Sialic Acid-containing Carbohydrate Research Articles

Study of the Efficacy and Safety of Darbepoetin Alfa versus Epoetin Alfa in the Treatment of Anemia in Pediatric Patients on Regular Hemodialysis

Abstract Background Anemia is a common comorbidity in patients with chronic kidney disease (CKD). That affects the vast majority and can have a significant impact on patient morbidity and mortality. Erythropoiesis-stimulating agents (ESAs) are the main lines in management of anemia in hemodialysis patients. Replacement therapy with recombinant human erythropoietin (r- HuEPO) has provided an effective treatment for patients with renal “Epoetin alfa” is a recombinant erythropoietin with the same amino acid sequence as human erythropoietin (EPO). In comparison to epoetin alfa, darbepoetin alfa (DPO) contains an increased amount of sialic acid-containing carbohydrate chains. It shows a much longer elimination half-life than epoetin alfa, with prolonged erythropoietic effect. Aim of the Work to evaluate whether darbpoetin and epopoetin alpha can be used interchangeably on the same patient, achieving the same desired effect and to detect any difference in the occurrence of side effects between both ESAs. Patient and Methods This was a crossover study that took place in the Pediatrics Dialysis unit, Children’s Hospital, Ain Shams University. The study was conducted on 40 children with end stage renal disease on regular hemodialysis over a period of 6 months. The patients were randomly put into two groups A and B, each group contained 20 patients. Each group received one type of ESA either ( darbpoetin alpha or epopoetin alpha )for 3 months and then shifted to the other type of ESA for another 3 months. The dosage of epoetin alfa used was (200 IU/kg/week) and a conversion rate of EPO: DPO, 300:1. A period of 6 weeks was agreed as a washout period after the first drug given in both groups in which no results were recorded but ESA was not stopped due to its essentiality. Blood tests (hemoglobin (Hb), serum urea, serum creatinine and iron profile; (S. iron, S. ferritin, TIBC, and transferrin saturation)) were withdrawn at the start and the end of the 3 months’ period of each drug. Results We found no difference on the Hb level with both drugs. The interchange between them did not yield any significant drop in the Hb levels. Hb target in both EPO and DPO was reached in 22.5% of total cases, and was not reached in 62.5%. This proved that both drugs give the same effect with nearly negligible difference. No serious Sside effect as convulsions, hypertension, and thrombosis were noticed in both groups. Conclusion The target Hb was not reached in most patients of both groups of ESRD on regular hemodialysis at the unit and none of both drugs showed superior results or less side effects than the other.

Exposure of Keratinocytes to Candida Albicans in the Context of Atopic Milieu Induces Changes in the Surface Glycosylation Pattern of Small Extracellular Vesicles to Enhance Their Propensity to Interact With Inhibitory Siglec Receptors.

Candida albicans (C. albicans) infection is a potential complication in the individuals with atopic dermatitis (AD) and can affect clinical course of the disease. Here, using primary keratinocytes we determined that atopic milieu promotes changes in the interaction of small extracellular vesicles (sEVs) with dendritic cells and that this is further enhanced by the presence of C. albicans. sEV uptake is largely dependent on the expression of glycans on their surface; modelling of the protein interactions indicated that recognition of this pathogen through C. albicans-relevant pattern recognition receptors (PRRs) is linked to several glycosylation enzymes which may in turn affect the expression of sEV glycans. Here, significant changes in the surface glycosylation pattern, as determined by lectin array, could be observed in sEVs upon a combined exposure of keratinocytes to AD cytokines and C. albicans. This included enhanced expression of multiple types of glycans, for which several dendritic cell receptors could be proposed as binding partners. Blocking experiments showed predominant involvement of the inhibitory Siglec-7 and -9 receptors in the sEV-cell interaction and the engagement of sialic acid-containing carbohydrate moieties on the surface of sEVs. This pointed on ST6 β-Galactoside α-2,6-Sialyltransferase 1 (ST6GAL1) and Core 1 β,3-Galactosyltransferase 1 (C1GALT1) as potential enzymes involved in the process of remodelling of the sEV surface glycans upon C. albicans exposure. Our results suggest that, in combination with atopic dermatitis milieu, C. albicans promotes alterations in the glycosylation pattern of keratinocyte-derived sEVs to interact with inhibitory Siglecs on antigen presenting cells. Hence, a strategy aiming at this pathway to enhance antifungal responses and restrict pathogen spread could offer novel therapeutic options for skin candidiasis in AD.

CA242 as a biomarker for pancreatic cancer and other diseases.

CA242 is a sialic acid-containing carbohydrate antigen attached to core proteins/lipids detected on the cell surface or in serum. Increased serum levels of CA242 have been used clinically as diagnostic biomarkers for pancreatic, colorectal, and other cancers. Since CA242 is overexpressed in malignant tumor tissues compared to that in normal tissues, it is believed that serum CA242 is a product of cancer cells. Thus far, the serum CA242 levels in patients suffering from different types of cancer and noncancerous diseases have not been systematically compared. In our current study, we acquired data of serum CA242 levels from 34,680 patients with 27 clinically defined diseases and from healthy controls (1317) in the clinical laboratory of our hospital over the past 5 years. The mean, median, and -log10p values were calculated. Our data showed that patients with pancreatic cancer, cervical cancer, and lymphoma had the highest median values of serum CA242, which were followed by esophagus cancer, colon cancer, ovarian cancer, type 2 diabetes mellitus, rectal cancer, coronary heart disease, breast cancer, diabetic nephropathy, acute myocardial infarction, and 15 other types of diseases compared to that of healthy controls. In contrast, patients suffering from sequela of brain damage and anemia had statistically lower median values than healthy controls. Based on the -log10p values, the increased serum CA242 levels could be used not only for diagnosis of pancreatic and other cancers, but also for diagnosis of type 2 diabetes mellitus and coronary heart disease, suggesting CA242 might be a systemic malfunction biomarker associated with cancers and other chronic diseases.

Cloning and characterization of a viral α2–3-sialyltransferase (vST3Gal-I) for the synthesis of sialyl Lewisx

Sialyl Lewis(x) (SLe(x), Siaα2-3Galβ1-4(Fucα1-3)GlcNAcβOR) is an important sialic acid-containing carbohydrate epitope involved in many biological processes such as inflammation and cancer metastasis. In the biosynthetic process of SLe(x), α2-3-sialyltransferase-catalyzed sialylation generally proceeds prior to α1-3-fucosyltransferase-catalyzed fucosylation. For the chemoenzymatic synthesis of SLe(x) containing different sialic acid forms, however, it would be more efficient if diverse sialic acid forms are transferred in the last step to the fucosylated substrate Lewis(x) (Le(x)). An α2-3-sialyltransferase obtained from myxoma virus-infected European rabbit kidney RK13 cells (viral α2-3-sialyltransferase (vST3Gal-I)) was reported to be able to tolerate fucosylated substrate Le(x). Nevertheless, the substrate specificity of the enzyme was only determined using partially purified protein from extracts of cells infected with myxoma virus. Herein we demonstrate that a previously reported multifunctional bacterial enzyme Pasteurella multocida sialyltransferase 1 (PmST1) can also use Le(x) as an acceptor substrate, although at a much lower efficiency compared to nonfucosylated acceptor. In addition, N-terminal 30-amino-acid truncated vST3Gal-I has been successfully cloned and expressed in Escherichia coli Origami™ B(DE3) cells as a fusion protein with an N-terminal maltose binding protein (MBP) and a C-terminal His(6)-tag (MBP-Δ30vST3Gal-I-His(6)). The viral protein has been purified to homogeneity and characterized biochemically. The enzyme is active in a broad pH range varying from 5.0 to 9.0. It does not require a divalent metal for its α2-3-sialyltransferase activity. It has been used in one-pot multienzyme sialylation of Le(x) for the synthesis of SLe(x) containing different sialic acid forms with good yields.

3DSDSCAR—a three dimensional structural database for sialic acid-containing carbohydrates through molecular dynamics simulation

The inherent flexibility and lack of strong intramolecular interactions of oligosaccharides demand the use of theoretical methods for their structural elucidation. In spite of the developments of theoretical methods, not much research on glycoinformatics is done so far when compared to bioinformatics research on proteins and nucleic acids. We have developed three dimensional structural database for a sialic acid-containing carbohydrates (3DSDSCAR). This is an open-access database that provides 3D structural models of a given sialic acid-containing carbohydrate. At present, 3DSDSCAR contains 60 conformational models, belonging to 14 different sialic acid-containing carbohydrates, deduced through 10 ns molecular dynamics (MD) simulations. The database is available at the URL: http://www.3dsdscar.org.

Hemoglobin increase in advanced/metastatic lung cancer patients receiving multicycle chemotherapy and weekly darbepoetin alfa: An initial ongoing experience

8250 Background: Recombinant human erythropoietin (rHuEpo) is efficient in treating chemotherapy-induced anemia, increasing mean hemoglobin (Hgb) levels by 1 g/dl at 4 weeks (w) (Gabrilove, JCO 2001). Darbepoetin alfa, a novel erythropoiesis stimulating protein, has a lower receptor-binding affinity than rHuEPO, due to its higher sialic acid-containing carbohydrate content, but a longer serum half-life, that should confer it a greater and faster in vivo potency at 2 and 4 w (Egrie, Exp Hematol 2003). Aim of this ongoing study was to evaluate whether in the treatment of anemic lung cancer patients, darbepoetin alfa had the same efficacy as that expected from rHuEpo. Methods:From July till October 2003, 24 patients with advanced-metastatic NSCLC receiving chemo- (22 pts) or radiotherapy (2 pts), mean age 64 yrs, ECOG 0–1, were treated for treatment-induced anemia with darbepoetin alfa, 150 mcg/w s.c. for 4 w. The treatment was started at Hgb levels ≤10.5 g/dl, or at Hgb >10.5 g/dl, but with a Hgb reduction ≥1.5 g/dl after the beginning of chemotherapy. Hgb response was assessed at 2 and 4 w of treatment. Results:Mean Hgb change was 0.54 g/dl and 1.14 g/dl at 2 and 4 w respectively. Eight/24 and 13/24 pts had a Hgb increase ≥1 g/dl at 2 and 4 w respectively, while 6/24 pts had a major response, with an increase ≥2g/dl at 4 w. Twenty-two patients were therefore stratified according to the schedule of chemotherapy they were receiving (platinum- vs non-platinum-based [59% vs 41%], oral vs i.v. [27% vs 73%]) and to pre-treatment vs no-pre-treatment with rHuEPO (27% vs 73%), and then correlated with the response to darbepoetin therapy (t-test). No statistical differences between the groups were observed. Conclusions: Compared with data from historical studies, in our cluster of patients darbepoetin alfa was equiactive with rHuEpo at 4 weeks, showing a similar rate of early responses at 2 w. Moreover, no significant differences in response were observed for those patients who were receiving less anemia-inducing treatments, such as non-platinum-based therapy or oral therapy (vinorelbine 4 pts, ZD-1839 2 pts). No significant financial relationships to disclose.

Inhibition of P-selectin-mediated cell adhesion by a sulfated derivative of sialic acid

P-selectin, a carbohydrate-binding cell adhesion molecule expressed on activated endothelial cells and platelets, plays a key role in the recruitment of leukocytes to inflammatory and hemorrhagic sites. It simultaneously recognizes a sialic acid-containing carbohydrate chain and the sulfated tyrosine residues of a specific counter-receptor expressed on the leukocyte surface. We examined the inhibitory effects of a synthetic sulfated derivative of sialic acid (NMSO3) on P-selectin-mediated cell adhesion and found the following: (1) P-selectin/IgG chimera bound to immobilized NMSO3. (2) The binding of P-selectin/IgG chimera to purified P-selectin glycoprotein ligand-1 was inhibited by soluble NMSO3. (3) The adhesion of HL60 cells to P-selectin-expressing CHO cells was inhibited by NMSO3. (4) NMSO3 inhibited P-selectin-induced tumor necrosis factor-α production in monocytes and activated platelet-induced generation of reactive oxygen species in neutrophils. In conclusion, NMSO3 acts as a specific inhibitor for P-selectin-mediated cell adhesion and for adhesion-dependent leukocyte activation.

Darbepoetin alfa: a new therapy for the management of anaemia associated with chronic kidney disease

Darbepoetin alfa (Aranesp®, Amgen, Inc., Thousand Oaks, California) is a new erythropoietic protein that corrects anaemia associated with chronic kidney disease (CKD) in the majority of patients. Darbepoetin alfa contains five N-linked carbohydrate chains compared with three in recombinant human erythropoietin (rHuEPO). The two additional sialic acid-containing carbohydrate chains prolong the serum half-life of darbepoetin alfa, resulting in greater biological activity and a reduced dosing frequency compared with rHuEPO. Clinical studies in patients with CKD have demonstrated that darbepoetin alfa is effective in correcting anaemia in rHuEPO-naive patients and in patients who have been converted from rHuEPO therapy. Darbepoetin alfa provides long-term maintenance of haemoglobin levels when administered once weekly or once every other week, with the possibility of once-monthly dosing in some patients. Darbepoetin alfa is well tolerated and has a safety profile similar to that of rHuEPO. Owing to its half-life being three times longer than rHuEPO, darbepoetin alfa can be administered at an extended dosing interval, without compromising efficacy. Lessfrequent dosing has potential benefits for both patients with CKD and healthcare providers. These benefits include reduced visits to the clinic, fewer injections and a reduced demand on staff and treatment facilities.

3-Aminobenzamide and 3-aminobenzoic acid, tags for capillary electrophoresis of complex carbohydrates with laser-induced fluorescent detection

The efficiencies in derivatization of reducing carbohydrates were compared by capillary electrophoresis using maltose as a model with nine monoaminobenzene derivatives by reductive amination in the presence of sodium cyanoborohydride. We found that aminobenzene derivatives substituted at the 3-position showed good reactivity with reducing carbohydrates as expected from the reaction mechanism, although the fluorescence intensities and molar absorptivities of these derivatives were not as high as those of 2- and 4-aminobenzene derivatives. The reagents, 3-aminobenzamide and 3-aminobenzoic acid, which showed the highest reactivity, were applied to the labeling of carbohydrate chains obtained from some sialic acid-containing glycoprotein samples, and also high-mannose and hybrid-type oligosaccharides. Capillary electrophoresis of these labeled carbohydrate chains in an inner surface-modified capillary with (50% phenyl)methylpolysiloxane allowed excellent separation of sialic acid-containing carbohydrate chains derived from fetuin and thyroglobulin as well as high mannose-type and hybrid-type carbohydrates derived from bovine pancreas ribonuclease B, soybean agglutinin and hen ovalbumin. The lower limit of calibration was as low as the 10 −16 mol (injected amount) with helium–cadmium laser induced detection.

L-selectin-mediated lymphocyte rolling on MAdCAM-1.

The L-selectin, a cell surface C-type lectin, directs lymphocyte traffic to lymph nodes, and contributes to lymphocyte homing to Peyer's patches and to leukocyte interactions with inflamed venules. Here we report that the mucosal vascular addressin MAdCAM-1, a mucosal endothelial adhesion molecule with immunoglobulin- and mucin-like domains, is a facultative ligand for L-selectin. MAdCAM-1 isolated from mesenteric lymph nodes, but not from cultured endothelioma cells, bears N-glycanase-resistant sialic acid-containing carbohydrate which supports adhesion of L-selectin-transfected lymphoid cells under shear. Interacting lymphoid cells display a 'rolling' behaviour similar to the selectin-dependent rolling of neutrophils observed in inflamed venules. MAdCAM-1 is also a ligand for the lymphocyte integrin homing receptor for Peyer's patches, alpha 4 beta 7 (ref. 7), and may be uniquely adapted to support both selectin-mediated lymphocyte rolling and integrin-mediated adhesion and arrest in vivo.

Purification and characterization of a membrane-bound and a secreted mucin-type glycoprotein carrying the carcinoma-associated sialyl-Lea epitope on distinct core proteins.

Two mucin-type glycoproteins detected by the monoclonal antibody C50, which reacts with the carcinoma-associated sialyl-Lewis a and sialyl-lactotetraose epitopes, were found in secreted and solubilized materials from the colon carcinoma cell line COLO 205. The larger glycoprotein (H-CanAg; heavy cancer antigen) was predominantly found in extracts of cells grown in vitro or as nude mice xenografts whereas the smaller species (L-CanAg; light cancer antigen) was the major component in spent culture medium and serum from grafted mice. Using detergent in the extraction buffer doubled the yield of H-CanAg, suggesting that this glycoprotein is membrane bound whereas the yield of L-CanAg was relatively unaffected. The two glycoproteins were purified from xenograft extracts and spent culture medium using perchloric acid precipitation, monoclonal antibody affinity purification, ion exchange chromatography, and gel filtration. Both glycoproteins were unaffected by reduction and alkylation in guanidine HCl. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, relative molecular masses were estimated to be 600-800 kDa for H-CanAg and 150-300 kDa for L-CanAg. Carbohydrate analysis revealed that the CanAg glycoproteins were highly glycosylated (81-89% carbohydrate by weight), carrying carbohydrate chains with average lengths of 13-18 sugars which were rich in fucose and sialic acid (2-3 residues/chain for each sugar). L-CanAg isolated from spent medium was glycosylated to a higher degree than its counterpart from xenograft extract. Immunochemical studies of the intact glycoproteins showed that both H-CanAg and L-CanAg expressed the monoclonal antibody-defined, sialic acid-containing carbohydrate epitopes CA203 and CA242 as well as the Lewis a blood group antigen whereas only H-CanAg appeared to carry the sialyl-Lewis x epitope. The amino acid compositions were typical of mucins, containing high amounts of serine, threonine (more than 25% together), and proline (11-18%). Significant differences in amino acid composition between H-CanAg and L-CanAg were found. A rabbit antiserum against the cytoplasmic C-terminal part of the MUC1 gene product, core protein of the carcinoma-associated polymorphic epithelial mucin (PEM) and DU-PAN-2, reacted with H-CanAg. After deglycosylation with trifluoromethanesulfonic acid, H-CanAg but not L-CanAg was recognized by the monoclonal antibodies SM-3 and HMFG-2, directed to the tandem repeat of the PEM apoprotein. However, these antibodies which react with PEM from mammary carcinomas without prior deglycosylation were unable to recognize intact H-CanAg, probably as a consequence of a more extensive glycosylation of this glycoprotein.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation and structural characterization of twenty-one sialyloligosaccharides from galactosialidosis urine. An intact N,N'-diacetylchitobiose unit at the reducing end of a diantennary structure.

Galactosialidosis urine was fractionated by gel-permeation chromatography on Bio-Gel P-6. The obtained sialic acid-containing carbohydrate fractions were purified by reversed-phase chromatography and separated according to charge by medium-pressure anion-exchange chromatography on Mono Q. The Mono Q fractions, being mixtures of sialyloligosaccharides differing mainly in sialic acid-linkage type (alpha 2-3/alpha 2-6), were subfractionated by high-performance liquid chromatography on Lichrosorb-NH2. The purified compounds were analysed by 500-MHz 1H-NMR spectroscopy. Twenty-one fully and partially sialylated N-acetyllactosamine-type compounds include mono-, di-, tri- and tetra-antennary structures. All structures have the sequence Man beta 1-4Glc-NAc at the reducing terminus in common, except one diantennary structure bearing an intact N,N'-diacetylchitobiose unit at the reducing end, which is a new feature in human glycoproteinosis urine.

THE CARBOHYDRATE-POLYPEPTIDE LINKAGES, THE AMINO ACID SEQUENCES OF THE PEPTIDES ADJACENT TO SOME OF THESE BONDS, AND THE COMPOSITION AND SIZE OF THE CARBOHYDRATE UNITS OF ALPHA-1-ACID GLYCOPROTEIN.

1. The glycopeptides derived from a proteolytic digest of sialic acid-free alpha(1)-acid glycoprotein were separated on a DEAE-cellulose column into five main fractions. 2. The average molecular weight of these glycopeptides was 2400, except for one fraction whose molecular weight was 3100. The average molecular weight of the sialic acid-free carbohydrate units was found to be 2200. From these data and the carbohydrate content of the native protein and the assumed molecular weight of 44000, it was concluded that alpha(1)-acid glycoprotein probably possesses five carbohydrate units. The sialic acid-containing carbohydrate units of this glycoprotein have an average molecular weight of 3000, except for one unit the molecular weight of which is significantly higher. 3. The N-, non-N- and C-terminal amino acids of the main glycopeptides were determined. Aspartic acid and threonine occur in most peptides. Alanine, glycine, proline, serine and lysine were present in varying amounts. Traces of other amino acids were also found. 4. The amino acid sequence of three main glycopeptides was established and indicated that these glycopeptides are located at different positions of the polypeptide chain of the glycoprotein. These sequences are: Asp(NH(2))-Pro-Lys; Thr-Asp(NH(2))-Ala; Asp(NH(2))-Gly-Thr. 5. From the results of a series of chemical reactions (periodate oxidation, hydrazinolysis, dinitrophenylation, mild acid hydrolysis) it was shown that the hydroxyl group of the N-terminal threonine and the in-amino group of lysine are free and that the beta-carboxyl group of aspartic acid is present as amide. It was concluded that this amide group is involved in the carbohydrate-polypeptide linkages of at least four carbohydrate units of alpha(1)-acid glycoprotein. 6. The carbohydrate composition of the sialic acid-free glycopeptides was determined in terms of moles of neutral hexoses, glucosamine and fucose/mole. 7. Fucose, at least to the larger part, is not linked to sialic acid, and its (glycosidic) linkage is significantly more stable toward acid hydrolysis than the bond of the sialyl residues. 8. Heterogeneity of the carbohydrate units of alpha(1)-acid glycoprotein was found with regard to size and to content of fucose and sialic acid.