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Carbohydrate Histochemistry
Published in Joan Gil, Models of Lung Disease, 2020
Bradley A. Schulte, Russell A. Harley, Samuel S. Spicer
Elimination or a pronounced decrease of staining after exposure to a highly specific exo- or endoglycosidase serves in an important way to establish the specificity of the staining method. The validity of the enzyme digestion obviously depends on the purity of the enzyme preparation and its freedom from other enzymatic activities, especially of proteinases and other glycosidases. A control procedure against a false-positive result from digestion requires eliminating simple solubilization and extraction and entails simultaneous exposure to the same solution minus the enzyme or containing heat-inactivated enzyme. A false-negative result on the other hand, should be checked by testing activity of the enzyme preparation on a known positive site.
Intracellular Maturation of Acute Phase Proteins
Published in Andrzej Mackiewicz, Irving Kushner, Heinz Baumann, Acute Phase Proteins, 2020
Erik Fries, E. Mathilda Sjöberg
The carbohydrate processing and intracellular transport of secretory proteins can to some extent be monitored by pulse-chase experiments followed by SDS/PAGE. For many proteins, the increase in size due to the addition of galactose and sialic acid residues in the trans-Golgi results in an easily detectable decrease in electrophoretic mobility.65-67 Alternatively, the immunoprecipitated protein is treated with the enzyme endoglycosidase H before electrophoresis. This enzyme will remove all early (high-mannose) forms and thus produce a clear shift in the mobility of the protein;65-67 resistance to the enzyme marks the arrival of the pulse-labled protein in the medial Golgi.12
The N-Formylpeptide Chemotactic Receptor
Published in Richard Horuk, Chemoattractant Ligands and Their Receptors, 2020
The physical state of FPR is sensitive to several enzyme treatments that together strongly suggest that both predicted sites for asparagine-linked glycosylation in the N-terminal domain are used.63 Papain treatment of neutrophils yields a membrane-bound fragment that retains ligand binding activity, but appears as a sharp band with an apparent Mr ~ 31 kDa on SDS-PAGE, slightly smaller than the molecular mass calculated from the cloned ORE This is most consistent with a papain cleavage site just after the glycosylated N-terminal ectodomain of the receptor. After treatment of neutrophils with endoglycosidase F, which removes asparagine-linked oligosaccharides, the cross-linked receptor appears as a sharp band 33 kDa in size, slightly larger than the papain-treated form, but the two isoelectric forms persist. Treatment of HL-60 cells with dibutyryl cyclic AMP in the presence of tunicamycin, a fungal product that blocks N-linked glycosylation, results in two smaller affinity-labeled FPR forms, with apparent molecular masses of 38–48 kDa and 32 kDa on SDS-PAGE, suggesting the presence of at least two glycosylation sites.
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
EndoS2 D184M endoglycosidase. Sequence aa 1–843 of EndoS2 from S. pyogenes55 (containing the D184M mutation)33 and a C-terminal decahistidine tag was cloned into a pET-20b(+) vector and transformed into BL21 (DE3)pLysS E. coli (Fisher, Cat# PRL1195). E. coli containing the EndoS2 D184M vector was then grown in LB media (Fisher, Cat# BP1427–500) with ampicillin at 37°C. EndoS2 D184M was produced following induction with IPTG and cultured overnight with shaking at 16°C. Cells were then centrifuged at 4000 × g (4°C, 45 min), and the resulting pellet was resuspended in Binding Buffer (100 mM sodium phosphate, 10 mM Tris base buffer, pH 8.0) with 20 mM imidazole (Sigma, Cat# I202-100 G). Cells were lysed using freeze/thaw method as described above, and the supernatant was purified using Ni-NTA affinity chromatography. Following binding of the protein to the Ni-NTA resin (Sigma, Cat# 70691–4), the resin was washed with six column volumes of Binding Buffer with 20 mM imidazole, and then Binding Buffer with 60 mM imidazole. EndoS2 D184M was eluted using an elution buffer comprising 100 mM sodium phosphate, 10 mM Tris base buffer with 240 mM imidazole (pH 8.0) for four column volumes into separate fractions. SDS-PAGE (BioRad, 10% Mini-PROTEAN® TGX Stain FreeTM Protein Gels, Cat# 4568034 or 4,568,036) was used to confirm protein elution, and purified fractions were combined and buffer exchanged into phosphate-buffered saline (PBS) pH 6.0.
GlycoVHH: optimal sites for introducing N-glycans on the camelid VHH antibody scaffold and use for macrophage delivery
Published in mAbs, 2023
Loes van Schie, Wander Van Breedam, Charlotte Roels, Bert Schepens, Martin Frank, Ahmad Reza Mehdipour, Bram Laukens, Wim Nerinckx, Francis Santens, Simon Devos, Iebe Rossey, Karel Thooft, Sandrine Vanmarcke, Annelies Van Hecke, Xavier Saelens, Nico Callewaert
Intact protein MS of purified wildtype (WT) GBP returned a single set of two peaks, indicating the presence of GBP molecules with either an Nterminal glutamine or an N-terminal pyroglutamate (17 Da difference) (Supplementary D). In contrast, the spectra of GBP glycovariants showed an additional cluster of peaks at higher molecular weight. The 1216 Da shift of the major peak corresponds with the presence of a Man5GlcNAc2 glycan. Small peaks at incremental 162 Da intervals indicate the presence of additional hexoses on the N-glycan (representative example GBP-Q14N-P14A-G16T in Figure 2d; other glycovariants in Supplementary D). Endoglycosidase treatment leaves a single GlcNAc, allowing mass spectrometrical quantification of site occupancy. Peak quantification of endoglycosidase T (endoT) digested purified protein samples demonstrated high N-glycosylation site occupancies from 71% up to 96% (Figure 2d, Supplementary D and E and Table 1), but this variance is largely due to bias toward the non-glycosylated species during purification, as set out above, as the non-glycosylated species in the selected glycovariants were not or barely detectable on SDS-PAGE prior to purification.
Mass spectrometry analysis of glycoprotein biomarkers in human blood of hepatocellular carcinoma
Published in Expert Review of Proteomics, 2019
Kwang Hoe Kim, Jin Young Kim, Jong Shin Yoo
Lubman and coworkers developed an LC-MS-based approach to detecting core fucosylation (CF) of glycoproteins in the serum of patients with HCC [115]. Depletion of high-abundance glycoproteins was followed by trypsin digestion, iTRAQ labeling, and enrichment of core fucosylated peptides by LCA lectin. Finally, endoglycosidase F3 (Endo F3)-digested glycopeptides were detected by LC-MS/MS. Endo F3 generates the innermost N-acetylglucosamine and/or core-fucose bound to the peptide. A total of 1,300 CF peptides derived from 613 CF proteins was identified in serum samples. Among them, the levels of 20 and 26 CF peptides differed between ALC-related HCC and HCV-related HCC, respectively, compared to cirrhosis. The levels of three CF peptides from FN were elevated in ALC-related HCC compared to ALC-related cirrhosis (AUROC = 0.890 at site 1007; sensitivity = 92.9%, specificity 85.7%) (Table 2). When combined with the serum AFP level, the AUROC value increased to 0.92,0 with a sensitivity of 100% and specificity of 92.9% (Table 3). The CF of FN has potential as a biomarker for early stage HCC.