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Serum Keratan Sulfate Concentration as a Measure of the Catabolism of Cartilage Proteoglycans
Published in Thomas F. Kresina, Monoclonal Antibodies, Cytokines, and Arthritis, 2020
Eugene J.-M. A. Thonar, James M. Williams, Brian A. Maldonado, Mary Ellen Lenz, Thomas J. Schnitzer, Giles V. Campion, Klaus E. Kuettner, M. Barry, E. Sweet
Because KS and some polylactosamines contain identical long linear segments of the repeat disaccharide (galactose acetylglucosamine), a deficiency in a glycosidase used in the synthesis of a polylactosamine chain can result in an abnormality in KS synthesis. A patient with decreased amounts of membrane-bound forms of galactosyltransferase in microsomal membranes from mononuclear cells was recently found to have an abnormally low level of serum KS (42). Further the shape of the inhibition curve produced by the serum KS was different from that obtained with normal sera. These results strongly suggest that the connective tissue cells in this patient are also affected by the galactosyltransferase defect and that the deficiency in synthesis of polylactosamines is generalized. As measurement of serum KS is relatively easy to perform, it could prove useful in diagnosing abnormalities in polylactosamine metabolism and in learning more, in each case, about the nature of the deficiency.
The Use of Tracers in Transport Studies
Published in Joan Gil, Models of Lung Disease, 2020
Maya Simionescu, Nicolae Ghinea
Horseradish peroxidase has been used predominantly in permeability studies and for quantification of fluid-phase pinocytosis (Steinman and Cohn, 1972). However, in some cells, like rat alveolar macrophages, HRP (a polymannose glycoprotein) is adsorbed to the plasma membrane, presumably to a mannose receptor; its binding was specifically inhibited by 10−3M mannose or 10−3M mannan (Sly and Stahl, (1978). Binding to the plasma membrane was followed by adsorptive endocytosis. In addition, HRP binds strongly to the cell surface of several cultured mammalian cells. Evidence was obtained that HRP may be bound to a glycosyltransferase at the cell surface since its presence was suppressed by 10 mM chitotriose and 10 mM UDP-galactose, an acceptor and donor, respectively, of galactose for the reactions catalyzed by galactosyltransferase (Straus and Keller, 1987). Thus, in some systems HRP is an adsorptive marker rather than a fluid-phase probe; the generality of these results must be tested for each cell under study.
Legume Nodule Biochemistry and Function
Published in Peter M. Gresshoff, Molecular Biology of Symbiotic Nitrogen Fixation, 2018
Robert B. Mellor, Dietrich Werner
The enzyme UDP-galactose-asialoagalactofetuin galactosyltransferase is found in both ER and Golgi76 and provides subterminal galactose for glycoconjugates. This activity is stimulated 800% in effective symbioses, which is less than the 1600% stimulation recorded for the N-acetylgalactasaminyltransferase, which is totally Golgi-located76 and provides terminal amino-sugar residues for glycoconjugates. Since PBM contains products of both the above enzymes, this provides biochemical support for the observations of Robertson and co-workers77,78 that the major pathway providing material for the PBM is over the Golgi. Of the putative Golgi marker enzyme, glucan synthetase I, is not measurable in infected cells79 although IDPase is easily detectable. In transmission electron micrographs dictyosome bundles are often seen adjacent end-on to peribacteroid membrane,80 and freeze-fracture studies reveal continuities between Golgi and PBM over vesicles.77
Bivalent non-human gal-α1-3-gal glycan epitopes in the Fc region of a monoclonal antibody model can be recognized by anti-Gal-α1-3-Gal IgE antibodies
Published in mAbs, 2023
Grayson Hatfield, Lioudmila Tepliakova, Jessica Tran, Huixin Lu, Michel Gilbert, Roger Y. Tam
β1–4-galactosyltransferase. Construct HP-21 is a recombinant version of the HP0826 β-1,4-galactosyltransferase from Helicobacter pylori cloned in plasmid pCWori+. The β-1,4-galactosyltransferase was produced in E. coli AD202 containing the construct HP-21 and grown in 2× YT medium using IPTG as the inducer. The cells were resuspended in 50 mM HEPES pH 7.5 and 0.5 M NDSB-201 (Calbiochem, Cat# 480005-25 GM). The cells were broken using an Avestin C5 Emulsiflex cell disruptor (Avestin, Ottawa) and the extract was centrifuged at 27,000 × g for 30 min at 4°C. The supernatant was then centrifuged at 208,000 × g for 1 h at 4°C. β-1,4-galactosyltransferase was partially purified by precipitation with (NH4)2SO4 at a final concentration of 1.65 M and incubation on ice for 100 min. The precipitate was recovered by centrifugation at 17,000 × g for 20 min at 4°C. The pellet was resuspended in 50 mM HEPES, pH 7.5, 0.1 M NaCl and dialyzed extensively against the same buffer.
A glyco-engineering approach for site-specific conjugation to Fab glycans
Published in mAbs, 2023
Maria L. Jaramillo, Traian Sulea, Yves Durocher, Mauro Acchione, Melissa J. Schur, Anna Robotham, John F. Kelly, Marie-France Goneau, Alma Robert, Yuneivy Cepero-Donates, Michel Gilbert
Cetuximab was first treated with a sialidase to remove the 2,3-linked sialic acids that are known to be present on the N-glycans of proteins produced in Chinese hamster ovary (CHO) cells.25 In order to enhance the level of galactosylation, the material was also incubated with UDP-Gal and the B4GalT galactosyltransferase. After purification, cetuximab was modified by the addition of Neu5NAz using either AST-03 or ST6Gal1, followed by addition of MB488-DBCO using click chemistry. For comparison, cetuximab was also randomly labeled by conjugating the NHS ester of Alexa Fluor® 488 to the primary amines (surface-accessible lysines). An antibody directed against a viral protein was used as a negative (nonspecific, NS) control antibody, to determine levels of non-targeted binding. Flow cytometry was used to test cell surface binding of the glyco-conjugated cetuximab antibodies on SKOV3 cells naturally overexpressing the epidermal growth factor receptor (EGFR) and on U87 glioblastoma cells engineered to overexpress EGFR. Both types of glyco-conjugated cetuximab antibodies showed similar affinity for SKOV3 and EGFR-overexpressing U87 glioblastoma cells relative to the non-targeted antibody (Figure S3). Furthermore, anti-EGFR antibodies that were conjugated via their glycan residues (cetux Fab-MB488 and cetux Fc/Fab-MB488) were similar in binding affinity to target cells when compared to antibodies labeled on random lysine residues (Table 1).
Phagocytosis of platelets opsonized with differently glycosylated anti-HLA hIgG1 by monocyte-derived macrophages
Published in Platelets, 2023
Thijs L. J. van Osch, Juulke Steuten, Jan Nouta, Carolien A. M. Koeleman, Arthur E. H. Bentlage, Sebastiaan Heidt, Arend Mulder, Jan Voorberg, S. Marieke van Ham, Manfred Wuhrer, Anja ten Brinke, Gestur Vidarsson
The production and glycoengineering techniques of the anti-HLA mAbs used in this study have been described in detail [46–52]. In brief, the protein sequences of the variable regions of all anti-HLA mAbs were used to assemble pcDNA3.1 expression vectors encoding for full human IgG1 and PG LA LA Fc mutants (P329 G, L234A, and L235A), which are incapable of binding complement and FcγRs [53]. The expression vectors were used for the production of recombinant antibodies in our in-house HEK Freestyle system. For obtaining antibodies with certain desired glycan-profiles, the chemical inhibitor 2-deoxy-2-fluoro-L-fucose (2FF, Carbosynth) was used to decrease fc fucosylation and 5 mM D-galactose (Sigma Aldrich) and the constructs coding for the enzymes β-1,4 galactosyltransferase 1 (B4GALT1) and β-galactoside alpha-2,6-sialyltransferase 1 (ST6GALT1) were used prior/during transfection to increase galactosylation and sialylation. Monoclonal antibodies were purified 6 days post-transfection and subjected to liquid chromatography–mass spectrometry-based IgG Fc glycosylation analysis [46,47,54].