The chemical versatility of natural-product assembly lines

Microbial natural products of both polyketide and nonribosomal peptide origin have been and continue to be important therapeutic agents as antibiotics, immunosupressants, and antitumor drugs. Because the biosynthetic genes for these metabolites are clustered for coordinate regulation, the sequencing of bacterial genomes continues to reveal unanticipated biosynthetic capacity for novel natural products. The re-engineering of pathways for such secondary metabolites to make novel molecular variants will be enabled by understanding of the chemical logic and protein machinery in the producer microbes. This Account analyzes the chemical principles and molecular logic that allows simple primary metabolite building blocks to be converted to complex architectural scaffolds of polyketides (PK), nonribosomal peptides (NRP), and NRP-PK hybrids. The first guiding principle is that PK and NRP chains are assembled as thioseters tethered to phosphopantetheinyl arms of carrier proteins that serve as thiotemplates for chain elongation. The second principle is that gate keeper protein domains select distinct monomers to be activated and incorporated with positional specificity into the growing natural product chains. Chain growth is via thioclaisen condensations for PK and via amide bond formation for elongating NRP chains. Release of the full length acyl/peptidyl chains is mediated by thioesterases, some of which catalyze hydrolysis while others catalyze regiospecific macrocyclization to build in conformational constraints. Tailoring of PK and NRP chains, by acylation, alkylation, glycosylation, and oxidoreduction, occurs both during tethered chain growth and after thioesterase-mediated release. Analysis of the types of protein domains that carry out chain initiation, elongation, tailoring, and termination steps gives insight into how NRP and PK biosynthetic assembly lines can be redirected to make novel molecules.     

单克隆抗体糖基化修饰的研究现状及进展

糖基化是经酶定向控制下,在蛋白质或脂质特定位点附加寡糖链的共翻译和翻译后重要的修饰过程,发生于高尔基体和内质网。糖基化修饰是单克隆抗体药物结构形成、功能发挥及药代动力学等生物学特性的重要影响因素。糖型通过影响单克隆抗体的结构而对药物的半衰期、效应功能等产生影响。在特定的细胞表达系统中,不同的细胞培养工艺参数对糖基化异质性产生影响。本文就糖型结构异质性对抗体效应功能的影响、细胞培养条件对抗体糖基化的作用以及糖基化研究技术的现状作一综述。     

Engineering disulfide-linked single-chain Fv dimers [(sFv')2] with improved solution and targeting properties: anti-digoxin 26-10 (sFv')2 and anti-c-erbB-2 741F8 (sFv')2 made by protein folding and bonded through C-terminal cysteinyl peptides

Single-chain Fv fusions with C-terminal cysteinyl peptides (sFv')have been engineered using model sFv proteins based upon the26-10 anti-digoxin IgG and 741F8 anti-c-erbB-2 IgG monoclonalantibodies. As part of the 741F8 sFv construction process, thePCR-amplified 741F8 V_H gene was modified in an effort to correctpossible primer-induced errors. Genetic replacement of the N-terminalp-strandsequence of 741F8 VH with that from the FR1 of anti-c-erbB-2520C9 VH resulted in a dramatic improvement of sFv folding yields.Folding in urea-glutathione redox buffers produced active sFv'with a protected C-terminal sulfhydryl, presumably as the mixeddisulfide with glutathione. Disulfide-bonded (sFv')₂ homodimerswere made by disulfide interchange or oxidation after reductiveelimination of the blocking group. Both 26–10 (sFv')₂and 741F8 (sFv')₂ existed as stable dimers that were well behavedin solution, whereas 741F8 sFv and sFv' exhibited considerableself-association. The 741F8 sFv binds to the extracellular domain(ECD) of the c-erbB-2 oncogene protein, which is often overexpressedin breast cancer and other adenocarcinomas. The recombinantECD was prepared to facilitate the analysis of 741F8 bindingsite properties; the cloned ECD gene, modified to encode a C-terminalSer-Gly-His₆ peptide, was transfected into Chinese hamster ovarycells using a vector that also expressed dihydrofolate reductaseto facilitate methotrexate amplification. Optimized cell linesexpressed ECD-His₆ at high levels in a cell bioreactor; afterisolation by immobilized metal affinity chromatography, finalECD yields were as high as 47 mg/l. An animal tumor model complementedphysicochemical studies of 741F8 species and indicated increasedtumor localization of the targeted 741F8 (sFv')₂ over othermonovalent 741F8 species.     

A FRET Sensor to Monitor Bivalent SUMO–SIM Interactions in SUMO Chain Binding

The small ubiquitin‐like modifier (SUMO) can be assembled into polymeric chains as part of its diverse biochemical signal pattern upon conjugation to substrate proteins. SUMO chain recognition is facilitated by receptor proteins that contain at least two SUMO‐interacting motifs (SIMs). Little is known about the structure of SUMO chains, both in an unliganded form and upon complexation with multi‐SIM protein partners. A FRET sensor has been developed based on a linear dimer of human SUMO‐2 as a minimal SUMO chain analogue. The synthetic acceptor and donor dyes were conjugated by maleimide and copper‐catalyzed click chemistry to each of the two SUMO subunits. FRET changes were only observed in the presence of di‐ or multi‐SIM ligands. Alteration of the short linker sequence between SIMs 2 and 3 of RNF4 showed a great tolerance, and hence, structural flexibility, of the SUMO dimer for bivalent binding of adjacent SIMs. The di‐SUMO FRET sensor reports on the binding of SIM clusters of the proteins C5orf25 and SOBP; this suggest that these can bind to adjacent subunits of a SUMO chain. The developed FRET sensor will be a useful tool to study the importance of SIM and linker sequences, as well as biochemical and structural properties of SUMO chains and multi‐SIM proteins.     

Preparation and controlled release of degradable polymeric ketoprofen–saccharide conjugates

A facile and efficient enzymatic and polymerization process was used to prepare polymeric prodrugs of ketoprofen with saccharide side chains. The chains included branches that included glucose, mannose, galactose, and lactose, and these were synthesized through free radical reaction. The prodrugs were characterized by FT-IR, NMR, and GPC and drug-loading capacity was influenced by varying the ratios of initiator and monomers (range 32.13 and 68.56% w/w). In vitro release characteristics of the polymeric drugs were systematically evaluated over the pH range 1.2–8.0 and the release profiles indicated that the hydrolytic nature of polymers were strongly depended on the variation in saccharide content, carbon chain length, and pH. The outcomes from this study demonstrate the importance of carbohydrate structures and how these are linked to drug release.     

Thickness threshold of structural ordering in thin MEH-PPV films

The microstructure of thin poly-[2-methoxy-5-(2′-ethyl-hexyloxy)-p-phenylene vinylene] (MEH-PPV) films was investigated by photoluminescence measurements and X-Ray diffraction analysis over thickness from several tens up to several hundreds of nanometer. Obtained results were correlated with exciton diffusion length obtained by the photovoltage method. All measurements revealed that there is a threshold thickness for the microstructural order as well as for exciton diffusion length which are primary physical properties for function of any device made up from such films. Development of more ordered phase can be observed above the critical thickness of about 150 nm. Below the threshold, the polymer chains are preferentially in conformations promoting localized intrachain interactions. Above the threshold, two main features can be observed: (i) the conjugation length of polymer chain segments reaches its maximum and (ii) polymer stacking in more ordered and larger crystalline domains influences strongly the material properties, namely enhances the exciton diffusion length.     

Novel Insight in Idiopathic Normal Pressure Hydrocephalus (iNPH) Biomarker Discovery in CSF

Idiopathic normal pressure hydrocephalus (iNPH) is a potentially reversible neurological disease, causing motor and cognitive dysfunction and dementia. iNPH and Alzheimer’s disease (AD) share similar molecular characteristics, including amyloid deposition, t-tau and p-tau dysregulation; however, the disease is under-diagnosed and under-treated. The aim was to identify a panel of sphingolipids and proteins in CSF to diagnose iNPH at onset compared to aged subjects with cognitive integrity (C) and AD patients by adopting multiple reaction monitoring mass spectrometry (MRM-MS) for sphingolipid quantitative assessment and advanced high-resolution liquid chromatography–tandem mass spectrometry (LC–MS/MS) for proteomic analysis. The results indicated that iNPH are characterized by an increase in very long chains Cer C22:0, Cer C24:0 and Cer C24:1 and of acute-phase proteins, immunoglobulins and complement component fragments. Proteins involved in synaptic signaling, axogenesis, including BACE1, APP, SEZ6L and SEZ6L2; secretory proteins (CHGA, SCG3 and VGF); glycosylation proteins (POMGNT1 and DAG1); and proteins involved in lipid metabolism (APOH and LCAT) were statistically lower in iNPH. In conclusion, at the disease onset, several factors contribute to maintaining cell homeostasis, and the protective role of very long chains sphingolipids counteract overexpression of amyloidogenic and neurotoxic proteins. Monitoring specific very long chain Cers will improve the early diagnosis and can promote patient follow-up.     

Loops, linkages, rings, catenanes, cages, and crowders: entropy-based strategies for stabilizing proteins

A protein molecule exists in either a compact folded state or a variable and open unfolded state. Since the unfolded state is favored by chain entropy, restricting its entropy is an attractive mechanism for shifting the equilibrium toward the folded state. A number of entropy-based strategies have been engineered or used by natural proteins to increase the folding stability: (a) shortening of loop lengths, (b) covalent linkage of dimeric proteins, (c) backbone cyclization, (d) catenation, (e) spatial confinement, and (f) macromolecular crowding. Theoretical analyses demonstrate the importance of accounting for consequences on the folded as well as the unfolded state and provide guidance for further exploitation of these stabilization strategies.     

An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability

The effects of linker length on binding affinity and degreeof aggregation have been examined in the antifluorescein 4-4-20and anticarcinoma CC49 single-chain Fvs. Longer linkers in theantifluorescein sFvs have higher affinities for fluoresceinand aggregate less. A proteolytically susceptible site betweenLys8 and Ser9, in the previously reported 212 linker has beenidentified. A new linker sequence, 218 (GSTSGSGKPGSGEGSTKG)was designed in which a praline was placed at the C-terminalside of the proteolytic clip site in the 212 linker. The CC49sFv containing the 218 linker showed reduced aggregation andwas found to be more stable to proteolysis in vitro, when comparedto the CC49/212 sFv. The CC49 sFv with the longer 218 linkerhad higher affinity than CC49/212 sFv. An aggregated CC49/212sFv sample had higher affinity than CC49/218 sFv. The CC49/218and CC49/212 sFvs had similar blood clearances in mice, whilethe aggregated CC49/212 sFv remained in circulation significantlylonger. In mice bearing LS-174T human colon carcinoma xenografts,the CC49/218 sFv showed higher tumor uptake than the CC49/212sFv and lower tumor uptake than the aggregated CC49/212 sFv.The higher tumor uptake of the CC49/218 is most likely a resultof its higher resistance to proteolysis. The higher affinityand higher tumor uptake of the aggregated CC49/212 sFv are mostlikely due to the repetitive nature of the TAG-72 antigen andthe higher avidity of multivalent aggregates. When the sFvswere radiolabeled with a lutetium-chelate the CC49/218 sFv showeda lower accumulation in the liver and spleen compared to theaggregated CC49/212 sFv.     

3-氨基喹啉与常用基体在MALDI-TOFMS测定天然提取植物多糖的比较

采用3-氨基喹啉(3-AQ)、二元基体3-AQ/2,5-二羟基苯甲酸(DHB)及常用于糖类物质测定的DHB和二元基体DHB/1-羟基异喹啉(1-HIQ)对一天然提取植物糖样进行了基体辅助激光解吸/电离飞行时间质谱(MALDI-TOF MS)分析。实验结果表明:此植物糖样由线形和环形聚己糖组成,其最大分子量至少达3000 Da;3-AQ与常用基体DHB及DHB/HIQ相比较,能提供更准确、更全面的样品组成及分子结构信息,且具有优越于常用基体解吸电离环状糖分子的能力。     

Stability constraints and protein evolution: the role of chain length, composition and disulfide bonds

Stability of the native state is an essential requirement in protein evolution and design. Here we investigated the interplay between chain length and stability constraints using a simple model of protein folding and a statistical study of the Protein Data Bank. We distinguish two types of stability of the native state: with respect to the unfolded state (unfolding stability) and with respect to misfolded configurations (misfolding stability). Several contributions to stability are evaluated and their correlations are disentangled through principal components analysis, with the following main results. (1) We show that longer proteins can fulfil more easily the requirements of unfolding and misfolding stability, because they have a higher number of native interactions per residue. Consistently, in longer proteins native interactions are weaker and they are less optimized with respect to non-native interactions. (2) Stability against misfolding is negatively correlated with the strength of native interactions, which is related to hydrophobicity. Hence there is a trade-off between unfolding and misfolding stability. This trade-off is influenced by protein length: less hydrophobic sequences are observed in very long proteins. (3) The number of disulfide bonds is positively correlated with the deficit of free energy stabilizing the native state. Chain length and the number of disulfide bonds per residue are negatively correlated in proteins with short chains and uncorrelated in proteins with long chains. (4) The number of salt bridges per residue and per native contact increases with chain length. We interpret these observations as an indication that the constraints imposed by unfolding stability are less demanding in long proteins and they are further reduced by the competing requirement for stability against misfolding. In particular, disulfide bonds appear to be positively selected in short proteins, whereas they evolve in an effectively neutral way in long proteins.     

Design and construction of a hybrid immunoglobulin domain with properties of both heavy and light chain variable regions

The complementarity-determining regions (CDRs) of a human kappa light chain were replaced with CDRs from a murine gamma-1 heavy chain and, by use of molecular modeling, key heavy chain framework residues were identified and thus included to preserve the native conformation of the heavy chain CDRs. Co-expression of this hybrid human kappa chain (V[HB]C[L]) with a human kappa chain counterpart (V[L]C[L], engineered to contain murine light chain CDRs) resulted in the secretion of high levels of a heterodimeric protein (V[HB]C[L]::V[L]C[L]) termed 'kappabody'. This protein also had equivalent affinity for antigen as the Fab' of the parent murine IgG1. High-level secretion was also observed for the hybrid chain as homodimers (V[HB]C[L]::V[HB]C[L]), which is not observed for chimeric chains consisting of a heavy chain variable region and light chain constant region, i.e. V[H]C[L] homodimers or single chains are not secreted. This indicates that regions within the variable domain, required for secretion of light chains, reside outside of the hypervariable regions (CDRs) and that the heavy chain CDRs and supporting residues do not prevent secretion. These results demonstrate the possibility of designing small, single- domain molecules possessing a given binding activity which may be secreted at high levels from mammalian cells.      

Renal dysfunction accounts for the dose limiting toxicity of DT390anti- CD3sFv, a potential new recombinant anti-GVHD immunotoxin

The toxicity of a highly selective, recombinant fusion immunotoxin, DT390anti-CD3sFv, was examined in mice. The protein was expressed from a hybrid gene in which the single chain Fv of the anti-murine CD3 epsilon antibody cDNA was spliced to truncated diphtheria toxin cDNA. DT390anti-CD3sFv, previously shown to have significant anti-GVHD effects when administered to mice given transplants of allogeneic MHC- disparate donor T cells (Vallera et al., Blood 88, 2342-2353, 1996), preferentially localized to kidney and had profound renal toxicity as assessed by histology and serum levels of blood urea nitrogen (BUN), and creatinine. Kidney effects were more severe than liver effects at the maximum tolerated dose (MTD) of 10 microg/day BID given over a six day interval. Toxic injury was attributed in part to the toxin moiety since DT390 administered alone was more toxic than equivalent doses of DT390 given in the context of DT390anti-CD3sFv fusion protein. The presence of anti-CD3sFv ligand reduced toxicity since DT390anti-CD3sFv was twice as toxic to severe combined immunodeficiency disease (scid) mice which do not have CD3epsilon expressing T cells as compared to their normal counterparts. Together, these findings further our understanding of the toxicities limiting the in vivo administration of DT fusion immunotoxins in mice and provide a foundation for future genetic modifications which should be directed at reducing these effects.      

On Surface Modification of Polymeric Biomaterials

Surface modification with hydrophilic polymers has been beneficial in improving blood compatibility of biomaterials. Formation of dense and tightly-bonded surface layers may prevent plasma protein adsorption owing to steric repulsion. General conditions for formation of layers, protecting blood components from direct contacts with the surface, are discussed. It seems to be necessary to ensure a delicate balance between adsorption energy of the attached chains and their length. The crucial point is to get a high grafting density which is more influential than high chain length. Length should be calibrated to the size of protein molecules to meet both effective repulsion and high density of the protecting chains and to avoid chain displacement by plasma proteins.     

All solid-state polymer electrolytes prepared from a hyper-branched graft polymer using atom transfer radical polymerization

We propose an all solid-state (liquid free) polymer electrolyte (SPE) prepared from a hyper-branched graft copolymer. The graft copolymer consisting of a poly(methyl methacrylate) main chain and poly(ethylene glycol) methyl ether methacrylate side chains was synthesized by atom transfer radical polymerization changing the average chain distance between side chains, side chain length and branched chain length of the proposed structure of the graft copolymer. The ionic conductivity of the SPEs increases with increasing the side chain length, branched chain length and/or average distance between the side chains. The ionic conductivity of the SPE prepared from POEM₉ whose POEM content = 51 wt% shows 2 × 10⁻⁵ S/cm at 30 °C. The tensile strength of the SPEs decreases with increases the side chain length, branched chain length and/or average distance between the side chains. These results indicate that a SPE prepared from the hyper-branched graft copolymer has potential to be applied to an all-solid polymer electrolyte.     

Charge transfer of 1,3,4-oxadiazole derivative,its functional polymers and study of their different aggregation-induced emission enhancement behaviors

In this study, a symmetrical D-π-A-π-D initiator ViOXD, based on substituted amino donor (D) and 1,3,4-oxadiazole acceptor (A), with rigid stilbene π-bridge was synthesized. The photophysical investigation of ViOXD in solution and aggregate state showed its solvatochromic and aggregation-caused quenching characteristics, because of its strong charge transfer and the formation of H-aggregates, respectively. The introduction of peripheral polystyrene or carbazole-containing PVPCz chains via atom transfer radical polymerization not only recovered the fluorescence of the ViOXD core, but also tuned its maximum emission wavelength in aggregate state, due to the stronger electron-donating ability and better conjugation of PVPCz chains than polystyrene chains. On the other hand, the large conjugation length and symmetrical charge transfer from the ends to the cores of ViOXD facilitated the electronic delocalization and bestowed the end-functionalized polymers with good fluorescence in aggregate state, which we have applied in cellular imaging by preparation of their fluorescent nanoparticles.     

Study of tack properties of uncrosslinked natural rubber

The purpose of this study was to better understand the effect of non-rubber substances (mainly proteins and lipids) on adhesion (against glasses) and self-adhesion tack properties of uncrosslinked natural rubber (NR). Various types of NR, with or without non-rubber constituents, were prepared. Synthetic polyisoprene rubber was also used as a reference material. All the rubbers were first characterized by many techniques (FT-IR, DSC, GPC, etc.). Two experimental tests were specially utilized to measure the level of adhesion and self-adhesion: (i) at very short contact times (from a few milliseconds up to 0.1 s) the impact of a pendulum and (ii) for longer contact times (from 0.1 s to a few h) the contact of a probe using a tensile testing machine. The tack energy increased with contact time for all the rubbers studied. Natural rubber which did not contain proteins and lipids exhibited the highest adhesion and self-adhesion tack abilities. In contrast, whole natural rubber, containing both proteins and lipids, showed the lowest tack property. In each case, self-adhesion levels are higher than those of adhesion, presumably due to interdiffusion of macromolecular chains or chain segments at the interface.     

Protein Lipidation Types: Current Strategies for Enrichment and Characterization

Post-translational modifications regulate diverse activities of a colossal number of proteins. For example, various types of lipids can be covalently linked to proteins enzymatically or non-enzymatically. Protein lipidation is perhaps not as extensively studied as protein phosphorylation, ubiquitination, or glycosylation although it is no less significant than these modifications. Evidence suggests that proteins can be attached by at least seven types of lipids, including fatty acids, lipoic acids, isoprenoids, sterols, phospholipids, glycosylphosphatidylinositol anchors, and lipid-derived electrophiles. In this review, we summarize types of protein lipidation and methods used for their detection, with an emphasis on the conjugation of proteins with polyunsaturated fatty acids (PUFAs). We discuss possible reasons for the scarcity of reports on PUFA-modified proteins, limitations in current methodology, and potential approaches in detecting PUFA modifications.     

一种有效的甘露糖3,6位选择性保护和脱保护的新方法

文章报道了一种有效的甘露糖3,6位选择性保护和脱保护的新方法,该方法具有条件温和,选择性好,收率高等优点。这对于用甘露糖作为糖基给体或是糖基受体,以其合成在3,6位用不同糖基修饰支化的寡糖有着非常重要的意义。由于3,6位支化的甘露糖在生物糖蛋白中有着很高的表达。合成这类寡糖对于理解它们在生物体中的作用提供了先决条件。