cell-free-production-proteoliposomes-for-functional-analysis-antibody

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug ...

Ehime University

Although the contribution of Hedgehog (Hh) signalling to stem cell development and oncogenesis is well recognised, its importance for spermatogonial stem cells (SSCs) has not been established. Here we interrogate adult rat SSCs using an established model in which only undifferentiated spermatogonial cells remain in the testis at 15 weeks following irradiation, and spermatogonial differentiation is induced within 4 weeks by gonadotrophin-releasing hormone antagonist (GnRH-ant) administration. Synthesis of Hh pathway components in untreated adult rat testes was compared with that in irradiated testes prior to and after GnRH-ant exposure using in situ hybridization. In adult testes with complete spermatogenesis, the Desert Hedgehog ligand transcript, Dhh, was detected in Sertoli cells, some spermatogonia and in spermatocytes by in situ hybridization. Spermatogenic cells were identified as sites of Hh signalling through detection of transcripts encoding the Hh receptor, Ptc2 transcripts and proteins for the key downstream target of Hh signalling, Gli1 and the Hh transcriptional activator, Gli2. Remarkably, the undifferentiated spermatogonia present in irradiated adult rat testes contained Dhh in addition to Ptc2, Gli1 and Gli2, revealing the potential for an autocrine Hh signalling loop to sustain undifferentiated spermatogonial cells. These transcripts became undetectable by in situ hybridization following GnRH-ant induction of spermatogonial differentiation, however, detection of Gli1 protein in spermatogonia in all groups indicates that Hh signalling is sustained. This is the first evidence of active Hh signalling in mammalian male germline stem cells, as has been documented for some cancer stem cells.

Dynamic Hedgehog signalling pathway activity in germline stem cells.

The mammalian epididymis is an exceptionally long ductal system tasked with the provision of one of the most complex intraluminal fluids found in any exocrine gland. This specialized milieu is continuously modified by the combined secretory and absorptive of the surrounding epithelium and thus finely tuned for its essential roles in promoting sperm maturation and storage. While considerable effort has been focused on defining the composition of the epididymal fluid, relatively less is known about the intracellular trafficking machinery that regulates this luminal environment. Here, we characterize the ontogeny of expression of a master regulator of this machinery, the dynamin family of mechanoenzymes. Our data show that canonical dynamin isoforms were abundantly expressed in the juvenile mouse epididymis. However, in peripubertal and adult animals dynamin takes on a heterogeneous pattern of expression such that the different isoforms displayed both cell- and segment-specific localization. Thus, dynamin 1 and 3 were predominately localized in the distal epididymal segments (corpus and cauda), where they were found within clear and principal cells, respectively. In contrast, dynamin 2 was expressed throughout the epididymis, but localized to the Golgi apparatus of the principal cells in the proximal (caput) segment and the luminal border of these cells in more distal segments. These dynamin isoforms are therefore ideally positioned to play complementary, nonredundant roles in the regulation of the epididymal milieu. In support of this hypothesis, selective inhibition of dynamin altered the profile of proteins secreted from an immortalized caput epididymal cell line.

Developmental expression of the dynamin family of mechanoenzymes in the mouse epididymis.

There are many strategies to purify recombinant proteins of interest, and affinity purification utilizing monoclonal antibody that targets a linear epitope sequence is one of the essential techniques used in current biochemistry and structural biology. Here we introduce a new protein purification system using a very short CP5 tag. First, we selected anti-dopamine receptor D1 (DRD1) rabbit monoclonal antibody clone Ra62 (Ra62 antibody) as capture antibody, and identified its minimal epitope sequence as a 5-amino-acid sequence at C-terminal of DRD1 (GQHPT-COOH, D1CE sequence). We found that single amino acid substitution in D1CE sequence (GQHVT-COOH) increased dissociation rate up to 10-fold, and named the designed epitope sequence CP5 tag. Using Ra62 antibody and 2 peptides with different affinity, we developed a new affinity protein purification method, CP5 system. Ra62 antibody quickly captures CP5-tagged target protein, and captured CP5-tagged protein was eluted by competing with higher affinity D1CE peptide. By taking the difference of the affinity between D1CE and CP5, sharp elution under mild condition was achieved. Using CP5 system, we successfully purified deubiquitinase CYLD and E3 ubiquitin ligase MARCH3, and detected their catalytic activity. As to G protein-coupled receptors (GPCRs), 9 out of 12 cell-free synthesized ones were purified, demonstrating its purification capability of integral membrane proteins. CP5 tagged CHRM2 expressed by baculovirus-insect cell was also successfully purified by CP5 system. CP5 system offers several distinct advantages in addition to its specificity and elution performance. CP5 tag is easy to construct and handle because of its short length, which has less effect on protein characters. Mild elution of CP5 system is particulaly suitable for preparing delicate proteins such as enzymes and membrane proteins. Our data demonstrate that CP5 system provides a new promising option in protein sample preparation with high yield, purity and activity for downstream applications in functional and structural analysis.

CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag.

Does dynamin regulate human sperm acrosomal exocytosis?

Characterization of a novel role for the dynamin mechanoenzymes in the regulation of human sperm acrosomal exocytosis.

The testicular spermatozoa of all mammalian species are considered functionally immature owing to their inability to swim in a progressive manner and engage in productive interactions with the cumulus-oocyte complex. The ability to express these key functional attributes develops progressively during the cells' descent through the epididymis, a highly specialized ductal system that forms an integral part of the male reproductive tract. The functional maturation of the spermatozoon is achieved  continuous interactions with the epididymal luminal microenvironment and remarkably, occurs in the complete absence of  gene transcription or protein translation. Compositional analysis of the luminal fluids collected from the epididymis of a variety of species has revealed the complexity of this milieu, with a diversity of inorganic ions, proteins, and small non-coding RNA transcripts having been identified to date. Notably, both the quantitative and qualitative profile of each of these different luminal elements display substantial segment-to-segment variation, which in turn contribute to the regionalized functionality of this long tubule. Thus, spermatozoa acquire functional maturity in the proximal segments before being stored in a quiescent state in the distal segment in preparation for ejaculation. Such marked division of labor is achieved  the combined secretory and absorptive activity of the epithelial cells lining each segment. Here, we review our current understanding of the molecular mechanisms that exert influence over the unique intraluminal environment of the epididymis, with a particular focus on vesicle-dependent mechanisms that facilitate intercellular communication between the epididymal soma and maturing sperm cell population.

Characteristics of the Epididymal Luminal Environment Responsible for Sperm Maturation and Storage.

The production of antibodies against the extracellular regions (ECR) of multispanning membrane proteins is notoriously difficult because of the low productivity and immunogenicity of membrane proteins due to their complex structure and highly conserved sequences among species. Here, we introduce a new method to generate ECR-binding antibodies utilizing engineered liposomal immunogen prepared using a wheat cell-free protein synthesis system. We used claudin-5 (CLDN-5) as the target antigen, which is a notoriously difficult to produce and poorly immunogenic membrane protein with two highly conserved extracellular loops. We drastically improved the productivity of CLDN-5 in the cell-free system after suppressing and normalizing mRNA GC content. To overcome its low immunogenicity, two engineered antigens were designed and synthesized as proteoliposomes: a human/mouse chimeric CLDN-5, and a CLDN-5-based artificial membrane protein consisting of symmetrically arranged ECRs. Intraperitoneal immunization of both engineered CLDN-5 ECR antigens induced ECR-binding antibodies in mice with a high success rate. We isolated five monoclonal antibodies that specifically recognized CLDN-5 ECR. Antibody clone 2B12 showed high affinity (<10 nM) and inhibited CLDN-5-containing tight junctions. These results demonstrate the effectiveness of the methods for monoclonal antibody development targeting difficult-to-produce membrane proteins such as CLDNs.

Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5.

Antibodies specifically recognizing integral membrane protein are essential tool for functional analysis, diagnosis, and therapeutics targeting membrane proteins. However, development of antibodies against membrane protein remains a big challenge, because mass production of membrane protein is difficult. Recently, we developed a highly efficient cell-free production method of proteoliposome antigen using cell-free protein synthesis method with liposome and dialysis cup. Here we introduce practical and efficient integrated procedures to produce large amount of proteoliposome antigen for anti-membrane protein antibody development.

Production of Immunizing Antigen Proteoliposome Using Cell-Free Protein Synthesis System.

Alzheimer's disease is a neurodegenerative disease characterized by the interstitial deposition of amyloid β (Aβ) plaque, which is thought to be related to chronic neuroinflammation. Aβ is known to make fibrils via oligomers from monomers. Aβ has been reported to activate the NLRP3 inflammasome in infiltrated macrophages. NLRP3, an intracellular pattern recognition receptor, has been reported to recognize numerous pathogens and/or metabolites and form complexes with adopter protein ASC to make the inflammasome, an interleukin (IL)-1β-processing platform. Although reactive oxygen species from mitochondria have been reported to be involved in the activation of the NLRP3 inflammasome in microglial cells upon the deposition of Aβ, whether Aβ directly or indirectly activates the NLRP3 inflammasome remains unclear.

Amyloid β directly interacts with NLRP3 to initiate inflammasome activation: identification of an intrinsic NLRP3 ligand in a cell-free system.

During the COVID-19 pandemic, central venous access line teams were implemented at many hospitals throughout the world to provide access for critically ill patients. The objective of this study was to describe the structure, practice patterns, and outcomes of these vascular access teams during the COVID-19 pandemic.

Managing central venous access during a health care crisis.

To explore the feasibility of an automatic machine-learning algorithm-based quality control system for the practice of diagnostic radiography, performance of a convolutional neural networks (CNN)-based algorithm for identifying radiographic (X-ray) views at different levels was examined with a retrospective, HIPAA-compliant, and IRB-approved study performed on 15,046 radiographic images acquired between 2013 and 2018 from nine clinical sites affiliated with our institution. Images were labeled according to four classification levels: level 1 (anatomy level, 25 classes), level 2 (laterality level, 41 classes), level 3 (projection level, 108 classes), and level 4 (detailed level, 143 classes). An Inception V3 model pre-trained with ImageNet dataset was trained with transfer learning to classify the image at all levels. Sensitivity and positive predictive value were reported for each class, and overall accuracy was reported for each level. Accuracy was also reported when we allowed for "reasonable errors". The overall accuracy was 0.96, 0.93, 0.90, and 0.86 at levels 1, 2, 3, and 4, respectively. Overall accuracy increased to 0.99, 0.97, 0.94, and 0.88 when "reasonable errors" were allowed. Machine learning algorithms resulted in reasonable model performance for identifying radiographic views with acceptable accuracy when "reasonable errors" were allowed. Our findings demonstrate the feasibility of building a quality-control program based on machine-learning algorithms to identify radiographic views with acceptable accuracy at lower levels, which could be applied in a clinical setting.

Generalized Radiographic View Identification with Deep Learning.

Aberrant subclavian artery (ASA) and Kommerell's diverticulum (KD) are rare vascular anomalies that may be associated with lifestyle-limiting and life-threatening complications. The aim of this study is to report contemporary outcomes after invasive treatment of ASA/KD using a large international dataset.

Contemporary outcomes after treatment of aberrant subclavian artery and Kommerell's diverticulum.

Aberrant subclavian artery (ASA) with or without Kommerell's diverticulum (KD) is a rare anatomic aortic arch anomaly that can cause dysphagia and/or life-threatening rupture. The objective of this study is to compare outcomes of ASA/KD repair in patients with a left versus right aortic arch.

International Multi-Institutional Experience with Presentation and Management of Aortic Arch Laterality in Aberrant Subclavian Artery and Kommerell's Diverticulum.

Article	Year
Dynamic Hedgehog signalling pathway activity in germline stem cells. Andrology\| PubMed ID: 24574096	2014
Developmental expression of the dynamin family of mechanoenzymes in the mouse epididymis. Biology of reproduction\| PubMed ID: 28395327	2017
CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag. PloS one\| PubMed ID: 28542437	2017
Characterization of a novel role for the dynamin mechanoenzymes in the regulation of human sperm acrosomal exocytosis. Molecular human reproduction\| PubMed ID: 29044420	2017
Characteristics of the Epididymal Luminal Environment Responsible for Sperm Maturation and Storage. Frontiers in endocrinology\| PubMed ID: 29541061	2018
Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5. Scientific reports\| PubMed ID: 29849184	2018
Production of Immunizing Antigen Proteoliposome Using Cell-Free Protein Synthesis System. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30244454	2018
Amyloid β directly interacts with NLRP3 to initiate inflammasome activation: identification of an intrinsic NLRP3 ligand in a cell-free system. Inflammation and regeneration\| PubMed ID: 30459926	2018
Managing central venous access during a health care crisis. Journal of vascular surgery\| PubMed ID: 32682063	2020
Generalized Radiographic View Identification with Deep Learning. Journal of digital imaging\| PubMed ID: 33263143	2021
Contemporary outcomes after treatment of aberrant subclavian artery and Kommerell's diverticulum. Journal of vascular surgery\| PubMed ID: 36657501	2023
International Multi-Institutional Experience with Presentation and Management of Aortic Arch Laterality in Aberrant Subclavian Artery and Kommerell's Diverticulum. Annals of vascular surgery\| PubMed ID: 37236537	2023

Wei Zhou

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