N-glycosylation of High Mobility Group Box 1 protein (HMGB1) modulates the interaction with glycyrrhizin: A molecular modeling study

High Mobility Group Box 1 protein (HMGB1) is an abundant protein with multiple functions in cells, acting as a DNA chaperone and damage-associated molecular pattern molecule. It represents an attractive target for the treatment of inflammatory diseases and cancers. The plant natural product glycyrrhizin (GLR) is a well-characterized ligand of HMGB1 and a drug used to treat diverse liver and skin diseases. The drug is known to bind to each of the two adjacent HMG boxes of the non-glycosylated protein. In cells, HMGB1 is N-glycosylated at three asparagine residues located in boxes A and B, and these N-glycans are essential for the nucleocytoplasmic transport of the protein. But the impact of the N-glycans on drug binding is unknown. Here we have investigated the effect of the N-glycosylation of HMGB1 on its interaction with GLR using molecular modelling, after incorporation of three N-glycans on a Human HMGB1 structure (PDB code 2YRQ). Sialylated bi-antennary N-glycans were introduced on the protein and exposed in a folded or an extended conformation for the drug binding study. The docking of the drug was performed using both 18α- and 18β-epimers of GLR and the conformations and potential energy of interaction (ΔE) of the different drug-protein complexes were compared. The N-glycans do not shield the drug binding sites on boxes A and B but can modulate the drug-protein interaction, via both direct and indirect effects. The calculations indicate that binding of 18α/β-GLR to the HMG box is generally reduced when the protein is N-glycosylated vs. the non-glycosylated protein. In particular, the N-glycans in an extended configuration significantly weaken the binding of GLR to box-B. The effects of the N-glycans are mostly indirect, but in one case a direct contact with the drug, via a carbohydrate-carbohydrate interaction, was observed with 18β-GLR bound to Box-B of glycosylated HMGB1. For the first time, it is shown (at least in silico) that N-glycosylation, one of the many post-translational modifications of HMGB1, can affect drug binding.     

Virtual Screening and In Vitro Evaluation of PD-1 Dimer Stabilizers for Uncoupling PD-1/PD-L1 Interaction from Natural Products

Genetic mutations accumulated overtime could generate many growth and survival advantages for cancer cells, but these mutations also mark cancer cells as targets to be eliminated by the immune system. To evade immune surveillance, cancer cells adopted different intrinsic molecules to suppress immune response. PD-L1 is frequently overexpressed in many cancer cells, and its engagement with PD-1 on T cells diminishes the extent of cytotoxicity from the immune system. To resume immunity for fighting cancer, several therapeutic antibodies disrupting the PD-1/PD-L1 interaction have been introduced in clinical practice. However, their immunogenicity, low tissue penetrance, and high production costs rendered these antibodies beneficial to only a limited number of patients. PD-L1 dimer formation shields the interaction interface for PD-1 binding; hence, screening for small molecule compounds stabilizing the PD-L1 dimer may make immune therapy more effective and widely affordable. In the current study, 111 candidates were selected from over 180,000 natural compound structures through virtual screening, contact fingerprint analysis, and pharmacological property prediction. Twenty-two representative candidates were further evaluated in vitro. Two compounds were found capable of inhibiting the PD-1/PD-L1 interaction and promoting PD-L1 dimer formation. Further structure optimization and clinical development of these lead inhibitors will eventually lead to more effective and affordable immunotherapeutic drugs for cancer patients.     

Discovery of Novel Small-Molecule Inhibitors of PD-1/PD-L1 Interaction via Structural Simplification Strategy

Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction is currently the focus in the field of cancer immunotherapy, and so far, several monoclonal antibodies (mAbs) have achieved encouraging outcomes in cancer treatment. Despite this achievement, mAbs-based therapies are struggling with limitations including poor tissue and tumor penetration, long half-life time, poor oral bioavailability, and expensive production costs, which prompted a shift towards the development of the small-molecule inhibitors of PD-1/PD-L1 pathways. Even though many small-molecule inhibitors targeting PD-1/PD-L1 interaction have been reported, their development lags behind the corresponding mAb, partly due to the challenges of developing drug-like small molecules. Herein, we report the discovery of a series of novel inhibitors targeting PD-1/PD-L1 interaction via structural simplification strategy by using BMS-1058 as a starting point. Among them, compound A9 stands out as the most promising candidate with excellent PD-L1 inhibitory activity (IC₅₀ = 0.93 nM, LE = 0.43) and high binding affinity to hPD-L1 (K_D = 3.64 nM, LE = 0.40). Furthermore, A9 can significantly promote the production of IFN-γ in a dose-dependent manner by rescuing PD-L1 mediated T-cell inhibition in Hep3B/OS-8/hPD-L1 and CD3-positive T cells co-culture assay. Taken together, these results suggest that A9 is a promising inhibitor of PD-1/PD-L1 interaction and is worthy for further study.     

PD-1与单克隆抗体残基特异性结合机制的计算丙氨酸扫描研究

通过分子动力学模拟检测了2种程序性细胞死亡蛋白(PD-1)/单克隆抗体(Pembrolizumab和Nivolumab)复合物,并使用高效的计算丙氨酸扫描方法预测了单抗与PD-1的结合热点,将它们与对PD-1/PD-L1结合重要的热点残基进行对比分析.结果显示, Pembrolizumab以类似于PD-L1的方式与PD-1结合,而Nivolumab则以不同的方式与PD-1结合. 2个PD-1/mAb复合物中共有的热点只有PD-1K131.同时发现,与PD-1K131结合的单抗的关键残基通常都受范德华(vdW)能量控制. 2种单克隆抗体上热点的自由能贡献都以vdW能量为主,这表明在下一代PD-1新抗体的设计中需要提高静电型热点残基的数量.     

Total Chemical Synthesis and Biological Activities of Glycosylated and Non‐Glycosylated Forms of the Chemokines CCL1 and Ser‐CCL1

CCL1 is a naturally glycosylated chemokine protein that is secreted by activated T‐cells and acts as a chemoattractant for monocytes. 1 Originally, CCL1 was identified as a 73 amino acid protein having one N‐glycosylation site, 1 and a variant 74 residue non‐glycosylated form, Ser‐CCL1, has also been described. 2 There are no systematic studies of the effect of glycosylation on the biological activities of either CCL1 or Ser‐CCL1. Here we report the total chemical syntheses of both N‐glycosylated and non‐glycosylated forms of (Ser‐)CCL1, by convergent native chemical ligation. We used an N‐glycan isolated from hen egg yolk together with the Nbz linker for Fmoc chemistry solid phase synthesis of the glycopeptide‐^αthioester building block. 3 Chemotaxis assays of these glycoproteins and the corresponding non‐glycosylated proteins were carried out. The results were correlated with the chemical structures of the (glyco)protein molecules. To the best of our knowledge, these are the first investigations of the effect of glycosylation on the chemotactic activity of the chemokine (Ser‐)CCL1 using homogeneous N‐glycosylated protein molecules of defined covalent structure.     

Biphenyl Ether Analogs Containing Pomalidomide as Small-Molecule Inhibitors of the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction

New biphenyl-based chimeric compounds containing pomalidomide were developed and evaluated for their activity to inhibit and degrade the programmed cell death-1/programmed cell death- ligand 1 (PD-1/PD-L1) complex. Most of the compounds displayed excellent inhibitory activity against PD-1/PD-L1, as assessed by the homogenous time-resolved fluorescence (HTRF) binding assay. Among them, compound 3 is one of the best with an IC₅₀ value of 60 nM. Using an ex vivo PD-1/PD-L1 blockade cell line bioassay that expresses human PD-1 and PD-L1, we show that compounds 4 and 5 significantly restore the repressed immunity in this co-culture model. Western blot data, however, demonstrated that these anti-PD-L1/pomalidomide chimeras could not reduce the protein levels of PD-L1.     

Modification of the thermal unfolding pathways of myoglobin upon drug interaction in different aqueous media

In this work, we have analyzed the influence of two structurally related phenothiazine drugs, promazine and triflupromazine hydrochlorides, when bound to myoglobin, a model protein, and how the drug concentration and solution conditions may affect the denaturation process of this protein. In this manner, we derive the thermodynamic quantities of the unfolding process by using a spectroscopic technique such as UV-vis spectroscopy at different drugs concentrations and at pH 2.5, 5.5, and 9.0. To do this, a thermodynamic model was used which included experimental data corresponding to the pre- and post-transition into the observable transition. It has been found that both drugs play a destabilizing role for the protein, at least at low concentrations. In addition, at acidic pH and higher drug concentrations, a stabilizing effect can be observed, which may be related to the formation of some type of protein refolding, subsequent aggregation, or both. The reason for this behavior has been suggested to be the different protein conformations at acidic pH, the increase of solvent-exposed hydrophobic and hydrophilic residues after denaturation and/or binding, and the different strength of drug-protein interactions when changing the solution conditions. For this reason, thermodynamic quantities such as Gibbs energies, DeltaG, and entropies of unfolding, DeltaS(m), increase as the solution pH increases provided that additional solvent-exposed hydrophobic residues are present, which were previously buried at room temperature. Moreover, the larger binding affinity at pH 9.0 due to enhanced electrostatic interactions between protein and drug molecules (drug and protein differ in their net electrical charge) additionally collaborates to this residue exposition to solvent as a consequence of the alteration of protein conformation as due to drug binding. Comparison of thermodynamic data between promazine and triflupromazine hydrochlorides also shows that drug-protein affinity and hydrophobicity also affect the thermodynamic denaturation parameters.     

Context-dependent effects of asparagine glycosylation on Pin WW folding kinetics and thermodynamics

Asparagine glycosylation is one of the most common and important post-translational modifications of proteins in eukaryotic cells. N-glycosylation occurs when a triantennary glycan precursor is transferred en bloc to a nascent polypeptide (harboring the N-X-T/S sequon) as the peptide is cotranslationally translocated into the endoplasmic reticulum (ER). In addition to facilitating binding interactions with components of the ER proteostasis network, N-glycans can also have intrinsic effects on protein folding by directly altering the folding energy landscape. Previous work from our laboratories (Hanson et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 109, 3131-3136; Shental-Bechor, D.; Levy, Y. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 8256-8261) suggested that the three sugar residues closest to the protein are sufficient for accelerating protein folding and stabilizing the resulting structure in vitro; even a monosaccharide can have a dramatic effect. The highly conserved nature of these three proximal sugars in N-glycans led us to speculate that introducing an N-glycosylation site into a protein that is not normally glycosylated would stabilize the protein and increase its folding rate in a manner that does not depend on the presence of specific stabilizing protein-saccharide interactions. Here, we test this hypothesis experimentally and computationally by incorporating an N-linked GlcNAc residue at various positions within the Pin WW domain, a small β-sheet-rich protein. The results show that an increased folding rate and enhanced thermodynamic stability are not general, context-independent consequences of N-glycosylation. Comparison between computational predictions and experimental observations suggests that generic glycan-based excluded volume effects are responsible for the destabilizing effect of glycosylation at highly structured positions. However, this reasoning does not adequately explain the observed destabilizing effect of glycosylation within flexible loops. Our data are consistent with the hypothesis that specific, evolved protein-glycan contacts must also play an important role in mediating the beneficial energetic effects on protein folding that glycosylation can confer.     

Selective in vitro glycosylation of recombinant proteins: semi-synthesis of novel homogeneous glycoforms of human erythropoietin

BACKGROUND: A natural glycoprotein usually exists as a spectrum of glycosylated forms, where each protein molecule may be associated with an array of oligosaccharide structures. The overall range of glycoforms can have a variety of different biophysical and biochemical properties, although details of structure-function relationships are poorly understood, because of the microheterogeneity of biological samples. Hence, there is clearly a need for synthetic methods that give access to natural and unnatural homogeneously glycosylated proteins. The synthesis of novel glycoproteins through the selective reaction of glycosyl iodoacetamides with the thiol groups of cysteine residues, placed by site-directed mutagenesis at desired glycosylation sites has been developed. This provides a general method for the synthesis of homogeneously glycosylated proteins that carry saccharide side chains at natural or unnatural glycosylation sites. Here, we have shown that the approach can be applied to the glycoprotein hormone erythropoietin, an important therapeutic glycoprotein with three sites of N-glycosylation that are essential for in vivo biological activity. RESULTS: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His(10)-WThEPO, His(10)-Asn24Cys, His(10)-Asn38Cys, His(10)-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l(-1) from Escherichia coli. Chemical glycosylation with glycosyl-beta-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. CONCLUSIONS: Erythropoietin expressed in E. coli bearing specific Asn-->Cys mutations at natural glycosylation sites can be glycosylated using beta-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins.     

Homogeneous Human Complex‐Type Oligosaccharides in Correctly Folded Intact Glycoproteins: Evaluation of Oligosaccharide Influence On Protein Folding,Stability, and Conformational Properties

The N‐glycosylation of proteins is generated at the consensus sequence NXS/T (where X is any amino acid except proline) by the biosynthetic process, and occurs in the endoplasmic reticulum and Golgi apparatus. In order to investigate the influence of human complex‐type oligosaccharides on counterpart protein conformation, crambin and ovomucoide, which consist of 46 and 56 amino acid residues, respectively, were selected for synthesis of model glycoproteins. These small glycoproteins were intentionally designed to be glycosylated at the α‐helix (crambin: 8 position), β‐sheet (crambin: 2 position) and loop position between the antiparallel β‐sheets (ovomucoide: 28 position), and were synthesized by using a peptide‐segment coupling strategy. After preparation of these glycosylated polypeptide chains, protein folding experiments were performed under redox conditions by using cysteine–cystine. Although the small glycoproteins bearing intentional glycosylation at the α‐helix and β‐sheet exhibited a suitable folding process, glycosylation at the loop position between the antiparallel β‐strands caused multiple products. The conformational differences in the isolated homogeneous glycoproteins compared with non‐glycosylated counterparts were evaluated by circular dichroism (CD) and NMR spectroscopy. These analyses suggested that this intentional N‐glycosylation did not result in large conformational changes in the purified protein structures, including the case of glycosylation at the loop position between the antiparallel β‐strands. In addition to these experiments, the conformational properties of three glycoproteins were evaluated by CD spectroscopy under different temperatures. The oligosaccharides on the protein surface fluctuated considerably; this was dependent on the increase in the solution temperature and was thought to disrupt the protein tertiary structure. Based on the measurement of the CD spectra, however, the glycoproteins bearing three disulfide bonds did not exhibit any change in their protein tertiary structure. These results suggest that the oligosaccharide conformational fluctuations were not disruptive to protein tertiary structure, and the tertiary structure of glycoproteins might be stabilized by the disulfide bond network.     

Design,Synthesis, Biological Activity and Molecular Docking Study of Coumarin Derivatives Bearing 2-Methylbiphenyl Moiety

A hybrid pharmacophore approach was used to design and synthesize a series of coumarin derivatives bearing 2-methylbiphenyl moiety, which were evaluated for their in vitro anticancer activities against four cancer cell lines(MCF-75 A549, H460 and HT29) and PD-1/PD-L1 inhibitory activities. Moreover, several compounds with excellent anticancer activities were selected to evaluate the cytotoxicities against one normal cell line(HEK-293). The most promising compound llo showed the best anticancer activities against the four tested cancer cell lines with the IC50 values of 6.45, 8.65, 6,57 and 8.13 gmol/L, respectively, and displayed weak cytotoxicity on the normal cell(HEK-293). Furthermore, screening of PD-1 /PD-L1 inhibitory activity revealed that compound llo could effectively inhibit the binding of PD-1/PD-L1, and the binding interactions of compound llo with PD-L1 protein were explored by molecular docking. All above evidences showed that compound llo might be worthy of further study as a valuable leading compound for the treatment of cancer.     

A Simple Strategy to Eliminate Hexosylation Bias in the Relative Quantification of N‐Glycosylation in Biopharmaceuticals

N‐glycosylation may affect the safety and efficacy of biopharmaceuticals and is thus monitored during manufacturing. Mass spectrometry of the intact protein is increasingly used to reveal co‐existing glycosylation variants. However, quantification of N‐glycoforms via this approach may be biased by single hexose residues as introduced by glycation or O‐glycosylation. Herein, we describe a simple strategy to reveal actual N‐glycoform abundances of therapeutic antibodies, involving experimental determination of glycation levels followed by computational elimination of the “hexosylation bias”. We show that actual N‐glycoform abundances may significantly deviate from initially determined values. Indeed, glycation may even obscure considerable differences in N‐glycosylation patterns of drug product batches. Our observations may thus have implications for biopharmaceutical quality control. Moreover, we solve an instance of the problem of isobaricity, which is fundamental to mass spectrometry.     

Zinc(II) complexes of ubiquitin: speciation, affinity and binding features

Intraneuronal inclusions consisting of hypermetallated, (poly-)ubiquitinated proteins are a hallmark of neurodegeneration. To highlight the possible role played by metal ions in the dysfunction of the ubiquitin-proteasome system, here we report on zinc(II)/ubiquitin binding in terms of affinity constants, speciation, preferential binding sites and effects on protein stability and self-assembly. Potentiometric titrations allowed us to establish that at neutral pH only two species, ZnUb and Zn(2)Ub, are present in solution, in line with ESI-MS data. A change in the diffusion coefficient of ubiquitin was observed by NMR DOSY experiments after addition of Zn(II) ions, and thus indicates metal-promoted formation of protein assemblies. Analysis of (1)H, (15)N, (13)Cα and (13)CO chemical-shift perturbation after equimolar addition of Zn(II) ions to ubiquitin outlined two different metal-binding modes. The first involves a dynamic equilibrium in which zinc(II) is shared between a region including Met1, Gln2, Ile3, Phe4, Thr12, Leu15, Glu16, Val17, Glu18, Ile61 and Gln62 residues, which represent a site already described for copper binding, and a domain comprising Ile23, Glu24, Lys27, Ala28, Gln49, Glu51, Asp52, Arg54 and Thr55 residues. A second looser binding mode is centred on His68. Differential scanning calorimetry evidenced that addition of increasing amounts of Zn(II) ions does not affect protein thermal stability; rather it influences the shape of thermograms because of the increased propensity of ubiquitin to self-associate. The results presented here indicate that Zn(II) ions may interact with specific regions of ubiquitin and promote protein-protein contacts.     

Identification of N-linked glycosylation sites in human nephrin using mass spectrometry

Nephrin is a type-1 transmembrane glycoprotein and the first identified principal component of the glomerular filtration barrier. Ten potential asparagine (N)-linked glycosylation sites have been predicted within the ectodomain of nephrin. However, it is not known which of these potential sites are indeed glycosylated and what type of glycans are involved. In this work, we have identified the terminal sugar residues on the ectodomain of human nephrin and utilized a straightforward and reliable mass spectrometry-based approach to selectively identify which of the ten predicted sites are glycosylated. Purified recombinant nephrin was subjected to peptide-N-glycosidase F (PNGase F) to enzymatically remove all the N-linked glycans. Since PNGase F is an amidase, the asparagine residues from which the glycans have been removed are deaminated to aspartic acid residues, resulting in an increase in the peptide mass with 1 mass unit. Following trypsin digestion, deglycosylated tryptic peptides were selectively identified by MALDI-TOF MS and their sequence was confirmed by tandem TOF/TOF. The 1 Da increase in peptide mass for each asparagine-to-aspartic acid conversion, along with preferential cleavage of the amide bond carboxyl-terminal to aspartic acid residues in peptides where the charge is immobilized by an arginine residue, was used as a diagnostic signature to identify the glycosylated peptides. Thus, nine of ten potential glycosylation sites in nephrin were experimentally proven to be modified by N-linked glycosylation.     

Antioxidant Properties and Aldehyde Reactivity of PD-L1 Targeted Aryl-Pyrazolone Anticancer Agents

Small molecules targeting the PD-1/PD-L1 checkpoint are actively searched to complement the anticancer arsenal. Different molecular scaffolds have been reported, including phenyl-pyrazolone derivatives which potently inhibit binding of PD-L1 to PD-1. These molecules are structurally close to antioxidant drug edaravone (EDA) used to treat amyotrophic lateral sclerosis. For this reason, we investigated the capacity of five PD-L1-binding phenyl-pyrazolone compounds (1–5) to scavenge the formation of oxygen free radicals using electron spin resonance spectroscopy with DPPH/DMPO probes. In addition, the reactivity of the compounds toward the oxidized base 5-formyluracil (5fU) was assessed using chromatography coupled to mass spectrometry and photodiode array detectors. The data revealed that the phenyl-pyrazolone derivatives display antioxidant properties and exhibit a variable reactivity toward 5fU. Compound 2 with a N-dichlorophenyl-pyrazolone moiety cumulates the three properties, being a potent PD-L1 binder, a robust antioxidant and an aldehyde-reactive compound. On the opposite, the adamantane derivative 5 is a potent PD-L1 binding with a reduced antioxidant potential and no aldehyde reactivity. The nature of the substituent on the phenyl-pyrazolone core modulates the antioxidant capacity and reactivity toward aromatic aldehydes. The molecular signature of the compound can be adapted at will, to confer additional properties to these PD-L1 binders.     

(Quasi‐)Racemic X‐ray Structures of Glycosylated and Non‐Glycosylated Forms of the Chemokine Ser‐CCL1 Prepared by Total Chemical Synthesis

Our goal was to obtain the X‐ray crystal structure of the glycosylated chemokine Ser‐CCL1. Glycoproteins can be hard to crystallize because of the heterogeneity of the oligosaccharide (glycan) moiety. We used glycosylated Ser‐CCL1 that had been prepared by total chemical synthesis as a homogeneous compound containing an N‐linked asialo biantennary nonasaccharide glycan moiety of defined covalent structure. Facile crystal formation occurred from a quasi‐racemic mixture consisting of glycosylated L ‐protein and non‐glycosylated‐D ‐protein, while no crystals were obtained from the glycosylated L ‐protein alone. The structure was solved at a resolution of 2.6–2.1 Å. However, the glycan moiety was disordered: only the N‐linked GlcNAc sugar was well‐defined in the electron density map. A racemic mixture of the protein enantiomers L ‐Ser‐CCL1 and D ‐Ser‐CCL1 was also crystallized, and the structure of the true racemate was solved at a resolution of 2.7–2.15 Å. Superimposition of the structures of the protein moieties of L ‐Ser‐CCL1 and glycosylated‐L ‐Ser‐CCL1 revealed there was no significant alteration of the protein structure by N‐glycosylation.     

Ezrin Modulates the Cell Surface Expression of Programmed Cell Death Ligand-1 in Human Cervical Adenocarcinoma Cells

Cancer cells employ programmed cell death ligand-1 (PD-L1), an immune checkpoint protein that binds to programmed cell death-1 (PD-1) and is highly expressed in various cancers, including cervical carcinoma, to abolish T-cell-mediated immunosurveillance. Despite a key role of PD-L1 in various cancer cell types, the regulatory mechanism for PD-L1 expression is largely unknown. Understanding this mechanism could provide a novel strategy for cervical cancer therapy. Here, we investigated the influence of ezrin/radixin/moesin (ERM) family scaffold proteins, crosslinking the actin cytoskeleton and certain plasma membrane proteins, on the expression of PD-L1 in HeLa cells. Our results showed that all proteins were expressed at mRNA and protein levels and that all ERM proteins were highly colocalized with PD-L1 in the plasma membrane. Interestingly, immunoprecipitation assay results demonstrated that PD-L1 interacted with ERM as well as actin cytoskeleton proteins. Furthermore, gene silencing of ezrin, but not radixin and moesin, remarkably decreased the protein expression of PD-L1 without affecting its mRNA expression. In conclusion, ezrin may function as a scaffold protein for PD-L1; regulate PD-L1 protein expression, possibly via post-translational modification in HeLa cells; and serve as a potential therapeutic target for cervical cancer, improving the current immune checkpoint blockade therapy.     

Membrane assembly: synthesis and intracellular processing of the vesicular stomatitis viral glycoprotein.

The glycoprotein (G) of vesicular stomatitis virus (VSV) is synthesized on membrane-bound polyribosomes. Approximately 30 min after its synthesis, it reaches the surface plasma membrane where it is incorporated into budding virus. The first part of this paper focuses on the 2 intracellular, membrane-bound, glycosylated forms of the glycoprotein which are intermediates in its biogenesis. All glycosylation and processing is completed in the smooth microsome fraction before the protein reaches the surface. Next, we turn to the mechanism by which G is synthesized on membrane-bound polyribosomes. All of the G mRNA is bound to membranes, and studies with puromycin suggest that this attachment of G mRNA is mediated by the nascent glycoprotein chain. After its synthesis G is a transmembrane protein with about 30 amino acids at the carboxyl terminus remaining on the cytoplasmic side of the endoplasmic reticulum. Since 95% of the glycoprotein, containing the carbohydrate residues, is resistant to attack by external proteases, it appears to be within the lumen of the endoplasmic reticulum or embedded within the lipid bilayer. Finally, we show that synthesis, glycosylation, and proper asymmetric insertion of G into the ER can be achieved in cell-free extracts. Both glycosylation of G and proper insertion into the ER membrane in this cell-free system require concomitant protein synthesis.     

N-糖基化蛋白质组样品富集策略的研究进展

蛋白质的糖基化是生物体内重要的蛋白质翻译后修饰之一,但其丰度通常较低,糖基化蛋白质酶解肽段中仅有2%~5%为糖基化肽段,因此,为实现糖基化蛋白质组的深度覆盖分析,对糖基化蛋白质/肽段进行富集是非常必要的。该文对糖基化蛋白质组样品不同富集方法的原理、特点以及最新研究进展进行了综述,同时也对N-糖基化蛋白质组学富集策略的发展前景进行了展望。     

Studies on the interaction of total saponins of panax notoginseng and human serum albumin by Fourier transform infrared spectroscopy

Total saponins of panax notoginseng (TPNS), isolated from the roots of panax notoginseng (Burk) F.H. Chen, have been considered as the main active components of San-Chi and have various therapeutical actions. Their interactions with human serum albumin have been investigated by Fourier transformed infrared spectrometry and fluorescence methods. The results showed that TPNS combined with HSA through C=O and C-N groups of polypeptide chain. The drug-protein combination caused the significant loss of alpha-helix structure and the microenvironment changes of the tyrosine residues in protein at higher drug concentration. Combining the curve-fitting results of amide I and amide III bands, the alterations of protein secondary structure after drug complexation were quantitatively determined. The alpha-helix structure has a decrease of approximately 6%, from 55 to 49% and the beta-sheet increased approximately 3%, from 23 to 26% at high drug concentration. However, no major alterations were observed for the beta-turn and random coil structures up on drug-protein binding.