RESUMEN
Neuritin plays an important role in promoting nerve injury repair and maintaining synaptic plasticity, making it a potential therapeutic target for the treatment of nerve injury and neurodegenerative diseases. The present study aimed to obtain an active, unlabeled neuritin protein. Initially, a neuritin protein expression system with an enterokinase site was constructed in Escherichia coli. After optimizing induction conditions and screening for high expression, a neuritin recombinant protein with purity exceeding 85 % was obtained through Ni-affinity chromatography. Subsequently, unlabeled neuritin with a molecular weight of 11 kDa was obtained through the enzymatic cleavage of the His label using an enterokinase. Furthermore, a neuritin recombinant protein with purity exceeding 95 % was obtained using gel chromatography. Functional investigations revealed that neurite outgrowth of PC12 cells was stimulated by the isolated neuritin. This study establishes a method to obtain active and unlabeled neuritin protein, providing a foundation for subsequent research on its biological functions.
Asunto(s)
Escherichia coli , Proteínas Ligadas a GPI , Proteínas Recombinantes , Animales , Células PC12 , Ratas , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Neuropéptidos/genética , Neuropéptidos/química , Neuropéptidos/metabolismo , Enteropeptidasa/metabolismo , Enteropeptidasa/genética , Enteropeptidasa/química , Cromatografía de Afinidad , Proyección Neuronal/efectos de los fármacos , Cromatografía en Gel , Expresión GénicaRESUMEN
GPI-anchored proteins (GPI-APs) are ubiquitous and essential but exist in low abundances on the cell surface, making their analysis and investigation especially challenging. To tackle the problem, a new method to detect and study GPI-APs based upon GPI metabolic engineering and DNA-facilitated fluorescence signal amplification was developed. In this context, cell surface GPI-APs were metabolically engineered using azido-inositol derivatives to introduce an azido group. This allowed GPI-AP coupling with alkyne-functionalized multifluorophore DNA assemblies generated by hybridization chain reaction (HCR). It was demonstrated that this approach could significantly improve the detection limit and sensitivity of GPI-APs, thereby enabling various biological studies, including the investigation of live cells. This new, enhanced GPI-AP detection method has been utilized to successfully explore GPI-AP engineering, analyze GPI-APs, and profile GPI-AP expression in different cells.
Asunto(s)
ADN , Hibridación de Ácido Nucleico , Humanos , ADN/química , Proteínas Ligadas a GPI/metabolismo , Animales , Glicosilfosfatidilinositoles/metabolismo , Glicosilfosfatidilinositoles/química , Colorantes Fluorescentes/química , Azidas/químicaRESUMEN
Adjuvant is an integral part of all vaccine formulations but only a few adjuvants with limited efficacies or application scopes are available. Thus, developing more robust and diverse adjuvants is necessary. To this end, a new class of adjuvants having α- and ß-rhamnose (Rha) attached to the 1- and 6'-positions of monophosphoryl lipid A (MPLA) was designed, synthesized, and immunologically evaluated in mice. The results indicated a synergistic effect of MPLA and Rha, two immunostimulators that function via interacting with toll-like receptor 4 and recruiting endogenous anti-Rha antibodies, respectively. All the tested MPLA-Rha conjugates exhibited potent adjuvant activities to promote antibody production against both protein and carbohydrate antigens. Overall, MPLA-α-Rha exhibited better activities than MPLA-ß-Rha, and 6'-linked conjugates were slightly better than 1-linked ones. Particularly, MPLA-1-α-Rha and MPLA-6'-α-Rha were the most effective adjuvants in promoting IgG antibody responses against protein antigen keyhole limpet hemocyanin and carbohydrate antigen sTn, respectively.
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Lípido A , Ramnosa , Lípido A/análogos & derivados , Lípido A/química , Lípido A/farmacología , Lípido A/inmunología , Animales , Ramnosa/química , Ramnosa/inmunología , Ramnosa/farmacología , Ratones , Adyuvantes de Vacunas/química , Adyuvantes de Vacunas/farmacología , Femenino , Adyuvantes Inmunológicos/farmacología , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/síntesis química , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina G/sangre , Ratones Endogámicos BALB C , Hemocianinas/química , Hemocianinas/inmunologíaRESUMEN
In this study, we developed a novel type of dibenzocyclooctyne (DBCO)-functionalized microbubbles (MBs) and validated their attachment to azide-labelled sialoglycans on human pluripotent stem cells (hPSCs) generated by metabolic glycoengineering (MGE). This enabled the application of mechanical forces to sialoglycans on hPSCs through molecularly specific acoustic tweezing cytometry (mATC), that is, displacing sialoglycan-anchored MBs using ultrasound (US). It was shown that subjected to the acoustic radiation forces of US pulses, sialoglycan-anchored MBs exhibited significantly larger displacements and faster, more complete recovery after each pulse than integrin-anchored MBs, indicating that sialoglycans are more stretchable and elastic than integrins on hPSCs in response to mechanical force. Furthermore, stimulating sialoglycans on hPSCs using mATC reduced stage-specific embryonic antigen-3 (SSEA-3) and GD3 expression but not OCT4 and SOX2 nuclear localization. Conversely, stimulating integrins decreased OCT4 nuclear localization but not SSEA-3 and GD3 expression, suggesting that mechanically stimulating sialoglycans and integrins initiated distinctive mechanoresponses during the early stages of hPSC differentiation. Taken together, these results demonstrated that MGE-enabled mATC uncovered not only different mechanical properties of sialoglycans on hPSCs and integrins but also their different mechanoregulatory impacts on hPSC differentiation, validating MGE-based mATC as a new, powerful tool for investigating the roles of glycans and other cell surface biomolecules in mechanotransduction.
Asunto(s)
Células Madre Pluripotentes , Humanos , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Microburbujas , Ingeniería MetabólicaRESUMEN
As new methods to interrogate glycan organization on cells develop, it is important to have a molecular level understanding of how chemical fixation can impact results and interpretations. Site-directed spin labeling technologies are well suited to study how the spin label mobility is impacted by local environmental conditions, such as those imposed by cross-linking effects of paraformaldehyde cell fixation methods. Here, we utilize three different azide-containing sugars for metabolic glycan engineering with HeLa cells to incorporate azido glycans that are modified with a DBCO-based nitroxide moiety via click reaction. Continuous wave X-band electron paramagnetic resonance spectroscopy is employed to characterize how the chronological sequence of chemical fixation and spin labeling impacts the local mobility and accessibility of the nitroxide-labeled glycans in the glycocalyx of HeLa cells. Results demonstrate that chemical fixation with paraformaldehyde can alter local glycan mobility and care should be taken in the analysis of data in any study where chemical fixation and cellular labeling occur.
RESUMEN
Glycosylphosphatidylinositols (GPIs) need to interact with other components in the cell membrane to transduce transmembrane signals. A bifunctional GPI probe was employed for photoaffinity-based proximity labelling and identification of GPI-interacting proteins in the cell membrane. This probe contained the entire core structure of GPIs and was functionalized with photoreactive diazirine and clickable alkyne to facilitate its crosslinking with proteins and attachment of an affinity tag. It was disclosed that this probe was more selective than our previously reported probe containing only a part structure of the GPI core for cell membrane incorporation and an improved probe for studying GPI-cell membrane interaction. Eighty-eight unique membrane proteins, many of which are related to GPIs/GPI-anchored proteins, were identified utilizing this probe. The proteomics dataset is a valuable resource for further analyses and data mining to find new GPI-related proteins and signalling pathways. A comparison of these results with those of our previous probe provided direct evidence for the profound impact of GPI glycan structure on its interaction with the cell membrane.
Asunto(s)
Glicosilfosfatidilinositoles , Polisacáridos , Glicosilfosfatidilinositoles/química , Membrana Celular/metabolismo , Polisacáridos/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de SeñalRESUMEN
A glycosylphosphatidylinositol (GPI) derivative with biotin linked to its mannose III 6-O-position was prepared by a convergent strategy. This biotinylated GPI was demonstrated to bind avidinated proteins readily through biotin-avidin interaction and, therefore, can serve as a universal platform to access various biologically significant GPI-anchored protein analogues.
Asunto(s)
Biotina , Glicosilfosfatidilinositoles , Glicosilfosfatidilinositoles/metabolismo , Proteínas Ligadas a GPIRESUMEN
As new methods to interrogate glycan organization on cells develop, it is important to have a molecular level understanding of how chemical fixation can impact results and interpretations. Site-directed spin labeling technologies are well suited to study how the spin label mobility is impacted by local environmental conditions, such as those imposed by cross-linking effects of paraformaldehyde cell fixation methods. Here, we utilize three different azide-containing sugars for metabolic glycan engineering with HeLa cells to incorporate azido glycans that are modified with a DBCO-based nitroxide moiety via click reaction. Continuous wave X-band electron paramagnetic resonance spectroscopy is employed to characterize how the chronological sequence of chemical fixation and spin labeling impacts the local mobility and accessibility of the nitroxide-labeled glycans in the glycocalyx of HeLa cells. Results demonstrate that chemical fixation with paraformaldehyde can alter local glycan mobility and care should be taken in the analysis of data in any study where chemical fixation and cellular labeling occur.
RESUMEN
Sialoglycans on HeLa cells were labeled with a nitroxide spin radical through enzymatic glycoengineering (EGE)-mediated installation of azide-modified sialic acid (Neu5Ac9N3) and then click reaction-based attachment of a nitroxide spin radical. α2,6-Sialyltransferase (ST) Pd2,6ST and α2,3-ST CSTII were used for EGE to install α2,6- and α2,3-linked Neu5Ac9N3, respectively. The spin-labeled cells were analyzed by X-band continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy to gain insights into the dynamics and organizations of cell surface α2,6- and α2,3-sialoglycans. Simulations of the EPR spectra revealed average fast- and intermediate-motion components for the spin radicals in both sialoglycans. However, α2,6- and α2,3-sialoglycans in HeLa cells possess different distributions of the two components, e.g., a higher average population of the intermediate-motion component for α2,6-sialoglycans (78%) than that for α2,3-sialoglycans (53%). Thus, the average mobility of spin radicals in α2,3-sialoglycans was higher than that in α2,6-sialoglycans. Given the fact that a spin-labeled sialic acid residue attached to the 6-O-position of galactose/N-acetyl-galactosamine would experience less steric hindrance and show more flexibility than that attached to the 3-O-position, these results may reflect the differences in local crowding/packing that restrict the spin-label and sialic acid motion for α2,6-linked sialoglycans. The studies further suggest that Pd2,6ST and CSTII may have different preferences for glycan substrates in the complex environment of the extracellular matrix. The discoveries of this work are biologically important as they are useful for interpreting the different functions of α2,6- and α2,3-sialoglycans and indicate the possibility of using Pd2,6ST and CSTII to target different glycoconjugates on cells.
Asunto(s)
Ácido N-Acetilneuramínico , Óxidos de Nitrógeno , Humanos , Espectroscopía de Resonancia por Spin del Electrón/métodos , Células HeLa , Óxidos de Nitrógeno/química , Marcadores de SpinRESUMEN
Glycosylphosphatidylinositol (GPI) anchorage is one of the most common mechanisms to attach proteins to the plasma membrane of eukaryotic cells. GPI-anchored proteins (GPI-APs) play a critical role in many biological processes but are difficult to study. Here, a new method was developed for the effective and selective metabolic engineering and labeling of cell surface GPI-APs with an azide-modified phosphatidylinositol (PI) as the biosynthetic precursor of GPIs. It was demonstrated that this azido-PI derivative was taken up by HeLa cells and incorporated into the biosynthetic pathway of GPIs to present azide-labeled GPI-APs on the live cell surface. The azido group was used as a molecular handle to install other labels through a biocompatible click reaction to enable various biological studies, e.g., fluorescent imaging and protein pull-down, which can help explore the functions of GPI-APs and discover new GPI-APs.
Asunto(s)
Glicosilfosfatidilinositoles , Proteínas de la Membrana , Humanos , Proteínas de la Membrana/metabolismo , Células HeLa , Azidas , Ingeniería Metabólica , Membrana Celular/metabolismoRESUMEN
A novel method for spin labelling of sialoglycans on the cell surface is described. C9-Azido sialic acid was linked to glycans on live cells via CSTII-catalysed α2,3-sialylation utilizing azido-sialic acid nucleotide as a sialyl donor, which was followed by attachment of a spin label to the azide via click reaction. It enables the study of cell surface sialoglycans by EPR spectroscopy.
Asunto(s)
Azidas , Polisacáridos , Membrana Celular/metabolismo , Espectroscopía de Resonancia por Spin del Electrón/métodos , Polisacáridos/química , Marcadores de SpinRESUMEN
A new and efficient method was developed for the synthesis of C3-substituted sialyl glycals that are useful for novel sialidase inhibitor discovery. This method was based on the cross-coupling reactions of 3-iodo-sialyl glycal methyl ester with boronic acids, alkenes and alkynes to directly introduce various functional groups to the sialyl glycal C3-position. A series of C3-aryl, alkyl, alkenyl, and alkynyl derivatives of sialyl glycal were efficiently and conveniently synthesized for the first time by this method, which has demonstrated its wide application scope.
Asunto(s)
Desoxiazúcares/química , Yodo/química , Ácidos Borónicos/química , Estructura MolecularRESUMEN
Tumor-associated carbohydrate antigens Lewis X (Lex), Lewis Y (Ley), and KH-1 are useful targets for cancer immunotherapy. In this regard, an insight into the structure-immunogenicity relationships of these antigens is important but this has not been systematically investigated yet. In the current study, Lex, Ley, and KH-1 antigens with a lactose unit at the reducing end as a spacer were synthesized and coupled with keyhole limpet hemocyanin (KLH) protein. Immunological evaluations of the resultant conjugates revealed that they all could elicit robust immune responses whilst the Ley conjugate could provoke the highest titers of total and IgG antibodies. The binding assays of their antisera to each antigen and to cancer cells showed that each antiserum had extensive cross-reaction with all three antigens as protein conjugates and strong but somewhat antigen-selective binding towards MCF-7 cancer cell. Moreover, none of these antisera had obvious binding to SKMEL-28 cancer cell that does not express Lex, Ley and KH-1. The results of assays of these antisera to mediate complement-dependent cytotoxicity (CDC) to MCF-7 and SKMEL-28 cancer cells were very similar to the results of binding assays. Thus, it was concluded that all three antigens could form effective conjugate vaccines whereas the Ley conjugate induced the most robust immune responses and the antiserum of Lex had the highest binding and cytotoxicity to target cancer cells. In addition, as the antibodies induced by each antigen had extensive cross-reaction with other two antigens, either Lex or Ley or the two combined can be utilized to formulate effective conjugate vaccines for cancer immunotherapy. Another paradigm-shifting discovery of this study is that the presentation of Lex, Ley, and KH-1 antigens on cancer cell can be different from that in synthetic conjugates, which should be taken into consideration during the design and optimization of related cancer vaccines or immunotherapies.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/inmunología , Antígenos del Grupo Sanguíneo de Lewis/inmunología , Animales , Conformación de Carbohidratos , Femenino , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos C57BLRESUMEN
The reactions of several ß-, γ-, and δ-azido alcohols with dibenzyl and dimethyl N,N-diisopropylphosphoramidites were examined. Detailed analysis of the intermediates and products formed from the reactions under different conditions provided useful information to gain insights into their mechanisms involving intramolecular Staudinger reaction, as well as the structure-reactivity relationships of both substrates. The reactions of γ- and δ-azido alcohols with dibenzyl N,N-diisopropylphosphoramidite could produce 6- and 7-membered cyclic phosphoramidates, thereby providing a new synthetic method for these biologically important molecules.
RESUMEN
Analogues of cancer-associated Lewis Y (Ley) antigen with varying structures at the reducing end were synthesized by a highly efficient strategy involving one-pot preactivation-based iterative glycosylation to obtain the key tetra-/pentasaccharide intermediates, which was followed by stereoselective fucosylation. After global deprotection, these oligosaccharides were coupled with carrier protein keyhole limpet hemocyanin. The resultant glycan-protein conjugates were subjected to immunological studies in mice. It was disclosed that the conjugate of the pentasaccharide analogue of Lewis Y antigen was more immunogenic than that of the hexasaccharide analogue, but the antisera of both conjugates could indiscriminately recognize each carbohydrate hapten. These results suggested that the short Lewis Y analogue may be utilized to develop functional conjugate cancer vaccines. More importantly, the results also proved that the reducing-end glucose residue in the hexasaccharide analogue of Lewis Y was probably not involved in its interaction with the immune system, whose discovery can have a broad impact on the design of new cancer vaccines.
Asunto(s)
Inmunoconjugados/química , Inmunoconjugados/inmunología , Antígenos del Grupo Sanguíneo de Lewis/química , Antígenos del Grupo Sanguíneo de Lewis/inmunología , Animales , Técnicas de Química Sintética , Reacciones Cruzadas , Femenino , Ratones , Ratones Endogámicos C57BL , Relación Estructura-ActividadRESUMEN
Type Ia group B Streptococcus (GBS) is one of the major causes of fatal infections in neonates. Its extracellular capsular polysaccharide (CPS) is a useful target for the development of anti-type Ia GBS vaccines. To explore the structure-activity relationships of type Ia GBS CPS and design more effective vaccines, a dimer of the branched pentasaccharide repeating unit of this CPS was synthesized by a highly convergent strategy highlighted by constructing the key intermediate via one-pot iterative glycosylation and imposing two side chains in one step via dual glycosylation. This represented the first total synthesis of a dimer of the repeating unit of any GBS CPS reported so far and the strategy should be applicable to higher oligomers of this repeating unit. The synthetic dimer and its monomeric analog were coupled with CRM197 carrier protein to generate conjugates that were evaluated in mice. Immunological results revealed that both carbohydrate antigens could induce robust total and IgG antibody responses and the elicited antibodies were cross-reactive with both carbohydrate antigens. It was concluded that both the monomeric and the dimeric repeating units may be employed as haptens for anti-type Ia GBS vaccine development.
RESUMEN
An efficient method was developed for the synthesis of a GM2 derivative suitable for the conjugation with various biomolecules. This GM2 derivative was covalently linked to keyhole limpet hemocyanin (KLH) and monophosphoryl lipid A (MPLA) to form novel therapeutic cancer vaccines. Immunological evaluations of the resultant conjugates in mice revealed that they elicited robust GM2-specific overall and IgG antibody responses. Moreover, the GM2-MPLA conjugate was disclosed to elicit strong immune responses without the use of an adjuvant, proving its self-adjuvant property. The antisera of both conjugates showed strong binding and mediated similarly effective complement-dependent cytotoxicity to GM2-expressing cancer cell line MCF-7. Based on these results, it was concluded that both GM2-MPLA and GM2-KLH are promising candidates as therapeutic cancer vaccines, whereas fully synthetic GM2-MPLA, which has homogeneous and well-defined structure and self-adjuvant property, deserves more attention and studies.
Asunto(s)
Adyuvantes Inmunológicos , Vacunas contra el Cáncer/inmunología , Gangliósido G(M2)/inmunología , Lípido A/análogos & derivados , Vacunas Sintéticas/inmunología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Vacunas contra el Cáncer/química , Proteínas del Sistema Complemento/inmunología , Modelos Animales de Enfermedad , Femenino , Gangliósido G(M2)/química , Lípido A/química , Lípido A/inmunología , Ratones , Estructura Molecular , Neoplasias/inmunología , Neoplasias/patología , Neoplasias/terapia , Vacunas Sintéticas/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A convergent strategy was developed for the first-time synthesis of biotin-labeled GPI core glycans. These GPI conjugates are useful for various biological studies showcased by their application in the scrutiny of pore-forming bacterial toxin-GPI interaction, revealing that the phosphate group at the GPI inositol 1-O-position had a significant impact on GPI-toxin binding.
Asunto(s)
Toxinas Bacterianas/metabolismo , Biotina/química , Glicosilfosfatidilinositoles/metabolismo , Anisoles/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/análisis , Biotina/metabolismo , Cerio/química , Ensayo de Inmunoadsorción Enzimática , Glicosilfosfatidilinositoles/química , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Streptococcus agalactiae/metabolismoRESUMEN
Mycothiol (MSH) is the predominant low molecular weight thiol produced by actinomycetes, and it plays a pivotal role in the bacterial detoxication process. 1L-myo-Inositol-1-phosphate (1L-Ins-1-P) α-N-acetylglucosaminyltransferase (GlcNAc-T), known as MshA, is the only glycosyltransferase involved in MSH biosynthesis. In this work, the MshA from Corynebacterium diphtheria, named as CdMshA, was expressed, purified, and studied in detail. Its enzymatic activity to transfer GlcNAc to 1L-Ins-1-P was confirmed by the isolation and rigorous characterization of its reaction product 3-phospho-1-d-myo-inositol-2-acetamido-2-deoxy-α-d-glucopyranoside. CdMshA was shown to accept only UDP-GlcNAc and 1L-Ins-1-P as its substrates among various tested glycosyl donors, such as UDP-GlcNAc, UDP-Gal, UDP-Glc, UDP-GalNAc and UDP-GlcA, and glycosyl acceptors, such as myo-inositol, 1L-Ins-1-P and 1D-Ins-1-P. The results have demonstrated the strict substrate selectivity of CdMshA. Furthermore, its reaction kinetics with UDP-GlcNAc and 1L-Ins-1-P as substrates were characterized, while site-directed mutagenesis of CdMshA disclosed that its amino acid residues N28, K81 and R157 were essential for its enzymatic activity.
Asunto(s)
Corynebacterium diphtheriae/metabolismo , Cisteína/biosíntesis , Glicopéptidos/biosíntesis , Inositol/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Dominio Catalítico , Inositol/biosíntesis , Cinética , Modelos Moleculares , Mutagénesis , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/genética , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The polysaccharide LbGp1 from Lycium barbarum L. was sulphated with sulphur trioxide-pyridine complex in DMF, yielding two sulphated polysaccharides, which were LbGp1-OL-SL with 13.7% sulphate content, and LbGp1-OL-SH with 27.4% sulphate content. The sulphation patterns were analysed using a GC-MS strategy. After a series of sequential chemical derivatisations, the sulphated polysaccharides were converted to partially methylated alditol acetates. All the sulphate groups were replaced by acetyl groups, maintaining the positional information of the original sulphation pattern. The number and position of sulphate substitutions were deduced by comparing the relative molar ratio from methylation analysis between native polysaccharide and sulphated derivatives. In LbGp1-OL-SL, 12.65% of sulphation located on C-5 of Ara, only 0.69% and 0.34% of sulphation occurred on C-4 and C-6 Gal, respectively; while in LbGp1-OL-SH, 24.96% of sulphate groups were found at C-5 of Ara, and 0.40% and 2.02% of sulphate groups were found at C-4 and C-6 Gal, respectively.