Analysis of N-linked oligosaccharides: progress towards the characterisation of glycoprotein-linked carbohydrates

The covalent attachment of carbohydrate to proteins is a very common co- or post-translational event in the biosynthesis of glycoproteins. The type and heterogeneity of these oligosaccharides can affect a range of physico-chemical and biological properties of a glycoprotein. Thus the development of sensitive, reliable and robust analytical methods for carbohydrate analysis is important in the pharmaceutical industry, especially in the recombinant production of experimental and therapeutic glycoproteins. In this report we have reviewed methodology for the in-gel enzymatic release of N-linked oligosaccharides from glycoproteins separated by electrophoresis. These oligosaccharides are derivatised by reductive amination using 3-acetamido-6-aminoacridine (AA-Ac), a novel, highly fluorescent probe. A major advantage of this technique is that glycan derivatives are amenable to analysis by an array of chromatographic and mass spectrometric methods, allowing the resolution and characterisation of a wide variety of glycan structures. It is hoped that in due course the methodology described will be applied to proteomics studies, especially in identifying the role of carbohydrate in protein function and disease.     

Expression, purification, and characterization of gp160e, the soluble, trimeric ectodomain of the simian immunodeficiency virus envelope glycoprotein, gp160

The envelope glycoprotein, gp160, of simian immunodeficiency virus (SIV) shares approximately 25% sequence identity with gp160 from the human immunodeficiency virus, type I, indicating a close structural similarity. As a result of binding to cell surface CD4 and co-receptor (e.g. CCR5 and CXCR4), both SIV and human immunodeficiency virus gp160 mediate viral entry by membrane fusion. We report here the characterization of gp160e, the soluble ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments.     

Structural evidence for recognition of a single epitope by two distinct antibodies

The structure of a complex between the hemagglutinin of influenza virus and the Fab of a neutralizing antibody was determined by X-ray crystallography at 2.8 A resolution. This antibody and another which has only 56% sequence identity bind to the same epitope with very similar affinities and in the same orientation. One third of the interactions is conserved in the two complexes; a significant proportion of the interactions that differ are established by residues of the H3 complementarity-determining regions (CDR) which adopt distinct conformations in the two antibodies. This demonstrates that there is a definite flexibility in the selection of antibodies that bind to a given epitope, despite the high affinity of their complexes. This flexibility allows the humoral immune response to be redundant, a feature that may be useful in achieving longer lasting protection against evolving viral pathogens.     

Plasticity of influenza haemagglutinin fusion peptides and their interaction with lipid bilayers

A detailed molecular dynamics study of the haemagglutinin fusion peptide (N-terminal 20 residues of the HA2 subunits) in a model bilayer has yielded useful information about the molecular interactions leading to insertion into the lipids. Simulations were performed on the native sequence, as well as a number of mutant sequences, which are either fusogenic or nonfusogenic. For the native sequence and fusogenic mutants, the N-terminal 11 residues of the fusion peptides are helical and insert with a tilt angle of approximately 30 degrees with respect to the membrane normal, in very good agreement with experimental data. The tilted insertion of the native sequence peptide leads to membrane bilayer thinning and the calculated order parameters show larger disorder of the alkyl chains. These results indicate that the lipid packing is perturbed by the fusion peptide and could be used to explain membrane fusion. For the nonfusogenic sequences investigated, it was found that most of them equilibrate parallel to the interface plane and do not adopt a tilted conformation. The presence of a charged residue at the beginning of the sequence (G1E mutant) resulted in a more difficult case, and the outcomes do not fall straightforwardly into the general picture. Sequence searches have revealed similarities of the fusion peptide of influenza haemagglutinin with peptide sequences such as segments of porin, amyloid alpha eta peptide, and a peptide from the prion sequence. These results confirm that the sequence can adopt different folds in different environments. The plasticity and the conformational dependence on the local environment could be used to better understand the function of fusion peptides.     

The post-translational modifications of the nuclear encoded subunits of complex I from bovine heart mitochondria

Bovine complex I is an assembly of 46 different proteins. Seven of them are encoded in mitochondrial DNA, and the rest are nuclear gene products that are imported into the organelle. Fourteen of the nuclear encoded subunits have modified N termini. Many of these post-translational modifications have been deduced previously from intact protein masses. These assignments have been verified by mass spectrometric analysis of peptides. Thirteen of them are N-alpha-acetylated, and a 14th, subunit B18, is N-alpha-myristoylated. Subunit B18 forms part of the membrane arm of the complex, and the myristoyl group may attach subunit B18 to the membrane. One subunit, B12, has a particularly complex pattern of post-translational modification that has not been analyzed before. It is a mixture of the N-alpha-acetylated form and the form with a free N terminus. In addition, it has one, two, or three methyl groups attached to histidine residues at positions 4, 6, and 8 in various combinations. The predominant form is methylated on residues 4 and 6. There is no evidence for the methylation of histidine 2. Subunit B12 is also part of the membrane arm of complex I, and it probably spans the membrane once, but as its orientation is not known, the methylation sites could be in either the matrix or the intermembrane space. These experiments represent another significant step toward establishing the precise chemical composition of mammalian complex I.     

A chimeric protein of simian immunodeficiency virus envelope glycoprotein gp140 and Escherichia coli aspartate transcarbamoylase

The envelope glycoproteins of the human immunodeficiency virus and the related simian immunodeficiency virus (SIV) mediate viral entry into host cells by fusing viral and target cell membranes. We have reported expression, purification, and characterization of gp140 (also called gp160e), the soluble, trimeric ectodomain of the SIV envelope glycoprotein, gp160 (B. Chen et al., J. Biol. Chem. 275:34946-34953, 2000). We have now expressed and purified chimeric proteins of SIV gp140 and its variants with the catalytic subunit (C) of Escherichia coli aspartate transcarbamoylase (ATCase). The fusion proteins (SIV gp140-ATC) bind viral receptor CD4 and a number of monoclonal antibodies specific for SIV gp140. The chimeric molecule also has ATCase activity, which requires trimerization of the ATCase C chains. Thus, the fusion protein is trimeric. When ATCase regulatory subunit dimers (R(2)) are added, the fusion protein assembles into dimers of trimers as expected from the structure of C(6)R(6) ATCase. Negative-stain electron microscopy reveals spikey features of both SIV gp140 and SIV gp140-ATC. The production of the fusion proteins may enhance the possibilities for structure determination of the envelope glycoprotein either by electron cryomicroscopy or X-ray crystallography.     

Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.     

Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis

Background

In the last years, the biotechnological production of platform chemicals for fuel components has become a major focus of interest. Although ligno-cellulosic material is considered as suitable feedstock, the almost inevitable pretreatment of this recalcitrant material may interfere with the subsequent fermentation steps. In this study, the fungus Ustilago maydis was used to produce itaconic acid as platform chemical for the synthesis of potential biofuels such as 3-methyltetrahydrofuran. No studies, however, have investigated how pretreatment of ligno-cellulosic biomass precisely influences the subsequent fermentation by U. maydis. Thus, this current study aims to first characterize U. maydis in shake flasks and then to evaluate the influence of three exemplary pretreatment methods on the cultivation and itaconic acid production of this fungus. Cellulose enzymatically hydrolysed in seawater and salt-assisted organic-acid catalysed cellulose were investigated as substrates. Lastly, hydrolysed hemicellulose from fractionated beech wood was applied as substrate.

Results

U. maydis was characterized on shake flask level regarding its itaconic acid production on glucose. Nitrogen limitation was shown to be a crucial condition for the production of itaconic acid. For itaconic acid concentrations above 25 g/L, a significant product inhibition was observed. Performing experiments that simulated influences of possible pretreatment methods, U. maydis was only slightly affected by high osmolarities up to 3.5 osmol/L as well as of 0.1 M oxalic acid. The production of itaconic acid was achieved on pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction of pretreated beech wood.

Conclusion

The fungus U. maydis is a promising producer of itaconic acid, since it grows as single cells (yeast-like) in submerged cultivations and it is extremely robust in high osmotic media and real seawater. Moreover, U. maydis can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this fungus combines important advantages of yeasts and filamentous fungi. Nevertheless, the biomass pretreatment does indeed affect the subsequent itaconic acid production. Although U. maydis is insusceptible to most possible impurities from pretreatment, high amounts of salts or residues of organic acids can slow microbial growth and decrease the production. Consequently, the pretreatment step needs to fit the prerequisites defined by the actual microorganisms applied for fermentation.     

Clinical features and predictors for disease natural progression in adults with Pompe disease: a nationwide prospective observational study

Background

Due partly to physicians’ unawareness, many adults with Pompe disease are diagnosed with great delay. Besides, it is not well known which factors influence the rate of disease progression, and thus disease outcome. We delineated the specific clinical features of Pompe disease in adults, and mapped out the distribution and severity of muscle weakness, and the sequence of involvement of the individual muscle groups. Furthermore, we defined the natural disease course and identified prognostic factors for disease progression.

Methods

We conducted a single-center, prospective, observational study. Muscle strength (manual muscle testing, and hand-held dynamometry), muscle function (quick motor function test), and pulmonary function (forced vital capacity in sitting and supine positions) were assessed every 3–6 months and analyzed using repeated-measures ANOVA.

Results

Between October 2004 and August 2009, 94 patients aged between 25 and 75 years were included in the study. Although skeletal muscle weakness was typically distributed in a limb-girdle pattern, many patients had unfamiliar features such as ptosis (23%), bulbar weakness (28%), and scapular winging (33%). During follow-up (average 1.6 years, range 0.5-4.2 years), skeletal muscle strength deteriorated significantly (mean declines of ?1.3% point/year for manual muscle testing and of ?2.6% points/year for hand-held dynamometry; both p<0.001). Longer disease duration (>15 years) and pulmonary involvement (forced vital capacity in sitting position <80%) at study entry predicted faster decline. On average, forced vital capacity in supine position deteriorated by 1.3% points per year (p=0.02). Decline in pulmonary function was consistent across subgroups. Ten percent of patients declined unexpectedly fast.

Conclusions

Recognizing patterns of common and less familiar characteristics in adults with Pompe disease facilitates timely diagnosis. Longer disease duration and reduced pulmonary function stand out as predictors of rapid disease progression, and aid in deciding whether to initiate enzyme replacement therapy, or when.