolipid and glycoproteins found in hormone and enzymes in serum and tissues and high serum SA levels are observed in diabetic patients. Serum SA levels were investigated in three groups, namely 61 normal pregnant women with normal 50 gr glucose loading test (group 1: gr 1), 36 patients with high 50 gr test values and normal 100 gr test (group 2: gr 2: group with impaired glucose tolerance test, IGT), and 8 patients with diagnosis of GDM (group 3:gr 3). According to Kruskal-Wallis test, significant difference was observed in SA levels between gr1 and gr2-gr3 (p = 0.001). Difference was observed between groups with respect to age (p < 0.001), the number of pregnancies (p = 0.001), BMI (p = 0.001), and diabetes history in the family (p = 0.001) (Table 1). Table 1 Summary of results and statistical relationship Variable Group 1 (n = 61) Group 2 (n = 36) Group 3 (n = 8) p Age 24 (18-38) 28 (19-38) 31 (20-38) <0.001 Pregnancy week 26 (24-29) 26 (24-32) 26,5 (24-29) 0.126 Pregnancy number 2 (0-6) 2 (1-5) 3.5 (1-5) <0.001 BMI 23.7 (17.9-38.2) 27.6 (17.04-41.14) 30.8 (21.9-43.8) <0.001 Weight taken 7 (1-13) 6 (2-12) 6 (4-10) 0.954 Sialic acids 2.66 (1.2-4.59) 3.22 (2.34-5.04) 3.05 (2.67-3.49) <0.001 Fetal weight 3,380 (2,310-4,290) 3,400 (2,650-4,600) 3,655 (3,270-3,960) 0.092 Smoking 5 (%8.2) 2 (%5.6) 0 (%0) 1 Family history 22 (%36.1) 10 (%27.8) 8 (%100) <0.001. Our study demonstrated that SA values are increased in GDM and IGT groups, which demonstrates that inflammatory disorders may occur also in IGT group, which is considered like normal pregnancy, and that this group may be considered in the same way as GDM.

Increased expression of sialic acid in cervical biopsies with squamous intraepithelial lesions

BackgroundAltered sialylation has been observed during oncogenic transformation. Sialylated oligosaccharides of glycoproteins and glycolipids have been implicated in tumor progression and metastases. In the cervical cancer high levels of sialic acid have been reported in the patients serum, and an increased of total sialic acid concentration has been reported for the cervical neoplasia and cervical cancer. This study investigates the changes in expression and distribution of α2,3-linked sialic acid and α2,6- linked sialic acid in low and high squamous intraepithelial lesions and in normal tissue.MethodsLectin histochemistry was used to examine the expression and distribution of sialic acid in different grades of cervical neoplasia. We applied Maackia amurensis lectin, which interacts with α2,3-linked sialic acid and Sambucus nigra lectin specific for α2,6-linked sialic acid.ResultsThe histochemical analysis showed that α2,3-linked sialic acid and α2,6- linked sialic acid increased in intensity and distribution in concordance with the grade of squamous intraepithelial lesion (SIL). These results are in concordance with a previous study that reports increased RNAm levels of three sialyltransferases.ConclusionsThese results show that the change in sialylation occurs before cancer development and may play an important role in cellular transformation. These findings provide the basis for more detailed studies of the possible role of cell surface glycoconjugates bearing sialic acid in the cellular cervix transformation.

Glycan Characterization of PSA 2-DE Subforms from Serum and Seminal Plasma

Prostate-specific antigen (PSA) two-dimensional electrophoresis (2-DE) subforms (F1-F5) have been described to be altered in prostate cancer (PCa) compared to benign prostatic hyperplasia (BPH). To understand their molecular differences, characterization of these subforms from PCa serum and seminal plasma, namely, at the glycan level, was performed. PSA 2-DE subforms from two serum PCa samples and seminal plasma were analyzed by N-glycan sequencing using high-performance liquid chromatography (HPLC) combined with exoglycosidase array digestions and by mass spectrometry. F1, F2, and F3 subforms showed the same N-glycan pattern, which contained higher levels of sialic acid than the F4 subform, whereas the F5 subform was unglycosylated. When comparing PSA subforms from PCa with seminal plasma, a decrease in sialylation was observed. Furthermore, the analysis of F3, the more abundant PSA subform, showed a higher proportion of alpha 2-3 sialic acid and a decrease in core fucosylated glycans in the PCa sample. These N-glycan changes in PCa PSA subforms highlight the importance of glycosylation as an indicator of PCa disease.

Levels of serum sialic acid and thiobarbituric acid reactive substances in subjects with impaired glucose tolerance and type 2 diabetes mellitus

Cardiovascular disease is a common cause of death for diabetic patients. High sialic acid levels (SA) and increased oxidative stress are important factors for cardiovascular diseases. We aimed to research whether SA and thiobarbituric acid reactive substances (TBARS) levels are associated with the degree of the diabetic regulation and investigate if SA and TBARS levels can be controlled with the regulation of the blood glucose levels. A total of 179 subjects were included in the study. Three groups, which were comprised of subjects with type 2 diabetes mellitus (DM) (DM group [DMG], n=149), impaired glucose tolerance (IGT) (IGT group [IGTG], n=15), and normal oral glucose tolerance (NGT) (NGTgroup [NGTG], n=15) were constituted. Glucose, cholesterol, high-density lipoprotein (HDL) and glycated hemoglobin (HbA1C), SA, and TBARS were measured in the sera of the patients. SA and TBARS levels were significantly increased in subjects with type 2 DM (P<0.001 for both). SA concentrations showed significant correlation with triglycerides (r=0.229; P<0.05), fasting glucose (r=0.508; P<0.01), 2-hr postprandial glucose (r=0.455; P<0.01), and HbA1C (r=0.467; P<0.01), and there was a positive correlation between TBARS and HbA1C (r=0.251; P<0.01). Diabetic patients were found to have higher risk for inflammation and oxidative stress. The regulation of blood glucose levels may contribute to the decline of both SA and TBARS levels.

Percentage of body fat and plasma glucose predict plasma sialic acid concentration in type 2 diabetes mellitus

Circulating sialic acid is an independent risk factor for cardiovascular disease and is higher in people with type 2 diabetes mellitus. Sialic acid is associated with body mass index, but it is uncertain whether body fat contributes to the higher levels of sialic acid in type 2 diabetes mellitus. Therefore, we have investigated whether the higher levels of sialic acid observed in type 2 diabetes mellitus persist when controlling for fatness. Fasting plasma samples were collected from 24 individuals with type 2 diabetes mellitus and 24 controls. Percentage of body fat was measured by bioelectrical impedance. Plasma sialic acid was quantified by an enzymatic method. Plasma sialic acid was higher in the group with type 2 diabetes mellitus than controls (602 ± 14 vs 545 ± 14 mg/L, P = .007). Percentage of body fat was associated with plasma sialic acid concentration in both the control group ( r = 0.481, P = .020) and the group with type 2 diabetes mellitus ( r = 0.527, P = .007). Fasting glucose was also associated with plasma sialic acid in the group with type 2 diabetes mellitus ( r = 0.700, P < .001). Adjustment for percentage of body fat accounted for the higher levels of sialic acid in type 2 diabetes mellitus. Using linear regression, 54.3% of the variation of plasma sialic acid was explained by percentage of body fat and glucose concentrations in the whole group. Seventy-four percent of sialic acid variation was explained by the same model in type 2 diabetes mellitus. In conclusion, this is the first study to show that percentage of body fat predicts plasma sialic acid concentration and contributes toward higher levels of sialic acid in type 2 diabetes mellitus.

Cerebrospinal fluid free sialic acid and aspartate transaminase levels in meningitis.

Sialic acid (SA) is a nine carbon sugar derived from mannosamine and pyruvate. High levels of sialic acid and aspartate transaminase (AST) levels in cerebrospinal fluid have been described in pyogenic meningitis (PM) compared to tubercular meningitis (TBM). To evaluate the levels of CSF free SA in PM and TBM and to assess the correlation between CSF free SA and CSF glucose or total protein levels. A total of 122 subjects were studied and divided into children and adults. Further, these have been subdivided into controls, PM and TBM. CSF free SA was estimated by thiobarbituric acid assay of Warren and AST by Reitmann and Frankel method. CSF free SA and AST levels in children and adults were significantly high in PM (p<0.001) as compared to TBM and controls. A very high CSF free SA and AST were found to be characteristic of PM, making them useful parameters to differentiate PM from TBM.

Biosynthesis of sialylated lipooligosaccharides in Haemophilus ducreyi is dependent on exogenous sialic acid and not mannosamine. Incorporation studies using N-acylmannosamine analogues, N-glycolylneuraminic acid, and 13C-labeled N-acetylneuraminic acid.

Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted disease. Cell surface lipooligosaccharides (LOS) of H. ducreyi are thought to play important biological roles in host infection. The vast majority of H. ducreyi strains contain high levels of sialic acid (N-acetylneuraminic acid, NeuAc) in their LOS. Here we investigate the biosynthetic origin of H. ducreyi sialosides by metabolic incorporation studies using a panel of N-acylmannosamine and sialic acid analogues. Incorporation of sialosides into LOS was assessed by matrix-assisted laser desorption and electrospray ionization mass spectrometry. A Fourier transform ion cyclotron resonance mass spectrometer provided accurate mass measurements, and a quadrupole time-of-flight instrument was used to obtain characteristic fragment ions and partial carbohydrate sequences. Exogenously supplied N-acetylmannosamine analogues were not converted to LOS-associated sialosides at a detectable level. In contrast, exogenous (13)C-labeled N-acetylneuraminic acid ([(13)C]NeuAc) and N-glycolylneuraminic acid (NeuGc) were efficiently incorporated into LOS in a dose-dependent fashion. Moreover, approximately 1.3 microM total exogenous sialic acid was sufficient to obtain about 50% of the maximum production of sialic acid-containing glycoforms observed under in vitro growth conditions. Together, these data suggest that the expressed levels of sialylated LOS glycoforms observed in H. ducreyi are in large part controlled by the exogenous concentrations of sialic acid and at levels one might expect in vivo. Moreover, these studies show that to properly exploit the sialic acid biosynthetic pathway for metabolic oligosaccharide engineering in H. ducreyi and possibly other prokaryotes that share similar pathways, precursors based on sialic acid and not mannosamine must be used.

The role of sialic acid and Ca 2+ ions in rabies virus-infected foxes brain

The receptors for rabies virus on the neuron membranes are polysialogangliosides arranged in multicomponent gelate structures. The impulse conductivity at the synapse membranes also depends on the interactions between negatively charged sialic acid from the oligosaccharide parts of gangliosides and positively charged Ca ions. From individual brain regions of healthy foxes and foxes naturally infected with rabies virus glycolipoprotein preparations were isolated. In them, the presence of polysialogangliosides and the concentration of sialic acid, Ca ions, and proteins were evidenced. The concentration of sialic acid was increased in all tested brain regions of the infected foxes. In the preparations from the cortex cerebri or cornu Ammonis of the infected foxes, 20-fold higher levels of sialic acid and the decreased concentrations of Ca ions were recorded. The increase in sialic acid levels is directly responsible for the augmented negative charge at synaptic membranes and, consequently, for a much higher permeability of these membranes, which results in a persistent stimulation followed by the release of neuro-transmitters.

Sialic acid level in maternal and neonatal lymphocytes and sera correlated to birth order and sex of the neonate.

Sialic acid (N-acetylneuraminic acid) was determined 1 h after normal term deliveries on peripheral blood lymphocytes from 42 mother-neonate pairs and in 29 maternal and neonatal sera. Results were evaluated according to maternal parity and sex of the neonate. The cases were divided into two groups: primiparae, and secundi- and multiparae. In primiparae the sialic acid level on lymphocytes from male neonates and from their mothers was by 23-30% decreased as compared to female neonatal and maternal cells. In the higher parity group, a significantly increased sialic acid level was found on lymphocytes from male as compared to female neonates, and maternal serum sialic acid concentration, unrelated to the newborns' sex, was by 17-20% increased as compared to primiparae. The results suggest that with increasing parity higher levels of sialic acid on male neonatal cells may possibly contribute to mask fetal male-specific histocompatibility antigens. Increased sialic acid levels in maternal sera from secundi- and multiparae suggest its possible contribution to an increased serum blocking effect.

High sialic acid content slows prourokinase turnover in rabbits

The clearance of natural and recombinant prourokinase (proUK) from the blood of rabbits was studied by means of a double-isotope method which allowed the differential removal of two distinct proUK species to be monitored when simultaneously administered to an individual animal. In initial experiments, proUK expressed in different cell lines contained between 0 and 2.5 molecules of sialic acid per molecule of protein. A slight trend toward slower clearance of proUK with higher sialic acid content was observed but rate differences were not statistically significant. Recombinant proUK produced in CHO cells grown in flow reactors, contained unusually high levels of sialic acid in excess of 3 moles/mole protein. Controlled exposure to immobilized neuraminidase was used to remove sialic acid from this protein in defined amounts. The clearance of the parent material was biphasic with average alpha and beta half-lives of 1.7 min and 16.7 min respectively. The AUC of the parent material was only slightly lowered upon removal of 30% of the original sialic acid. Species with 60% or 90% removal of sialylate were much more rapidly cleared from the circulation respectively yielding AUCs equal to 56% and 41% of that observed with the parent material. Thus proUK containing 2.5–3.5 sialic acid molecules per molecule of protein turned over significantly more slowly in rabbits than did less sialylated proUK. The clearance rate was relatively insensitive to sialic acid content between 0 and 1.5 sialic acid residues per proUK molecule.

Characterization of a sialic‐acid‐rich mucus glycoprotein secreted by a premalignant human colorectal adenoma cell line

The human colonic cell line PC/AA, derived from an adenoma, retains in vitro colonic cell differentiation, notably the production of mucus glycoproteins. The PC/AA adenoma cells produce an extracellular gel layer in culture. The PC/AA gel could be isolated by extraction of the cell cultures with guanidine hydrochloride. The extracted material was purified by gel filtration and caesium chloride density-gradient centrifugation and showed properties typical of mucus glycoproteins, namely, a carbohydrate content above 60% of dry weight rich in N-acetylgalactosamine and sialic acid and low in mannose; an amino acid composition with high serine threonine and proline content; a molecular weight above 1,000 kDa on Sepharose CL 4B chromatography and on SDS-polyacrylamide gel electrophoresis under reducing conditions (greater than 200 kDa); a buoyant density of approximately 1.48 g/ml and the release of oligosaccharides by the alkaline beta-elimination reaction. Comparison of the gel mucus glycoprotein purified from premalignant PC/AA cells with normal human colon mucin showed that it has a higher sialic acid content. This suggests that higher sialic acid levels may precede the development of malignancy.

Screening of type Ia and Ib Streptococcus agalactiae strains with high sialic acid levels by determination of susceptibility to tetracyclines.

The type-specific capsular polysaccharide antigen of Streptococcus agalactiae is recognized to be an antiphagocytic factor in strains having large amounts of it. In the present study, it was indicated that vaginal isolates of types Ia and Ib could be classified into two groups on the basis of both their levels of the sialic acid, which occupies the terminal side chains of the polysaccharide, and their susceptibility to tetracyclines: one group comprised strains with low sialic acid levels (less than 9 micrograms/mg of cell dry weight) as well as with susceptibility to tetracyclines (MIC, less than or equal to 0.5 micrograms/ml), and the other comprised strains with higher sialic acid levels (greater than or equal to 9 micrograms/mg) and resistance to tetracyclines (MIC, greater than or equal to 8 micrograms/ml). A few isolates were found to have low levels of sialic acid and to be resistant to tetracyclines, but no isolates that were both relatively high in sialic acid and susceptible to tetracyclines were ever detected. Among strains of those serotypes, the MICs of tetracyclines were not in proportion to the sialic acid levels and were not affected when the sialic acid levels of each strain were altered by using Todd-Hewitt broth with various concentrations of Na2HPO4 and glucose. It was, therefore, apparent that the correlation of sialic acid levels with susceptibility to tetracyclines was not related directly to the sialic acid content or to the amount of the capsular polysaccharide. Since no plasmid DNAs were detected among representative strains that were tetracycline resistant, it was apparent that at least for the strains tested, resistance was chromosomal gene associated. In strains of S. agalactiae of types of Ia and Ib, the determination of susceptibility to tetracyclines was considered to be useful for screening strains with higher sialic acid levels.