Consequences Of Glycosylation Research Articles

C-Terminal Domain of Hemocyanin, a Major Antimicrobial Protein from Litopenaeus vannamei: Structural Homology with Immunoglobulins and Molecular Diversity.

Invertebrates rely heavily on immune-like molecules with highly diversified variability so as to counteract infections. However, the mechanisms and the relationship between this variability and functionalities are not well understood. Here, we showed that the C-terminal domain of hemocyanin (HMC) from shrimp Litopenaeus vannamei contained an evolutionary conserved domain with highly variable genetic sequence, which is structurally homologous to immunoglobulin (Ig). This domain is responsible for recognizing and binding to bacteria or red blood cells, initiating agglutination and hemolysis. Furthermore, when HMC is separated into three fractions using anti-human IgM, IgG, or IgA, the subpopulation, which reacted with anti-human IgM (HMC-M), showed the most significant antimicrobial activity. The high potency of HMC-M is a consequence of glycosylation, as it contains high abundance of α-d-mannose relative to α-d-glucose and N-acetyl-d-galactosamine. Thus, the removal of these glycans abolished the antimicrobial activity of HMC-M. Our results present a comprehensive investigation of the role of HMC in fighting against infections through genetic variability and epigenetic modification.

Glycosylation of Cellulases: Engineering Better Enzymes for Biofuels.

Cellulose in plant cell walls is the largest reservoir of renewable carbon on Earth. The saccharification of cellulose from plant biomass into soluble sugars can be achieved using fungal and bacterial cellulolytic enzymes, cellulases, and further converted into fuels and chemicals. Most fungal cellulases are both N- and O-glycosylated in their native form, yet the consequences of glycosylation on activity and structure are not fully understood. Studying protein glycosylation is challenging as glycans are extremely heterogeneous, stereochemically complex, and glycosylation is not under direct genetic control. Despite these limitations, many studies have begun to unveil the role of cellulase glycosylation, especially in the industrially relevant cellobiohydrolase from Trichoderma reesei, Cel7A. Glycosylation confers many beneficial properties to cellulases including enhanced activity, thermal and proteolytic stability, and structural stabilization. However, glycosylation must be controlled carefully as such positive effects can be dampened or reversed. Encouragingly, methods for the manipulation of glycan structures have been recently reported that employ genetic tuning of glycan-active enzymes expressed from homogeneous and heterologous fungal hosts. Taken together, these studies have enabled new strategies for the exploitation of protein glycosylation for the production of enhanced cellulases for biofuel production.

Context-Dependent Effects of Asparagine Glycosylation on Pin WW Folding Kinetics and Thermodynamics

In extracting folding and unfolding rate information from our apparent rate constant data, and in fitting the extracted folding rates as a function of temperature to Kramer's model, we inadvertently used the pre-jump equilibrium temperature instead of the post-jump temperature: a difference of 12 °C. The absolute folding and unfolding rate values for each WW variant in Tables 3 and ​and44 are different than reported in the original paper by 2-fold, but most of the folding and unfolding rate ratios (comparing the folding or unfolding rates of the glycosylated vs. non-glycosylated WW variants) are similar to the values presented in the original paper. The changes to the data do not change the conclusion of our paper that specific, evolved protein-glycan contacts must also play a role in mediating the beneficial energetic effects on protein folding that glycosylation can confer. Table 3 Experimentally Measured and Computationally Predicted Folding Rates for Pin WW Variants Having Either Asn or Asn-GlcNAc at the Indicated Positionsa Table 4 Experimentally Measured and Computationally Predicted Unfolding Rates for Pin WW Variants Having Either Asn or Asn-GlcNAc at the Indicated Positionsa The corrected data appear in Tables 3 and ​and44 below. The corrected versions of Figures S44 to S55 (the data that Tables 3 and ​and44 summarize) are provided in the Supporting Information. The following sentences in the Results section text referring to our kinetic data (pp. 15364): The modest stabilizing effect of the Asn to Asn-GlcNAc substitution at positions 20 and 30 appears to be a result, in part, of an increased folding rate. Glycosylation at position 20 (compare 20 with 20g) increases the folding rate ⍰1.1-fold (Table 3). Glycosylation at position 30 (compare 30 with 30g) has a similar effect. These small folding rate increases agree with the predictions of the computational model and could be consistent with a small amount of denatured-state destabilization as a consequence of glycosylation. However, the decreased unfolding rate of 20g relative to 20 (also predicted by the model) could indicate that glycosylation at position 20 actually stabilizes the folding transition state and native state simultaneously. are changed to: The modest stabilizing effect of the Asn to Asn-GlcNAc substitution at position 20 appears to be primarily due to an increased folding rate (20g folds 1.5 times faster than 20). This folding rate increase agrees with the predictions of the computational model, and could be consistent with a small amount of denatured-state destabilization as a consequence of glycosylation. However, the unfolding rates of 20g and 20 are indistinguishable, which disagrees with the predicted decrease in unfolding rate upon glycosylation. The stabilizing effect of glycosylation at position 30 appears to come primarily from a decrease in unfolding rate (30g unfolds 0.7 times as fast as non-glycosylated Pin WW), as predicted by the model. This decrease compensates for an unexpected decrease in folding rate: 30g folds 0.8 times as fast as Pin WW whereas the model predicted a large increase in folding rate. Results for 33 and 33g are similarly inconsistent with the predictions of the model. In addition, the following sentence in the Discussion section text about our kinetic data (pp. 15366): For example, the observed increased stability and increased folding rate of 20g relative to 20, and of 30g relative to 30, could be the result of simultaneous transition state and native state stabilization (rather than destabilization of the denatured ensemble), reflecting the presence of favorable glycan-protein contacts at these positions. is changed to: For example, the observed increased stability of 20g and 30g relative to 20 and 30, respectively, could be the result of favorable native-state GlcNAc-protein contacts at these positions.

Analysis of lysine clipping of a humanized Lewis-Y specific IgG antibody and its relation to Fc-mediated effector function

During the analytical characterization of the humanized Lewis-Y specific monoclonal antibody IGN311 (IgG1/κ) used for passive anti-cancer therapy in humans, isoelectric focusing (IEF) experiments revealed that IGN311 batches produced in serum-containing and serum-free medium, respectively, displayed different banding patterns. The additional bands in the IEF pattern correlated with additional peaks observed by subsequent cation exchange (CEX)-HPLC analysis. Since the IEF pattern is one of the specification criteria in the quality control of monoclonal antibodies and a non-matching pattern may be indicative for lot-to-lot inconsistency, this phenomenon was investigated in detail. First, we investigated whether a difference in antibody glycosylation was the cause for the observed charge heterogeneity. De- N-glycosylation experiments demonstrated that charge heterogeneity observed in the IEF pattern is not a consequence of glycosylation. In contrast, sample treatment by carboxypeptidase B, removing the carboxy-terminal lysine residues from the two heavy chains of the antibody, resulted in reduced charge heterogeneity eliminating the two most basic bands observed in IEF. These data were supported by reversed phase HPLC–MALDI-TOF-MS analysis of enzymatically cleaved peptides of the antibody as well as by carboxy-terminal sequencing of the heavy chains. It was demonstrated that the differences in the IEF banding pattern were due to lysine clipping occurring during the production of the antibody. The antibody batch produced under serum-free conditions was less affected by lysine clipping. Both antibody variants – clipped and unclipped – elicited the same potency in a complement dependent cytotoxicity (CDC) assay demonstrating that lysine clipping of IGN311 does not impair Fc-mediated effector functions.

Biocatalysed synthesis of β- O-glucosides from 9-fluorenon-2-carbohydroxyesters. Part 3: IFN-inducing and anti-HSV-2 properties

In pursuing research on the antiviral, interferon (IFN)-inducing tilorone congeners, a new series of fluoren-carboxyhydroxyesters has been prepared and biologically explored. These esters have subsequently been used as sugar acceptors in the enzymatic transglycosylation reaction using the ‘retaining’ β-glycosidase from the archaeon Sulfolobus solfataricus (Ssβ-Gly). Both aglycones ( 1– 6) and corresponding β-glucosides ( β- glu 1– β-glu 6) have been screened for cytotoxicity, interferon-stimulating and antiviral properties against HSV-2. It was found that the addition of compounds β- glu 5, β- glu 6 and β- glu 4 to HSV-2 infected U937 cells downregulates viral replication and triggers cells to release IFN-α/β. Taken together, the results showed improved pharmacological profiles as a consequence of glycosylation. A molecular modelling study carried out on this series of compounds completed the structural characterisation of the novel compounds.

Polypeptide Characteristics and Immunological Properties of Exo- and Endoglucanases Purified from Maize Coleoptile Cell Walls

Summary Cereal coleoptile cell walls have exo- and endoglucanases capable of mediating the hydrolysis of non-cellulosic β-(l,3)(l,4)-glucan in situ . A purified exoglucanase (EC 3.2.1.58) resolved as a single band at 73.5 kDa, while endoglucanase isozymes consistently appeared as two bands at 32.9 and 34.3 kDa when subjected to SDS-PAGE. HPLC analysis of the native proteins by gel-permeation chromatography revealed molecular weights of ca. 55 and 29 kDa for the exo- and endoglucanases, respectively. The exoglucanase has an isoelectric focusing point at pi 7.2 and the endoglucanase isozymes appeared as two major bands, one at pi 7.8 and another at 7.3. Deglycosylation of the native proteins followed by SDS-PAGE demonstrated that sugars accounted for ca. 6.5 % of the exoglucanase and were 12.5 and 8.8 % of the two endoglucanase isozymes, respectively. After deglycosylation the two endoglucanases converged at 30.0 kDa, suggesting polypeptide homology and that divergence in electrophoretic mobility was a consequence of glycosylation. Antibodies raised against intact exo- and endoglucanases recognized the polypeptide of the corresponding enzymes, irrespective of glycosylation. The N-terminal amino acid sequence supported the conclusion that the exo- and endoglucanase have different polypeptide structures.

Purification and characterization of the messenger RNA for the heavy chain of rat immunoglobulin E.

The isolation and translational properties of rat immunoglobulin E (IgE) heavy chain mRNA are described. The mRNA has a sedimentation coefficient of approximately 18S, a chain length of about 2000 nucleotides and directs the synthesis in vitro of a polypeptide of 65000 molecular weight in an mRNA-dependent rabbit reticulocyte lysate. Inclusion of dog pancreatic microsomes in the cell-free translation system resulted in a heavy chain product of about 75000 molecular weight, presumably as a consequence of glycosylation in vitro. This species co-migrated in an SDS polyacrylamide gel with mature IgE heavy chain. Substantial purification of heavy chain mRNA was achieved by denaturing sucrose gradient centrifugation and agarose gel electrophoresis.