Hydrophobic Glycoproteins Research Articles

Short Amphiphiles or Micelle Peptides May Help to Fight Against COVID-19.

COVID-19 is a global threat as a result of the incessant spread of SARS-CoV- 2, necessitating the rapid availability of effective antiviral medications to protect our society. For SARSCoV- 2, a group of peptides has already been indicated, although their effectiveness has yet to be shown. SARS-CoV-2 is an enveloped virus with hydrophobic fusion protein and spike glycoproteins. Here, we have compiled a list of amphiphilic peptides that have been published, as well as their in-silico docking studies with the SARS-CoV-2 spike glycoprotein. The findings demonstrated that spike protein and amphiphilic peptides with increased binding affinity create a complex. It was also observed that PalL1 (ARLPRTMVHPKPAQP), 10AN1 (FWFTLIKTQAKQPARYRRFC), THETA defensin (RCICGRGICRLL), and mucroporin M1 (LFRLIKSLIKRLVSAFK) showed the binding free energy of more than -1000 kcal/mol. Molecular pI and hydrophobicity are also important factors of peptides to enhance the binding affinity with spike protein of SARS-CoV-2. In light of these findings, it is crucial to compare the in-vitro to in-vivo efficacy of amphiphilic peptides in order to produce an efficient anti-SARS-CoV-2 peptide therapy that might assist control the present pandemic scenario.

Characterization of Murine Brain Membrane Glycoproteins by Detergent Assisted Lectin Affinity Chromatography

Membrane glycoproteins play vital roles in many fundamental physiological and pathophysiological processes in the central nervous system and represent important targets for pharmaceuticals and biomarker discovery. However, their isolation and characterization have been greatly limited. Lectin affinity chromatography (LAC) has evolved as a powerful method to enrich glycoproteins in biofluid and cell/tissue lysate. However, its use in the hydrophobic fraction of the samples has rarely been explored. In this study, we have conducted a systematic investigation on the lectin binding efficiency in the presence of four commonly used detergents. We have found that, under certain concentrations, detergents can minimize nonspecific binding and facilitate the elution of hydrophobic glycoproteins. With the detergent assisted lectin affinity chromatography (DALAC), a total of 1491 proteins were identified with low numbers of false positives from two lectins. Proteins (699) were identified with at least two unique peptides, of which 219 are membrane glycoproteins. Compared to the traditional methods, the DALAC approach significantly increased the recovery of plasma membrane and glycoproteins. NP-40 is recommended as a well rounded detergent for DALAC, but the conditions for enriching certain target proteins need to be empirically determined. This study represents the first global identification of the murine brain glycoproteome.

Genetic evidence that apolipoprotein E4 is not a relevant susceptibility factor for cholelithiasis in two high-risk populations

Apolipoprotein E (apoE) isoforms are genetic determinants of interindividual variations in lipid metabolism. To assess whether apoE is a genetic risk factor for cholesterol gallstone disease (GD), we analyzed apoE variants in populations from Chile and Germany, two countries with very high prevalence rates of this disease. ApoE genotypes were determined in Chilean gallstone patients (n = 117) and control subjects (n = 122) as well as in German gallstone patients (n = 184) and matched controls (n = 184). In addition, we studied apoE variants in subgroups of Chilean patients with strong differences in their susceptibility to acquire gallstones: 50 elderly subjects without gallstones in spite of well-known risk factors for this disease (gallstone-resistant) and 32 young individuals with gallstones but without risk factors (gallstone-susceptible). Furthermore, correlation analysis of apoE genotypes with cholesterol crystal formation times, biliary cholesterol saturation index (CSI), and gallstone cholesterol contents was performed in 81 cholecystectomized patients. In this study analyzing the largest sample set available, apoE4 genotype was not associated with an increased frequency of GD in either population. Moreover, in the Chilean population after adjusting for risk factors such as gender, age, body mass index, serum lipids, and glucose, the odds ratio for the association of the apoE4 allele and GD was significantly (P < 0.05) <1. Also, genotypes were not correlated with cholesterol crystal formation time, CSI, or gallstone cholesterol content. In contrast to previous smaller studies, apoE polymorphisms were not associated with susceptibility to cholesterol GD in high-risk populations.

Regulation of Wnt Signalling by Receptor-mediated Endocytosis

Wnts compromise a large family of secreted and hydrophobic glycoproteins that control a variety of developmental and adult processes in all metazoan organisms. Recent advances in the field of Wnt signalling have revealed that Wnt activates multiple intracellular cascades, resulting in the regulation of cellular proliferation, differentiation, migration and polarity. However, it is not clear how Wnt activates these pathways after it binds to the receptors. It has been shown that Wnt and its antagonist Dickkopf are internalized with their receptors. This review highlights distinct endocytic pathways correlate with specificity of Wnt signalling events.

Identification of the hydrophobic glycoproteins of Caenorhabditis elegans

Hydrophobic proteins such as integral membrane proteins are difficult to separate, and therefore to study, at a proteomics level. However, the Asn-linked (N-linked) carbohydrates (N-glycans) contained in membrane glycoproteins are important in differentiation, embryogenesis, inflammation, cancer and metastasis, and other vital cellular processes. Thus, the identification of these proteins and their sites of glycosylation in a well-characterized model organism is the first step toward understanding the mechanisms by which N-glycans and their associated proteins function in vivo. In this report, a proteomics method recently developed by our group was applied to identify 117 hydrophobic N-glycosylated proteins of Caenorhabditis elegans extracts by analysis of 195 glycopeptides containing 199 Asn-linked oligosaccharides. Most of the proteins identified are involved in cell adhesion, metabolism, or the transport of small molecules. In addition, there are 18 proteins for which no function is known or predictable by sequence homologies and two proteins which were previously predicted to exist only on the basis of genomic sequences in the C. elegans database. Because N-glycosylation is initiated in the lumen of the endoplasmic reticulum (ER), our data can be used to reassess the previously predicted subcellular localizations of these proteins. As well, the identification of N-glycosylation sites helps establish the membrane topology of the associated glycoproteins. Caenorhabditis elegans strains are presently available with mutations in 17 of the genes we have identified. The powerful genetic tools available for C. elegans can be used to make other strains with mutations in genes encoding N-glycosylated proteins and thereby determine N-glycan function.

Accumulation of retinol in the liver after prolonged hyporetinolemia in the vitamin A-sufficient rat

We assessed the effects of prolonged reduction of plasma retinol concentrations (hyporetinolemia) on the distribution of tissue vitamin A (VA) and of its active compounds using a model of continuous recombinant human interleukin-6 (rhIL-6) infusion via osmotic minipumps in VA-sufficient male rats. Plasma retinol and retinol-binding protein (RBP) concentrations remained decreased and lower in rhIL-6-treated rats compared with controls from 7.5 h throughout 7 days of infusion (P < 0.001). This reduction was accompanied by a 68% increase in hepatic retinol concentration by 7 days (P < 0.05). Hepatic and renal retinyl palmitate and retinoic acid concentrations did not change, and renal megalin content remained unchanged; hepatic RBP concentrations were 41% lower in rhIL-6-treated rats compared with controls (P < 0.05). These results indicate that instead of being lost, retinol accumulated in the liver during inflammation and that hyporetinolemia was attributable to a decrease in the availability of hepatic RBP. A plausible consequence of the effect of rhIL-6-induced hyporetinolemia is that by 7 days tissues that are dependent on plasma retinol may become deprived of VA. These results have important implications in understanding the mechanism by which measles infection induces hyporetinolemia and VA deficiency of extrahepatic tissues.

Towards crystallization of hydrophobic myelin glycoproteins: P0 and PASII/PMP22

The preparation of a pure and homogeneous protein sample at proper concentration is a prerequisite for success when attempting their crystallization for structural determination. The detergents suitable for solubilization particularly of membrane proteins are not always the best for crystallization. Myelin of the peripheral nervous system of vertebrates is the example of a membrane for which neutral or “gentle” detergents are not even strong enough to solubilize its proteins. In contrast, sodium- or lithium-dodecyl sulfate is very effective. We solubilized myelin membrane in 2% (w/v) sodium dodecyl sulfate, followed by chromatographic purification of the hydrophobic myelin glycoproteins P0 and PASII/PMP22, and finally, we have exchanged the sodium dodecyl sulfate bound to protein for other neutral detergents using ceramic hydroxyapatite column. Theoretically, we should easily exchange sodium dodecyl sulfate for any neutral detergent, but for some of them, the solubility of myelin glycoproteins is low. To monitor the potential variability in the secondary structure of glycoproteins, we have used circular dichroism. Sodium dodecyl sulfate seems to be the appropriate detergent for the purpose of purification of very hydrophobic glycoproteins, since it can be easily exchanged for another neutral detergent.

Cholesterol crystallization-promoting activity of aminopeptidase-N isolated from the vesicular carrier of biliary lipids

Different hydrophobic glycoproteins are associated to native biliary vesicles, which are the major carrier of biliary cholesterol. Some of these proteins promote cholesterol crystallization, a key step in cholesterol gallstone formation. This study was specifically conducted to identify the 130 kDa biliary vesicle-associated glycoprotein and to determine its in vitro effect on the cholesterol crystal formation time. The 130 kDa vesicular glycoprotein was identified as aminopeptidase-N by amino acid sequencing and specific enzymatic assay. Polyclonal antibodies raised against aminopeptidase-N allowed us to determine its concentration in human hepatic bile, which varied from 17.3 to 57.6 μg ml . Aminopeptidase-N showed a concentration-dependent cholesterol crystallization activity when it was added to supersaturated model bile at a concentration range usually found in native bile. Because of this promoting effect on in vitro cholesterol crystal formation, we suggest that biliary aminopeptidase-N may play a critical role in the pathogenesis of cholesterol gallstone disease.

Isolation and partial characterization of cholesterol pronucleating hydrophobic glycoproteins associated to native biliary vesicles

Cholesterol is transported both in unilamellar phosphatidylcholine vesicles and in bile salts-mixed micelles in native bile. The vesicular carrier of biliary lipids apparently has a well defined protein profile with a potent cholesterol crystallization-promoting activity. This study was conducted to identify and further characterize these vesicular proteins and to test the effect of isolated vesicular proteins on the cholesterol crystal formation in supersaturated model bile. The results confirmed that proteins are a constant component of highly purified biliary vesicles both in hepatic and gallbladder bile. Immunoglobulins (IgA, IgG and IgM) and albumin are associated to the purified hepatic biliary vesicles. Furthermore, four different hydrophobic glycoproteins with a molecular mass of 130, 114, 86, and 62–67 kDa were isolated. These glycoproteins showed no reactivity with anti-human whole serum or anti-immunoglobulin antibodies, suggesting that these proteins are biliary-specific. Isolated 130, 114 and 62–67 kDa vesicular glycoproteins significantly decreased the cholesterol nucleation time in artificial model bile. We concluded that some, but not all, vesicular-bound hydrophobic glycoproteins have cholesterol pronucleating activity and they may be involved in the pathogenesis of cholesterol gallstone disease.

Structural characterization of the dihydropyridine-sensitive calcium channel alpha 2-subunit and the associated delta peptides.

Upon disulfide bond reduction, the alpha 2-subunit of the dihydropyridine-sensitive Ca2+ channel undergoes a characteristic mobility shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with the concurrent appearance of the three delta peptides delta 1 (25,000 Da), delta 2 (22,000 Da), and delta 3 (17,000 Da). Densitometric scanning of Coomassie Blue-stained gels shows a stoichiometric ration of 1.0:0.31.47:0.08 for the alpha 2-subunit and the delta peptides 1, 2, and 3, respectively. Characterization of the delta peptides using antibodies, photoincorporation of a hydrophobic probe, and lectin staining shows tham to be antigenically similar hydrophobic glycoproteins. Amino-terminal sequence analysis of the delta peptides reveals three identical sequences that match the predicted amino acid sequence of the alpha 2-subunit starting at Ala935. Enzymatic deglycosylation of the reduced alpha 2.delta complex produces individual core peptides of 105,000 and 17,000 Da, respectively. Treatment of skeletal muscle membranes with high pH in the presence of reducing agents is able to extract the larger amino-terminal peptide but not the smaller carboxyl (delta) peptide, consistent with a single transmembrane domain in the carboxyl (delta) region. The data support a model of the alpha 2-subunit in which the propeptide is processed into two chains that remain attached through disulfide linkages.

Characterization and purification of Parafilaria bovicola antigens by chromatofocusing to enhance specificity in serodiagnosis.

A study was conducted to determine if the purification of Parafilaria bovicola antigens can increase the specificity of serodiagnosis of parafilariasis in enzyme-linked immunosorbent assay. Antigens released from adult worms of P. bovicola were separated by chromatofocusing on a polybuffer exchanger of the pH range 7.3-4.0 Polypeptide analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis showed the presence of four major polypeptides with MWs of 41, 36, 24 and 20 kDa. Additional biochemical characterization identified the 24- and 20-kDa polypeptides as hydrophobic glycoproteins. The chromatofocusing purification procedures were also applied for separation of a whole-worm extract. Again, the 41- and 36-kDa antigens were identified in separate peak fractions. Using ELISA, it was shown that the 41- and 35-kDa antigens were recognized by bovine antibodies specific for P. bovicola, but not by other sera collected from cattle infected by Onchocerca gutturosa, Onchocerca lienalis, Ostertagia ostertagi and Dictyocaulus viviparus. The serological evaluation strongly suggests that the 41- and 36-kDa antigens are P. bovicola specific.

High-performance concanavalin a affinity chromatography of liver and hepatoma membrane proteins

Although the separation of water-soluble glycoproteins by high-performance (HP) concanavalin A (ConA) affinity chromatography (AC) is feasible, irregularities may be encountered with hydrophobic glycoproteins. The separation of plasma membrane glycoproteins from liver and Morris hepatoma 7777, used as a model, showed that not only the interaction between the lectin and the oligosaccharide portion of the glycoproteins plays a role in the chromatographic process, but also the hydrophobic interactions between sample and lectin and between sample and support. In this, the characteristics of the support, such as surface hydrophobicity and pore size, play an important part. It was found that a portion of the ConA is not covalently bound to the column, especially when elution is carried out with buffers containing detergents. Moreover, some extremely hydrophobic proteins could only be eluted from the column when high concentrations of detergents [1% (w/v) or higher] were applied. Despite these difficulties, four membrane glycoproteins from the liver with apparent molecular weights of 60, 80, 100 and 110–120 kilodaltons could be highly enriched by ConA HPAC. These proteins were further fractionated according to their strength of binding to the ConA and their different hydrophobic characteristics, using various detergents as eluents.

Partikelelektrophoretische Charakterisierung der Oberflächeneigenschaften von alkanutilisierenden Hefezellen: Chemische Zusammensetzung und Tensidadsorption

AbstractBy using particle electrophoresis and quantitative analysis the interaction of anionic surfactants (sodium dodecylculfate, sodium decylbenzene sulfonate) with a hydrocarbon‐grown yeast and a commercial baker's yeast has been investigated to obtain further informations about the chemical composition of the surface region (3–6 nm) of the cell wall of this yeasts. A correlation is found between the chemical composition and the different adsorption behaviour of various batches of the same yeasts. It was found that surface‐localized hydrophobic glycoproteins, probably proteophosphomannan are responsible for the strong pH dependent adsorption behaviour of a typical Candida species.In contrast the cell surface of a typical Saccharomyces species was strongly hydrophilic and showed no surfactant adsorption.This can be explained by the presence of a polysaccharide probably phosphomannan in the surface region only.The implication of proteins in the adsorption process is confirmed by model experiments too.The models for the yeast cell wall proposed by LAMPEN and KIDBY et al. were critically discussed in terms of our results.

Efficacy of purified schistosoma japonicum egg antigens for ELISA serodiagnosis of human Schistosomiasis japonica: specificity and sensitivity.

At present, there is no consensus that purified schistosome egg antigens offer any advantage in the diagnosis of schistosomiasis by enzyme linked immunosorbent assay (ELISA). Previously, we demonstrated by multiple techniques that the major serologic antigens in Schistosoma japonicum soluble egg antigen (SEA) are glycoproteins, and that the glycoproteins with highest specificity and sensitivity are hydrophobes. We therefore tested these materials for their specificity, sensitivity and cost effectiveness in the ELISA. In this study we used five SEA fractions that varied in their purity and antigenicity. The order of immunologic specific activity in the ELISA, measured by titration of a standard sera pool, was: hydrophobic glycoproteins (highest), crude SEA glycoproteins, hydrophilic glycoproteins, crude SEA, and SEA proteins (lowest). Complexity (purity) of these materials were (in rank order), hydrophilic glycoproteins (purest), hydrophobic glycoproteins, crude glycoproteins, SEA proteins, and crude SEA (most complex). Epidemiologic sensitivity in the ELISA was tested on limited but well characterized populations. At high antigen coating concentration (0.5 microgram/well), the only antigen fraction with poor sensitivity was SEA proteins. There was little difference in epidemiologic sensitivity between the purer fractions with highest immunologic sensitivity (hydrophobic glycoproteins and crude SEA glycoproteins) and the crude SEA which possesses intermediate immunologic sensitivity. Differences in epidemiologic sensitivity were most pronounced when wells were coated at an antigen concentration (0.1 microgram/well) where crude SEA began to fail. Specificity for all preparations, assessed by reactivity with sera from patients with other trematode infections and with cestode and nematode infections, was excellent. The clinical sensitivity of the ELISA employing crude S. japonicum SEA is so high, and the specificity so good, that the increased immunologic sensitivity of partially purified antigens had little effect on epidemiologic sensitivity. This is not true for the S. mansoni ELISA where crude antigens had inferior sensitivity and specificity.