Purification and Characterization of Antibodies Directed against the α-Gal Epitope

The α-Gal epitope is an immunogen trisaccharide structure consisting of N-acetylglucosamine (GlcNAc)β1,4-galactose (Gal)α1,3-Gal. It is presented as part of complex-type glycans on glycoproteins or glycolipids on cell surfaces of non-primate mammalians. About 1% of all antibodies in human sera are specific toward α1,3-Gal and are therefore named as anti-α-Gal antibodies. This work comprises the purification and characterization of anti-α-Gal antibodies from human immunoglobulin G (IgG). A synthetically manufactured α Gal epitope affinity resin was used to enrich anti-α-Gal antibodies. Selectivity experiments with purified antibodies were carried out using enzyme-linked immunosorbent assays (ELISA), Western blotting, and erythrocyte agglutination. Furthermore, binding affinities toward α-Gal were determined by surface plasmon resonance (SPR) and the IgG distribution of anti α Gal antibodies (83% IgG2, 14% IgG1, 2% IgG3, 1% IgG4) was calculated applying ELISA and immunodiffusion. A range of isoelectric points from pH 6 to pH 8 was observed in 2D gel electrophoresis. Glycan profiling of anti α Gal antibodies revealed complex biantennary structures with high fucosylation grades (86%). Additionally, low amounts of bisecting GlcNAc (15%) and sialic acids (13%) were detected. The purification of anti-α-Gal antibodies from human IgG was successful, and their use as detection antibodies for α Gal-containing structures was evaluated

Comparative N-Glycosylation Analysis of the Fc Portions of a Chimeric Human Coagulation Factor VIII and Immunoglobulin G1

Prevention and treatment of bleeding in patients suffering from hemophilia A are inconvenient due to repeated intravenous infusions owing to the short half-life of coagulation factor VIII (FVIII) in circulation. Besides (glyco-)pegylation of the FVIII molecule, a bioengineering approach comprises the protein fusion to Fc-immunoglobulin (Ig)G that mediate protection from clearance or degradation via binding to the neonatal Fc receptor. While human-like N-glycosylation of recombinant FVIII is known to be crucial for the clotting factor’s quality and function, the particular glycosylation of the fused Fc portion has not been investigated in detail so far, despite its known impact on Fcγ receptor binding. Here, we analyzed the N-glycosylation of the Fc part of a chimeric FVIII-Fc protein compared to a commercial IgG1 purified from human plasma. Fc parts from both samples were released by enzymatic cleavage and were subsequently separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Corresponding protein bands were referred to PNGase F in-gel digestion in order to release the respective N-glycans. Analysis via matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed structural differences of both N-glycan patterns. Labeling with 2-aminobenzamide (2AB) and analysis via hydrophilic interaction liquid chromatography (HILIC) allowed a quantitative comparison of the respective N-glycosylation. Observed variations in Fc glycosylation of the chimeric FVIII fusion protein and human plasma-derived IgG1, e.g., regarding terminal sialylation, are discussed, focusing on the impact of the clotting factor’s properties, most notably its binding to Fcγ receptors

Purification and Characterization of Antibodies Directed against the α-Gal Epitope

The α-Gal epitope is an immunogen trisaccharide structure consisting of N-acetylglucosamine (GlcNAc)β1,4-galactose (Gal)α1,3-Gal. It is presented as part of complex-type glycans on glycoproteins or glycolipids on cell surfaces of non-primate mammalians. About 1% of all antibodies in human sera are specific toward α1,3-Gal and are therefore named as anti-α-Gal antibodies. This work comprises the purification and characterization of anti-α-Gal antibodies from human immunoglobulin G (IgG). A synthetically manufactured α Gal epitope affinity resin was used to enrich anti-α-Gal antibodies. Selectivity experiments with purified antibodies were carried out using enzyme-linked immunosorbent assays (ELISA), Western blotting, and erythrocyte agglutination. Furthermore, binding affinities toward α-Gal were determined by surface plasmon resonance (SPR) and the IgG distribution of anti α Gal antibodies (83% IgG2, 14% IgG1, 2% IgG3, 1% IgG4) was calculated applying ELISA and immunodiffusion. A range of isoelectric points from pH 6 to pH 8 was observed in 2D gel electrophoresis. Glycan profiling of anti α Gal antibodies revealed complex biantennary structures with high fucosylation grades (86%). Additionally, low amounts of bisecting GlcNAc (15%) and sialic acids (13%) were detected. The purification of anti-α-Gal antibodies from human IgG was successful, and their use as detection antibodies for α Gal-containing structures was evaluated

O-acetylation and de-O-acetylation of sialic acids in human colorectal carcinoma

Adecrease in the level ofO-acetylated sialic acids observed in colorectal carcinoma may lead to an increase in the expression of sialyl LewisX, a tumor-associated antigen, which is related to progression of colorectal cancer to metastasis. The underlying mechanism for this reduction is, however, not fully understood. Two enzymes are thought to be primarily responsible for the turnover of O-acetyl ester groups on sialic acids; sialate-O-acetyltransferase (OAT) and sialate-O-acetylesterase (OAE). We have previously reported the characterization of OAT activity from normal colon mucosa, which efficiently O-acetylates CMP-Neu5Ac exclusively in the Golgi apparatus prior to the action of sialyltransferase [Shen, Y., Tiralongo, J., Iwersen, M., Sipos, B., Kalthoff, H. & Schauer, R. (2002) Biol. Chem. 383, 307-317]. In this report we describe the identification of a lysosomal and a cytosolic OAE activity in human colonic mucosa that specifically hydrolyses 9-O-acetyl groups on sialic acid. Utilizingmatched resection margin and cancer tissue from colorectal carcinoma patients we provide strong evidence suggesting that the level of O-acetylated sialic acids present in normal and diseased human colon may be dependent on the relative activities of OAT to lysosomal OAE. Furthermore, we show that the level of free cytosolic Neu5,9Ac2 in human colon is regulated by the relative activity of the cytosolic OAE.No Full Tex

Purification and characterization of 9-O-acetylated sialoglycoproteins from leukemic cells and their potential as immunological tool for monitoring childhood acute lymphoblastic leukemia

Sialic acids as terminal residues of oligosaccharide chains play crucial roles in several cellular recognition events. Exploiting the selective affinity of Achatinin-H toward N-acetyl-9-Oacetylneuraminic acid-a2-6-GalNAc, we have demonstrated the presence of 9-O-acetylated sialoglycoproteins (Neu5,9Ac2- GPs) on lymphoblasts of 70 children with acute lymphoblastic leukemia (ALL) and on leukemic cell lines by fluorimetric HPLC and flow cytometric analysis. This study aims to assess the structural aspect of the glycotope of Neu5,9Ac2-GPsALL and to evaluate whether these disease-specific molecules can be used to monitor the clinical outcome of ALL. The Neu5,9Ac2- GPsALL were affinity-purified, and three distinct leukemiaspecific molecular determinants (135, 120, and 90 kDa) were demonstrated bySDS–PAGE, western blotting, and isoelectric focusing. The carbohydrate epitope of Neu5,9Ac2-GPsALL was confirmed by using synthetic sialic acid analogs. The enhanced presence of anti-Neu5,9Ac2-GPALL antibody in ALL patients prompted us to develop an antigen-ELISA using purified Neu5,9Ac2-GPsALL as coating antigens. Purified antigen was able to detect leukemia-specific antibodies at presentation of disease, which gradually decreased with treatment. Longitudinal monitoring of 18 patients revealed that in the early phase of the treatment patients with lower anti-Neu5,9Ac2-GPs showed a better prognosis. Minimal cross-reactivity was observed in other hematological disorders (n¼50) like chronic myeloid leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and non-Hodgkin’s lymphoma as well as normal healthy individuals (n¼21). This study demonstrated the potential of purified Neu5,9Ac2-GPsALL as an alternate tool for detection of anti-Neu5,9Ac2-GP antibodies to be helpful for diagnosis and monitoring of childhood ALL patients

Identification and characterization of adsorbed serum sialoglycans on Leishmania donovani promastigotes

Sialic acids as terminal residues of oligosaccharide chains play a crucial role in several cellular recognition events. The presence of sialic acid on promastigotes of Leishmania donovani, the causative organism of Indian visceral leishmani- asis, was demonstrated by fluorimetric high-performance liquid chromatography showing Neu5Ac and, to a minor extent, Neu5,9Ac2. The presence of Neu5Ac was confirmed by GC/MS analysis. Furthermore, binding with sialic acid- binding lectins Sambucus nigra agglutinin (SNA), Maackia amurensis agglutinin (MAA), and Siglecs showed the presence of both a2,3- and a2,6-linked sialic acids. No endo- genous biosynthetic machinery for Neu5Ac could be demon- strated in the parasite. Concomitant western blotting of parasite membranes and culture medium with SNA demon- strated the presence of common sialoglyconjugates (123, 90, and 70 kDa). Similarly, binding of MAA with parasite mem- brane and culture medium showed three analogous sialogly- cans corresponding to 130, 117, and 70 kDa, indicating that a2,3- and a2,6-linked sialoglycans are adsorbed from the fetal calf serum present in the culture medium. L. donovani promastigotes also reacted with Achatinin-H, a lectin that preferentially identifies 9-O-acetylated sialic acid in a2!6 GalNAc linkage. This determinant was evidenced on parasite cell surfaces by cell agglutination, ELISA, and flow cytome- try, where its binding was abolished by pretreatment of cells with a recombinant 9-O-acetylesterase derived from the HE1 region of the influenza C esterase gene. Additionally, binding of CD60b, a 9-O-acetyl GD3-specific monoclonal antibody, corroborated the presence of terminal 9-O-acetylated disialo- glycans. Our results indicate that sialic acids (a2!6 and a2!3 linked) and 9-O-acetyl derivatives constitute compo- nents of the parasite cell surface