Affinity Chromatography Research Articles

Electrolyte Reconfiguration by Cation/Anion Cross‐coordination for Highly Reversible and Facile Sodium Storage

Hard carbon (HC) materials are promising anodes for sodium‐ion batteries (SIBs) owing to low cost, high specific capacity and low working potential. However, the poor compatibility of the electrolyte with HC leads to low initial coulombic efficiency (ICE) and sluggish Na+ transport kinetics. Here, we propose an electrolyte reconfiguration strategy based on the hard and soft acid and base (HSAB) theory by introducing methyltriphenylphosphonium bromide (MTPPB). MTPPB can realize a spontaneous cross‐coordination solvation structure with NaPF6 by selective affinity, synchronously optimizing the interfacial chemistry and sodium storage process. The advantages of the chemical π‐π bridging of MTPP+‐HC and interaction of MTPP+‐PF6− contribute to preferential and oriented reduction of PF6−, forming a low‐resistance supramolecular SEI. Additionally, Na+‐Br− coordination weakens the Na+‐solvent interactions, facilitating Na+ de‐solvation kinetics. Consequently, the HC||Na cell achieves a superior ICE of 96.6%, desirable rate capability under 25 °C and invisible capacity decay after 500 cycles at 1 C under −20 °C. The Na4Fe3(PO4)2P2O7||HC pouch battery displays a high ICE of 90.3% and a 15% increment of energy density under 25 °C. This work provides a guidance through electrolyte reconfiguration engineering for designing practical HC‐based SIBs with high energy/power density and long‐life span in the extended operating‐temperature range.

Electrolyte Reconfiguration by Cation/Anion Cross-coordination for Highly Reversible and Facile Sodium Storage.

Hard carbon (HC) materials are promising anodes for sodium-ion batteries (SIBs) owing to low cost, high specific capacity and low working potential. However, the poor compatibility of the electrolyte with HC leads to low initial coulombic efficiency (ICE) and sluggish Na+ transport kinetics. Here, we propose an electrolyte reconfiguration strategy based on the hard and soft acid and base (HSAB) theory by introducing methyltriphenylphosphonium bromide (MTPPB). MTPPB can realize a spontaneous cross-coordination solvation structure with NaPF6 by selective affinity, synchronously optimizing the interfacial chemistry and sodium storage process. The advantages of the chemical π-π bridging of MTPP+-HC and interaction of MTPP+-PF6- contribute to preferential and oriented reduction of PF6-, forming a low-resistance supramolecular SEI. Additionally, Na+-Br- coordination weakens the Na+-solvent interactions, facilitating Na+ de-solvation kinetics. Consequently, the HC||Na cell achieves a superior ICE of 96.6%, desirable rate capability under 25 °C and invisible capacity decay after 500 cycles at 1 C under -20 °C. The Na4Fe3(PO4)2P2O7||HC pouch battery displays a high ICE of 90.3% and a 15% increment of energy density under 25 °C. This work provides a guidance through electrolyte reconfiguration engineering for designing practical HC-based SIBs with high energy/power density and long-life span in the extended operating-temperature range.

Mechanistic Studies of Small Molecule Ligands Selective to RNA Single G Bulges.

Small-molecule RNA binders have emerged as an important pharmacological modality. A profound understanding of the ligand selectivity, binding mode, and influential factors governing ligand engagement with RNA targets is the foundation for rational ligand design. Here, we report a novel class of coumarin derivatives exhibiting selective binding affinity towards single G RNA bulges. Harnessing the computational power of all-atom Gaussian accelerated Molecular Dynamics (GaMD) simulations, we unveiled a rare minor groove binding mode of the ligand with a key interaction between the coumarin moiety and the G bulge. This predicted binding mode is consistent with results obtained from structure-activity-relationship (SAR) studies and transverse relaxation measurements by NMR spectroscopy. We further generated 444 molecular descriptors from 69 coumarin derivatives and identified key contributors to the binding events, such as charge state and planarity, by lasso (least absolute shrinkage and selection operator) regression. Strikingly, small structure perturbations on these key contributors, such as the addition of a methyl group that disrupts the planarity of the ligand resulted in > 100-fold reduction in the binding affinity. Our work deepened the understanding of RNA-small molecule interactions and integrated a new generalizable platform for the rational design of selective small-molecule RNA binders.

Naked eye detection of anions: Dihydrazone receptor-based visual sensing

In this study, we designed and synthesized a novel colorimetric sensor for fluoride ions by integrating a thienodicarbohydrazide framework with salicylaldehyde, resulting in a unique bis-hydrazone-phenol receptor. This sensor exhibited exceptional selectivity for fluoride and acetate ions in nonaqueous environments, with detection visible to the naked eye. Upon interaction with fluoride ions, the receptor underwent a significant red shift in its UV–Visible absorption spectrum, moving from an absorption maximum at 332 nm–380 nm, signalling the recognition event. Acetate ions induced a similar shift. The binding interactions were thoroughly investigated, with a Jobs plot revealing a 1:1 receptor-anion complex formation with fluoride ions and a 1:2 complex with acetate ions. Further validation came from 1H NMR titration, which confirmed the presence of hydrogen bonding interactions between the receptor and the ions. The receptor demonstrated remarkable binding constants of 5.2 × 10⁴ M−2 for acetate and 1.6 × 10⁴ M−1 for fluoride ions, highlighting its strong and selective affinity. Fluorescence titrations further confirmed the receptor's potential as a chemo sensor, showing distinct emission changes in the presence of fluoride and acetate ions. This study not only underscores the receptor's capability for visual detection of fluoride ions in non-aqueous systems but also paves the way for its application in diverse analytical and environmental settings.

Optimisation of induction and purification protocols of recombinant 22.6kDa tegumental protein of Schistosoma spindale inprokaryotic vector

Schistosomosis is a prevalent zoonotic parasitic disease affecting both humans and animals on a global scale, with an estimated 165 million cattle and 200 million people impacted worldwide. Eventhough, serological methodologies designed for the identification of specific antibodies targeting parasitic antigens are esteemed for their high sensitivity, there are criticisms due to their incapacity to reliably indicate active infection, inability to correlate with the intensity of infection and lack of specificity. Enhancing the specificity of serological assays presents a significant challenge, primarily attributable to the identification and synthesis of specific antigens. In addressing these limitations, recombinant technology with specific immunogenic proteins as candidate antigens emerges as a viable alternative. In this study, induction of 22.6 kDa recombinant tegument protein of Schistosoma spindale was achieved using 0.6 mM concentration of IPTG at 37°C for four hours. Nickel chelating affinity chromatography was employed for protein purification, yielding maximum protein concentration at 75mM elution. Subsequently, the dialysis technique was employed to remove contaminants, while lyophilisation method was employed for protein concentration. The protein concentration post dialysis was measured at 0.220 mg/mL, while lyophilisation resulted in a concentration of 2mg/m Keywords: 22.6 kDa tegumental protein, Schistosoma spindale, dialysis, lyophilisation

Production of anti-rM14 and anti-rM42 antibodies and their application in the stidy of expression of metalloproteases M14 and M42 of keratinolytic strain of Bacillus paralicheniformis

In the present study, two metalloproteases M14 and M42 from a Kazakh strain of Bacillus paralicheniformis T7 with high proteolytic activity were successfully cloned, expressed and purified. Protease genes were amplified by polymerase chain reaction (PCR) using specific primers selected on the basis of full genome sequencing data of B. paralicheniformis strain N7 (GenBank accession #CP124861). The amplified fragments were cloned in the expression vector pET-28c(+). Recombinant analogs of the metalloproteases M14 and M42 were obtained by plasmid expression in Escherichia coli cells of the strain ArcticExpressRP(DE3). Using metal affinity chromatography, rM14 and rM42 proteins were purified and polyclonal antibodies were produced by immunization of rabbits. Using anti-rM14 and anti-rM42 antibodies, the expression level of native metalloproteases M14 and M42 in cell lysate and culture fluid of B. paralicheniformis T7 strain was examined after culturing the strain on keratin-containing media: chicken feather, horn, hoof, hide and wool of cattle. Substrate-dependent expression of metalloprotease genes of B. paralicheniformis strain T7 was established. The obtained results indicate the prospective use B. paralicheniformis strain T7 as a producer of metalloproteases M14 and M42.

Geranylgeranylated-chlorophyll-protein complexes in lhl3 mutant of the green alga Chlamydomonas reinhardtii.

Chlorophylls a and b (Chl a and b) are involved in light harvesting, photochemical reactions, and electron transfer reactions in plants and green algae. The core complexes of the photosystems (PSI and PSII) associate with Chl a, while the peripheral antenna complexes (LHCI and LHCII) bind Chls a and b. One of the final steps of Chl biosynthesis is the conversion of geranylgeranylated Chls (ChlsGG) to phytylated Chls by geranylgeranyl reductase (GGR). Here, we isolated and characterized a pale green mutant of the green alga Chlamydomonas reinhardtii that was very photosensitive and was unable to grow photoautotrophically. This mutant has a 16-bp deletion in the LHL3 gene, which resulted in the loss of LHL3 and GGR and accumulated only ChlsGG. The lhl3 mutant cells grown in the dark accumulated PSII and PSI proteins at 25-50% of WT levels, lacked PSII activity, and retained a decreased PSI activity. The PSII and PSI proteins were depleted to trace amounts in the mutant cells grown in light. In contrast, the accumulation of LHCI and LHCII was unaffected except for LHCA3. Our results suggest that the replacement of Chls with ChlsGG strongly affects the structural and functional integrity of PSII and PSI complexes but their associating LHC complexes to a lesser extent. Affinity purification of HA-tagged LHL3 confirmed the formation of a stable LHL3-GGR complex, which is vital for GGR stability. The LHL3-GGR complex contained a small amount of PSI complex assembly factors, suggesting a putative coupling between Chl synthesis and PSI complex assembly.

Structure and Antigenicity of the Porcine Astrovirus 4 Capsid Spike

Porcine astrovirus 4 (PoAstV4) has been recently associated with respiratory disease in pigs. In order to understand the scope of PoAstV4 infections and to support the development of a vaccine to combat PoAstV4 disease in pigs, we designed and produced a recombinant PoAstV4 capsid spike protein for use as an antigen in serological assays and for potential future use as a vaccine antigen. Structural prediction of the full-length PoAstV4 capsid protein guided the design of the recombinant PoAstV4 capsid spike domain expression plasmid. The recombinant PoAstV4 capsid spike was expressed in Escherichia coli, purified by affinity and size-exclusion chromatography, and its crystal structure was determined at 1.85 Å resolution, enabling structural comparisons to other animal and human astrovirus capsid spike structures. The recombinant PoAstV4 capsid spike protein was also used as an antigen for the successful development of a serological assay to detect PoAstV4 antibodies, demonstrating that the recombinant PoAstV4 capsid spike retains antigenic epitopes found on the native PoAstV4 capsid. These studies lay a foundation for seroprevalence studies and the development of a PoAstV4 vaccine for swine.

Calixarene incorporated molecular imprinting on covalent organic framework for supramolecular recognition and specific extraction of citrinin

Specific recognition and selective extraction of mycotoxin in environmental and food matrixes is significant to guarantee public health. Covalent organic frameworks (COFs) are promising adsorbents with tailorable functionality, but their low binding affinity and poor selectivity hamper their wide application for selective extraction of trace mycotoxin from complex matrix. Herein, we report calixarene incorporated molecular imprinting on COF to prepare molecularly imprinted calix[4]arene-containing COF (MICOF-CX4) for supramolecular recognition and specific adsorption of citrinin. Calixarene with host-guest chemistry was used as a functional monomer, while amine units with different topologies and function groups were selected to regulate MICOF-CX4 to match with citrinin. The complementary shape and supramolecular interactions of MICOF-CX4 gave highly selective recognition for citrinin. Moreover, MICOF-CX4 with vast accessible surface and plentiful imprinting sites exhibited faster adsorption kinetics and 4-fold higher adsorption capacity for citrinin adsorption than no-imprinted COF-CX4. Combination of MICOF-CX4 based solid-phase extraction with high-performance liquid chromatography-mass spectrometry allowed interference free determination of trace citrinin in real samples with a low detection limit of 0.03 ng mL−1, good precision of 4.5 % and quantitative recovery of 88.2 %−101.4 %. The cooperative functions of calixarene and molecular imprinting make COF promising adsorbent for specific adsorption of trace targets in complex matrixes.

Novel Insights into Ethanol-Soluble Oyster Peptide-Zinc-Chelating Agents: Structural Characterization, Chelation Mechanism, and Potential Protection on MEHP-Induced Leydig Cells.

Numerous studies have reported that mono-(2-ethylhexyl) phthalate (MEHP) (bioactive metabolite of Di(2-ethylhexyl) phthalate) has inhibitory effects on Leydig cells. This study aims to prepare an oyster peptide-zinc complex (PEP-Zn) to alleviate MEHP-induced damage in Leydig cells. Zinc-binding peptides were obtained through the following processes: zinc-immobilized affinity chromatography (IMAC-Zn2+), liquid chromatography-mass spectrometry technology (LC-MS/MS) analysis, molecular docking, molecular dynamic simulation, and structural characterization. Then, the Zn-binding peptide (PEP) named Glu-His-Ala-Pro-Asn-His-Asp-Asn-Pro-Gly-Asp-Leu (EHAPNHDNPGDL) was identified. EHAPNHDNPGDL showed the highest zinc-chelating ability of 49.74 ± 1.44%, which was higher than that of the ethanol-soluble oyster peptides (27.50 ± 0.41%). In the EHAPNHDNPGDL-Zn complex, Asn-5, Asp-7, Asn-8, His-2, and Asp-11 played an important role in binding to the zinc ion. Additionally, EHAPNHDNPGDL-Zn was found to increase the cell viability, significantly increase the relative activity of antioxidant enzymes and testosterone content, and decrease malondialdehyde (MDA) content in MEHP-induced TM3 cells. The results also indicated that EHAPNHDNPGDL-Zn could alleviate MEHP-induced apoptosis by reducing the protein level of p53, p21, and Bax, and increasing the protein level of Bcl-2. These results indicate that the zinc-chelating peptides derived from oyster peptides could be used as a potential dietary zinc supplement.

Structural basis of μ-opioid receptor targeting by a nanobody antagonist

The μ-opioid receptor (μOR), a prototypical G protein-coupled receptor (GPCR), is the target of opioid analgesics such as morphine and fentanyl. Due to the severe side effects of current opioid drugs, there is considerable interest in developing novel modulators of μOR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, represent alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the μOR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded μOR ligand and uncover the molecular basis for μOR antagonism by determining the cryo-EM structure of the NbE-μOR complex. NbE displays a unique ligand binding mode and achieves μOR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a β-hairpin loop formed by NbE that deeply protrudes into the μOR, we design linear and cyclic peptide analogs that recapitulate NbE’s antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes lower molecular weight μOR ligands that can serve as a basis for therapeutic developments.

Oriental covalent immobilization of N-glycan binding protein via N-terminal selective modification

Lectin affinity chromatography is one of powerful tools for the study of protein glycosylation. Different lectin proteins can recognize different structures of monosaccharides or oligosaccharide units, allowing for the selective separation of glycopeptides or glycoproteins containing different polysaccharide structures. However, the N-glycans were only partially captured by most of common lectins, reducing the coverage rate of identifying N-glycoconjugates. Recently, it has been reported that the engineering variant of glycan binding protein Fbs1 has a high affinity for innermost Man3GlcNAc2 structure and is able to bind diverse types of N-glycans, which can be suitable for the analysis of protein N-glycosylation. However, efficient immobilization of protein to separation matrix is particularly challenging as it requires the functionality and integrity of the protein to be preserved. Herein, we describe a simple and robust strategy for oriental covalent immobilization of proteins on magnetic nanoparticles by N-terminal selective labeling techniques. We inserted the enterokinase cleavage site to produce the specific N- terminal glycine of protein. Under physiological conditions, the protein was immobilized on the surface of the MNPs by this glycine tag, and the enrichment process could be completed within 30 min. A whole enrichment and purification of glycan and glycopeptides were completed and analyzed by MALDI TOF-MS. The functional materials achieved stable enrichment of glycan structure in different enzyme digestion systems or complex samples, showing excellent anti-interference and applicability.

Selective adsorption of cationic dye by κ-carrageenan-potato starch bio-hydrogel: Kinetics, isotherm, and thermodynamic studies

An eco-friendly κ-carrageenan/potato starch bio-hydrogel is designed for the efficient removal of methylene blue (MB) dye from water. The incorporation of potato starch was successfully confirmed through XRD, FT-IR, and SEM analysis, while TGA highlighted the hydrogel's thermal stability. Batch adsorption experiments demonstrated excellent MB removal efficiency, with a maximum adsorption capacity of 116.1 mg/g under optimal conditions (initial dye concentration: 100 mg/L, contact time: 180 min, temperature: 20 °C, adsorbent dosage: 1.6 g/L, and pH: 11). FT-IR analysis indicated that electrostatic interactions and hydrogen bonding primarily govern the adsorption process. The adsorption followed pseudo-second-order kinetics and fitted well with the Langmuir isotherm model. Thermodynamic studies revealed that the adsorption was exothermic and spontaneous. A key feature of this bio hydrogel is its selective affinity for the cationic dye MB, in a mixture with Acid Orange (AO) and other cationic dyes (Rhodamine B (Rh B) and crystal violet (CV)). The adsorbent also demonstrated impressive reusability, maintaining 93 % of its efficiency after five cycles, highlighting its potential for sustainable and cost-effective water treatment.

Combining mathematical modeling, in vitro data and clinical target expression to support bispecific antibody binding affinity selection: a case example with FAP-4-1BBL.

The majority of bispecific costimulatory antibodies in cancer immunotherapy are capable of exerting tumor-specific T-cell activation by simultaneously engaging both tumor-associated targets and costimulatory receptors expressed by T cells. The amount of trimeric complex formed when the bispecific antibody is bound simultaneously to the T cell receptor and the tumor-associated target follows a bell-shaped curve with increasing bispecific antibody exposure/dose. The shape of the curve is determined by the binding affinities of the bispecific antibody to its two targets and target expression. Here, using the case example of FAP-4-1BBL, a fibroblast activation protein alpha (FAP)-directed 4-1BB (CD137) costimulator, the impact of FAP-binding affinity on trimeric complex formation and pharmacology was explored using mathematical modeling and simulation. We quantified (1) the minimum number of target receptors per cell required to achieve pharmacological effect, (2) the expected coverage of the patient population for 19 different solid tumor indications, and (3) the range of pharmacologically active exposures as a function of FAP-binding affinity. A 10-fold increase in FAP-binding affinity (from a dissociation constant [KD] of 0.7 nM-0.07nM) was predicted to reduce the number of FAP receptors needed to achieve 90% of the maximum pharmacological effect from 13,400 to 4,000. Also, the number of patients with colon cancer that would achieve 90% of the maximum effect would increase from 6% to 39%. In this work, a workflow to select binding affinities for bispecific antibodies that integrates preclinical in vitro data, mathematical modeling and simulation, and knowledge on target expression in the patient population, is provided. The early implementation of this approach can increase the probability of success with cancer immunotherapy in clinical development.

Highly Efficient Separation of Intermediate-Size m-Xylene from Xylenes via a Length-Matched Metal-Organic Framework with Optimal Oxygen Sites Distribution.

Xylene separation is crucial but challenging, especially for the preferential separation of the intermediate-size m-xylene from xylene mixtures. Herein, exploiting the differences in molecular length and alkyl distribution among xylenes, we present a length-matched metal-organic framework, formulated as Al(OH)[O2C-C4H2O-CO2], featuring an effective pore size corresponding to m-xylene molecular length combined with multiple negative O hydrogen bond donors distribution, can serve as a molecular trap for efficient preferential separation of the intermediate-size m-xylene. Benchmark separation performance was achieved for separating m-xylene from a ternary mixture of m-xylene/o-xylene/p-xylene, with simultaneous record-high m-xylene uptake (1.3 mmol g-1) and m-xylene/p-xylene selectivity (5.3) in the liquid-phase competitive adsorption. Both vapor- and liquid-phase fixed-bed tests confirmed its practical separation capability with benchmark dynamic m-xylene/p-xylene and m-xylene/o-xylene selectivities, as well as excellent regenerability. The selective and strong m-xylene binding affinity among xylene molecules was further elucidated by simulations, validating the effectiveness of such a pore environment for the separation of intermediate-size molecules.

Development and Characterization of A Novel SpyTagged Modular Nanobody as A Detection Platform for CD22-Positive Cells.

CD22, as a surface protein of B cells, is used in the diagnosis and target-specific immunotherapy of B-cell malignancies. SpyTag and SpyCatcher, on the other hand, are two covalently coupled proteins capable of developing a bi- or multi-specific modular protein. The aim of this study was to develop FITC-conjugated SpyCatcher-SpyTagged anti-CD22 Nanobody (FITC-SpyC-SpyT-CD22Nb) to recognize CD22 on the surface of malignant B cells. In this experimental study, the SpyTag-CD22Nb construct was subcloned into a pET22 vector and expressed in E. coli BL21 (DE3). After purification using His-tag affinity chromatography, the size of the eluted protein was confirmed on a Western blot. In addition, the SpyCatcher protein, subcloned into pET28, was expressed in E. coli BL21 (DE3), purified by His-tag affinity chromatography and subjected to FITC labeling. FITC-SpyCatcher and SpyTag-CD22Nb were coupled in a 1:1 molar ratio. The specific binding of the produced FITC-SpyC-SpyT-CD22Nb was tested using CD22+ Raji and CD22- K562 cell lines and was evaluated by flow cytometry. SpyTag-CD22Nb and SpyCatcher were successfully expressed in E. coli BL21 (DE3). The 1:1 molar ratio of SpyTag-CD22Nb and FITC-SpyCatcher successfully formed FITC-SpyC-SpyT-CD22Nb at room temperature. The flow cytometry results showed that FITC-SpyC-SpyT-CD22Nb specifically binds to the CD22+ Raji cells, while there is no binding to the CD22- K562 control cells. The novel FITC-SpyC-SpyT-CD22Nb produced in the present study is capable of detecting the surficial expression of CD22. According to our findings, FITC-SpyC-SpyT-CD22Nb is applicable for specific targeting of CD22 in a therapeutic manner, i.e., chimeric antigen receptor (CAR)-T cell therapy and antibody drug conjugates (ADCs).

Synthesis of Multivalent Glycoside-Immobilized Carboxymethyl Cellulose Nanohydrogel Particles with Superadsorption Ability for Lectins.

Carboxymethyl cellulose (CMC) is a water-soluble cellulose derivative that is nontoxic, biocompatible, biodegradable, and nonallergenic. As developing an adsorbent material for carbohydrate-binding proteins is challenging, we aimed to synthesize CMC nanohydrogel particles (CMCGPs) with an extremely high lectin adsorption tendency in this study. CMCGPs were used as the backbone of an adsorption carrier that was synthesized by cross-linking CMC with ethylene glycol diglycidyl ether. A series of glycoside-immobilized CMCGPs were synthesized by binding two types of glycans (LacNAc and lactose) to the polyvalent carboxymethyl groups that are present on the CMCGP surface and act as reaction sites. These immobilized glycosides function as molecular recognition sites. Glycan moieties were incorporated into the CMCGP backbone at degrees of immobilization (DI) ranging from 8.7 to 21.0% by altering the reaction composition. LacNAc-CMCGP (3b) showed a 19.9% DI of LacNAc glycoside to the CMCGP carboxymethyl group; on average, its particle size swelled to 418 nm in phosphate-buffered saline, which is approximately 1.4 times its dry-state size. Analyzing the adsorbent properties of glyco-CMCGPs using a lectin-binding assay showed the high structural specificity of glyco-CMCGPs to lectins. The equilibrium isotherm data was explained by the Langmuir adsorption model. Notably, compound 3b adsorbed 1.95 ± 0.05 μg of wheat germ agglutinin (WGA) lectin per 1.0 μg-dry of 3b particles at an adsorption equilibrium time of a few minutes. Furthermore, solid-state 13C nuclear magnetic resonance analysis showed that WGA lectin retained its natural structure without denaturation after binding to LacNAc-CMCGP. These results were also supported by affinity purification experiments of WGA from raw wheat germ extract using LacNAc-CMCGP, demonstrating that glyco-CMCGP is capable of adsorbing and desorbing lectin while maintaining its biological activity. Thus, multivalent glycoside-immobilized CMCGPs that use woody biomass derivatives as the backbone are expected to be applied as biorefinery materials, which specifically and abundantly adsorb not just plant lectins but also pathogenic viruses and toxin proteins.

Biochemical plasticity of the Escherichia coli CRISPR Cascade revealed by in vitro reconstitution of Cascade activities from purified Cas proteins.

The most abundant clustered regularly interspaced short palindromic repeats (CRISPR) type I systems employ a multisubunit RNA-protein effector complex (Cascade), with varying protein composition and activity. The Escherichia coli Cascade complex consists of 11 protein subunits and functions as an effector through CRISPR RNA (crRNA) binding, protospacer adjacent motif (PAM)-specific double-stranded DNA targeting, R-loop formation, and Cas3 helicase-nuclease recruitment for target DNA cleavage. Here, we present a biochemical reconstruction of the E. coli Cascade from purified Cas proteins and analyze its activities including crRNA binding, dsDNA targeting, R-loop formation, and Cas3 recruitment. Affinity purification of 6His-tagged Cas7 coexpressed with untagged Cas5 revealed the physical association of these proteins, thus producing the Cas5-Cas7 subcomplex that was able to bind specifically to type I-E crRNA with an efficiency comparable to that of the complete Cascade. The crRNA-loaded Cas5-7 was found to bind specifically to the target dsDNA in a PAM-independent manner, albeit with a lower affinity than the complete Cascade, with both spacer sequence complementarity and repeat handles contributing to the DNA targeting specificity. The crRNA-loaded Cas5-7 targeted the complementary dsDNA with detectable formation of R-loops, which was stimulated by the addition of Cas8 and/or Cas11 acting synergistically. Cascade activity reconstitution using purified Cas5-7 and other Cas proteins showed that Cas8 was essential for specific PAM recognition, whereas the addition of Cas11 was required for Cas3 recruitment and target DNA nicking. Thus, although the core Cas5-7 subcomplex is sufficient for specific crRNA binding and basal DNA targeting, both Cas8 and Cas11 make unique contributions to efficient target recognition and cleavage.

Striatal cell-type-specific molecular signatures reveal therapeutic targets in a model of dystonia.

Striatal dysfunction is implicated in many forms of dystonia, including idiopathic, inherited and iatrogenic dystonias. The striatum is comprised largely of GABAergic spiny projection neurons (SPNs) that are defined by their long-range efferents. Direct SPNs (dSPNs) project to the internal globus pallidus/substantia nigra reticulata whereas indirect pathway SPNs (iSPNs) project to the external pallidum; the concerted activity of both SPN subtypes modulates movement. Convergent results from genetic, imaging and physiological studies in patients suggest that abnormalities of both dSPNs and iSPNs contribute to the expression of dystonia, but the molecular adaptations underlying these abnormalities are not known. Here we provide a comprehensive analysis of SPN cell-type-specific molecular signatures in a model of DOPA-responsive dystonia (DRD mice), which is caused by gene defects that reduce dopamine neurotransmission, resulting in dystonia that is specifically associated with striatal dysfunction. Individually profiling the translatome of dSPNs and iSPNs using translating ribosome affinity purification with RNA-seq revealed hundreds of differentially translating mRNAs in each SPN subtype in DRD mice, yet there was little overlap between the dysregulated genes in dSPNs and iSPNs. Despite the paucity of shared adaptations, a disruption in glutamatergic signaling was predicted for both dSPNs and iSPNs. Indeed, we found that both AMPA and NMDA receptor-mediated currents were enhanced in dSPNs but diminished in iSPNs in DRD mice. The pattern of mRNA dysregulation was specific to dystonia as the adaptations in DRD mice were distinct from those in parkinsonian mice where the dopamine deficit occurs in adults, suggesting that the phenotypic outcome is dependent on both the timing of the dopaminergic deficit and the SPN-specific adaptions. We leveraged the unique molecular signatures of dSPNs and iSPNs in DRD mice to identify biochemical mechanisms that may be targets for therapeutics, including LRRK2 inhibition. Administration of the LRRK2 inhibitor MLi-2 ameliorated the dystonia in DRD mice suggesting a novel target for therapeutics and demonstrating that the delineation of cell-type-specific molecular signatures provides a powerful approach to revealing both CNS dysfunction and therapeutic targets in dystonia.

6644 Glycosylation Patterns of the IgG Variable Region in Thyroglobulin Antibodies and Their Impact on Antigen Binding Affinity

Abstract Disclosure: S. Wang: None. Y. Cao: None. C. Zhao: None. C. Huang: None. K. Zhao: None. Y. Li: None. Y. Huang: None. Z. Ying: None. Y. Zhang: None. Y. Gao: None. Objective: Thyroglobulin antibodies (TgAb), key serological markers in Hashimoto's thyroiditis (HT) patients, are predominantly in immunoglobulin G (IgG) form. Prior research has highlighted the importance of increased glycosylation in TgAb IgG from HT patients. This study investigates the unique glycosylation patterns of F(ab')₂ fragments in TgAb IgG in HT patients, compared to healthy individuals. It focuses on how these patterns differ and their impact on binding to thyroglobulin (Tg). Exploring the role of N-glycosylation in the F(ab')₂ segment of TgAb IgG is crucial for understanding its effect on immune responses in HT, potentially unveiling key mechanisms of the disease. Methods: To assess serum TgAb levels in HT patients (n≥28), we used an electrochemiluminescence immunoassay, dividing patients into medium-level (mHT, n≥14) and high-level (hHT, n≥14) groups. TgAb IgG was isolated from serum of both HT and control groups (n = 14) using affinity chromatography. TgAb IgG was then cleaved with Ides enzyme to yield TgAb IgG F(ab’)2 and Fc fragments. The F(ab')2 fragment of TgAb IgG underwent MALDI-QIT-TOF-MS mass spectrometry for glycosylation profiling. Additionally, mixed TgAb IgG was separated using columns that recognize mannose and terminal sialic acid, yielding high mannose and high terminal sialic acid TgAb IgG. Different TgAb IgG glycoforms were prepared through enzymatic treatment with peptide-N-glycosidase F, β1-4 galactosidase, and α2-3,6,8 neuraminidase. The binding affinity of these TgAb IgG to Tg antigen was measured using Tg specific ELISA and surface plasmon resonance (SPR). Results: Using MALDI-TOF MS, we identified 22 IgG glycan types in both HT and control (Con) groups. In the hHT group, six glycan types (G0F, G1F, G2F, M5A1, A3G1F, A3F) were upregulated compared to Con, with statistical significance (p < 0.05). The mHT group showed increased levels of G1F and G2F compared to Con (p < 0.05). Between hHT and mHT, only A3F was significantly elevated (p < 0.05). Removal of N-glycosylation from IgG TgAb F(ab)2 reduced binding to Tg antigen. Presence of both mannose and terminal sialic acid in TgAb enhanced the binding to Tg. Conclusions: Most TgAb IgG in the variable region of HT patients contain N-glycans. The glycosylation patterns of TgAb IgG F(ab)2 in HT differ significantly from healthy controls, affecting antibody binding affinity. Our findings reveal the complex role of glycosylation patterns in TgAb IgG, providing new insights into HT pathogenesis and potential research directions. Presentation: 6/1/2024