Cell Surface Labeling Research Articles

Ultrafast and Chemoselective Biotinylation of Living Cell Surfaces for Time-Resolved Surfaceome Analysis.

Cell surface proteins participate in many important biological processes, such as cell-to-cell interaction, signal transduction, cell adhesion, and protein transportation. In-depth study of the cell surface protein group is of great significance. Nevertheless, detection and analysis of the surfaceome remain a significant challenge due to their low abundance and hydrophobicity. Herein, we reported an ultrafast and chemoselective labeling method using our newly developed trifunctional probe, the OPA-S-S-alkyne, which labeled cell surface lysine residues, and then established a novel cell surfaceome profiling approach. According to our experimental results, the OPA-S-S-alkyne probe can react extremely fast with living cells, labeling cells in only 1 min, while traditional NHS (labeling cell surface lysine with N-hydroxysuccinimide ester probe) and CSC (labeling cell surface glycan with hydrazide biotin probe) methods normally take longer time of more than 30 min and 1 h, respectively. Taking advantage of this ultrafast property of the method, we highlight the utility of this method by exploring the temporal dynamic changes of surfaceome upon EGF stimulation in living Hela cells and reported "early" and "late" EGF-regulated cell surface proteins, which are difficult to be distinguished by the current cell surface profiling approaches.

Individual cell modification with cell surface specific atom transfer radical polymerization for enhanced Cr(VI) removal

Modifying cells with polymers on the surface can enable them to gain or enhance function with various applications, wherein the atom transfer radical polymerization (ATRP) has garnered significant potential due to its biocompatibility. However, specifically initiating ATRP from the cell surface for in-situ modification remains challenging. This study established a bacterial surface-initiated ATRP method and further applied it for enhanced Cr(VI) removal. The cell surface specificity was facilely achieved by cell surface labelling with azide substrates, following alkynyl ATRP initiator specifically anchoring with azide-alkyne click chemistry. Then, the ATRP polymerization was initiated from the cell surface, and different polymers were successfully applied to in-situ modification. Further analysis revealed that the modification of Shewanella oneidensis with poly (4-vinyl pyridine) and sodium polymethacrylate improved the heavy metal tolerance and enhanced the Cr(VI) removal rate of 2.6 times from 0.088h-1 to 0.314h-1. This work provided a novel idea for bacterial surface modification and would extend the application of ATRP in bioremediation.

First-in-human infection imaging with 89Zr-labelled leukocytes and comparison of scan quality with [99mTc]Tc-HMPAO-labelled leukocytes.

Nuclear medicine infection imaging is routinely performed with the use of leukocytes radiolabelled with technetium-99m hexamethylpropyleneamine oxime ([99mTc]Tc-HMPAO) and single-photon emission computed tomography (SPECT). Positron emission tomography (PET) is more sensitive than SPECT and results in higher-quality images. Zirconium-89 (89Zr) is a positron emitter with a half-life of 78.4 h, which translates to the biological half-life and slow biodistribution of intact cells and allows delayed PET imaging for more accurate biodistribution of the labelled leukocytes to infection foci. A first-in-human study with [89Zr]Zr-oxine-leukocytes in four healthy volunteers was reported in 2022. Our first-in-human study utilising the cell surface labelling approach aimed to image infection in patients with the use of 89Zr-labelled leukocytes, using p-isothiocyanatobenzyl-desferrioxamine B (Df-Bz-NCS) as a bifunctional chelating agent, and to compare the scan quality and biodistribution of [89Zr]Zr-Df-Bz-NCS-labelled leukocytes on PET images to SPECT images obtained with [99mTc]Tc-HMPAO-labelled leukocytes. Leukocytes were isolated from whole-blood samples of eight patients with clinically and/or radiologically confirmed infection. Isolated leukocytes were labelled with [99mTc]Tc-HMPAO according to standardised methods, and [89Zr]Zr-Df-Bz-NCS according to our previously published radiolabelling method. Whole-body SPECT imaging was performed 2 and 18 h post injection of [99mTc]Tc-HMPAO-labelled leukocytes, and whole-body PET/CT was performed 3 and 24 h post injection of [89Zr]Zr-Df-Bz-NCS-labelled leukocytes in seven patients. Successful [89Zr]Zr-Df-Bz-NCS-leukocyte labelling was achieved. High labelling efficiencies were obtained (81.7% ± 3.6%; n = 8). A mean high viability of [89Zr]Zr-Df-Bz-NCS-labelled leukocytes was observed (88.98% ± 12.51%). The [89Zr]Zr-Df-Bz-NCS-leukocyte labelling efficiency was not significantly affected by the white blood cell count of the patient. The performance of [99mTc]Tc-HMPAO- and [89Zr]Zr-Df-Bz-NCS-labelled leukocytes, in terms of the ability to accurately detect infection, were similar in two out of seven patients, and [99mTc]Tc-HMPAO-labelled leukocytes outperformed [89Zr]Zr-Df-Bz-NCS-labelled leukocytes in one patient with femoral osteomyelitis. However, in two cases of pulmonary pathology, [89Zr]Zr-Df-Bz-NCS-labelled leukocytes demonstrated improved pathological uptake. No skeletal activity was observed in any of the patients imaged with [89Zr]Zr-Df-Bz-NCS-labelled leukocytes, illustrating the in vivo stability of the radiolabel. Although the [89Zr]Zr-Df-Bz-NCS-leukocyte labelling aspect of this study was noteworthy, infection imaging did not yield convincingly positive results due to the pulmonary trapping of intravenously administered [89Zr]Zr-Df-Bz-NCS-labelled leukocytes.

Structural and genetic basis for the binding of a mouse monoclonal antibody to Flavobacterium psychrophilum lipopolysaccharide.

A mouse monoclonal antibody (mAb FL100A) previously prepared against Flavobacterium psychrophilum (Fp) CSF259-93 has now been examined for binding to lipopolysaccharides (LPS) of this strain and Fp 950106-1/1. The corresponding O-polysaccharides (O-PS) of these strains are formed by identical trisaccharide repeats composed of l-Rhamnose (l-Rha), 2-acetamido-2-deoxy-l-fucose (l-FucNAc) and 2-acetamido-4-R1-2,4-dideoxy-d-quinovose (d-Qui2NAc4NR1) where R1 represents a dihydroxyhexanamido moiety. The O-PS loci of these strains are also identical except for the gene (wzy1 or wzy2) that encodes the polysaccharide polymerase. Accordingly, adjacent O-PS repeats are joined through d-Qui2NAc4NR1 and l-Rha by wzy2-dependent α(1-2) linkages in Fp CSF259-93 versus wzy1-dependent β(1-3) linkages in Fp 950106-1/1. mAb FL100A reacted strongly with Fp CSF259-93 O-PS and LPS but weakly or not at all with Fp 950106-1/1 LPS and O-PS. Importantly, it also labelled cell surface blebs on the former but not the latter strain. Additionally, mAb binding was approximately 5-times stronger to homologous Fp CSF259-93 LPS than to LPS from a strain with a different R-group gene. A conformational epitope for mAb FL100A binding was suggested from molecular dynamic simulations of each O-PS. Thus, Fp CSF259-93 O-PS formed a stable well-defined compact helix in which the R1 groups were displayed in a regular pattern on the helix exterior while unreactive Fp 950106-1/1 O-PS adopted a flexible extended linear conformation. Taken together, the findings establish the specificity of mAb FL100A for Wzy2-linked F. psychrophilum O-PS and LPS.

Podocyte glycocalyx plays an essential role in the function of GFB and the development of albumin leakage

The role of the cell surface layer, glycocalyx is emerging in various areas of cell biology including signaling, metabolism, and immune response. Although a decrease of podocyte glycocalyx depth in glomerular pathologies has been reported before, the specific role of specialized glycocalyx between the podocyte foot processes and the glomerular basement membrane (GBM) in the function and dysfunction of the glomerular filtration barrier (GFB) is elusive. Here we aimed to develop podocyte or endothelial glycocalyx targeting strategies to gain mechanistic understanding of the specific roles of the glycocalyx in the function of GFB and in the development of albumin leakage. Our overall hypothesis is that the specialized podocyte glycocalyx forms an important additional layer of the GFB and changes in the structure and composition of podocyte glycocalyx leads to GFB dysfunction and the development of albumin leakage. New genetic mouse models were generated by the knock-in of pCAG-Lox-STOP-Lox-EGFP-Sdc1 to generate animals with Cre-dependent expression of a syndecan-1 (Sdc1) fusion protein with N-terminal, extracellular GFP. Sdc1-GFPflox animals were crossed with either Podocin-Cre or Cdh5-Cre mice for the specific genetic labeling of the glycocalyx in podocytes or endothelial cells, respectively. Intravital multiphoton microscopy (MPM) was used to measure changes in the depth of glycocalyx and corresponding changes in GFB function in vivo over time. Endoglycosidase enzymes, such as hyaluronidase (50U/animal), heparinase (0.7 U/mouse), and chondroitinase (1 U/mouse) were used to acutely modify the composition of glycocalyx. Alexa-680 conjugated wheat germ agglutinin (WGA) lectin was used to label the glycosaminoglycan (GAG) component of the glycocalyx; Alexa-594 conjugated albumin was used to visualize GFB permeability. Classic phenotyping of Pod and Cdh5 Sdc1-GFP animals showed no significant difference in terms of systolic blood pressure, glomerular filtration rate, body and kidney weight between the Sdc1-GFP and wild type littermates. The co-localization of the genetic GFP and anti-podocin immunofluorescence co-staining confirmed the podocyte-specific expression of Sdc1-GFP. In vivo MPM imaging of Pod-Sdc1-GFP mouse kidneys revealed intense linear labeling of the podocyte cell surface including the apical and basolateral surfaces of the cell body as well as the surfaces of primary, secondary, and foot processes. Interestingly, the thickness and fluorescence intensity of Sdc1-GFP labeled glycocalyx was greater on the basal surface of the podocyte at the GBM as compared to the apical surface implying the role of glycocalyx in the function of GFB. In addition, labeling of the basolateral surface of the podocyte cell body and podocyte foot processes enabled the visualization of sub-podocyte space. Acute injection of endoglycosidase enzymes significantly (3-fold) reduced Sdc1-GFP fluorescence intensity and the thickness of the linear labeling suggesting the acute shedding of podocyte glycocalyx. Importantly, increased albumin permeability of the GFB was observed simultaneously with the shedding of podocyte glycocalyx, indicating the disruption of GFB charge and size-selective functions. In conclusion, the specialized podocyte glycocalyx at the GBM may function as an important new layer of the GFB and may play an important role in the disruption of GFB functions in disease. ASN Carl W. Gottschalk Research Scholar Grant University of Southern California Dean's Pilot Project Award DK123564. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Leucine Zipper Downregulated in Cancer-1 Interacts with Clathrin Adaptors to Control Epidermal Growth Factor Receptor (EGFR) Internalization and Gefitinib Response in EGFR-Mutated Non-Small Cell Lung Cancer.

The epidermal growth factor receptor (EGFR) is a common driver of non-small cell lung cancer (NSCLC). Clathrin-mediated internalization (CMI) sustains EGFR signaling. AXL is associated with resistance to EGFR-tyrosine kinase inhibitors (TKIs) in EGFR-mutated (EGFRM) NSCLC. We investigated the effects of Leucine zipper downregulated in cancer-1 (LDOC1) on EGFR CMI and NSCLC treatment. Coimmunoprecipitation, double immunofluorescence staining, confocal microscopy analysis, cell surface labelling assays, and immunohistochemistry studies were conducted. We revealed that LDOC1 interacts with clathrin adaptors through binding motifs. LDOC1 depletion promotes internalization and plasma membrane recycling of EGFR in EGFRM NSCLC PC9 and HCC827 cells. Membranous and cytoplasmic EGFR decreased and increased, respectively, in LDOC1 (-) NSCLC tumors. LDOC1 depletion enhanced and sustained activation of EGFR, AXL, and HER2 and enhanced activation of HER3 in PC9 and HCC827 cells. Sensitivity to first-generation EGFR-TKIs (gefitinib and erlotinib) was significantly reduced in LDOC1-depleted PC9 and HCC827 cells. Moreover, LDOC1 downregulation was significantly associated (p < 0.001) with poor overall survival in patients with EGFRM NSCLC receiving gefitinib (n = 100). In conclusion, LDOC1 may regulate the efficacy of first-generation EGFR-TKIs by participating in the CMI of EGFR. Accordingly, LDOC1 may function as a prognostic biomarker for EGFRM NSCLC.

Title: MLLT1 Is Required to Maintain Normal B-Lineage Hematopoiesis and Mitochondrial Function

MLLT1 (aka ENL) is an epigenetic reader protein that binds to crotonylated/acetylated lysine residues of histone 3 through its N-terminal YEATS domain. Multiple different positive and negative regulatory complexes compete for binding to the MLLT1 C-terminal domain. MLLT1 has been implicated in leukemic stem cell survival in MLL-rearranged cell lines but its role in normal hematopoiesis has not been investigated. Mllt1 is expressed in multiple bone marrow cell subsets, with high expression levels in multipotent and B-lineage progenitors. We generated conditional Mllt1 knockout ( Mllt1 del) mice and observed a B-lineage defect in bone marrow of Mllt1 deleted mice compared to control Mllt1 fl/fl mice. Mitochondrial activity has been linked to hematopoietic lineage fate decision choices. We performed seahorse mitochondrial stress test on lineage negative (stem and progenitor) bone marrow cells and found that lineage negative cells of Mllt1 del mice have lower mitochondrial activity (as a measure of oxygen consumption rate) than control Mllt1 fl/fl mice. In addition, we observed mitochondrial mass (as a quantitative measure of ratio of mtDNA gene CytB / nDNA gene ActB) to be decreased in lineage negative cells of Mllt1 del mice when compared to control Mllt1 fl/fl mice. Surface marker labeling was used to sort more defined subpopulations from bone marrow of Mllt1 del and Mllt1 fl/fl mice. We observed decreased mitochondrial mass in MPP4s and MEPs of Mllt1 del mice when compared to control Mllt1 fl/fl mice. Flow cytometry-based cell surface labeling to distinguish different bone marrow progenitors, plus mitochondria staining using MitoTracker Green (for mitochondrial mass) and MitoTracker Red CMXRos (for mitochondrial membrane potential) showed altered mitochondrial function of B-lineage progenitors in Mllt1 del mice when compared to control Mllt1 fl/fl mice. Thus, Mllt1 is essential for normal mitochondrial function, which may be necessary to maintain specific bone marrow hematopoietic subpopulations. Ongoing studies include gene expression profiling of sorted lymphoid progenitor populations and analysis of direct Mllt1 target genes that contribute to the altered phenotype in Mllt1 del mice.

Short-term Dendritic Dynamics of Neonatal Cortical Neurons Revealed by in vivo Imaging with Improved Spatiotemporal Resolution.

Individual neurons in sensory cortices exhibit specific receptive fields based on their dendritic patterns. These dendritic morphologies are established and refined during the neonatal period through activity-dependent plasticity. This process can be visualized using two-photon in vivo time-lapse imaging, but sufficient spatiotemporal resolution is essential. We previously examined dendritic patterning from spiny stellate (SS) neurons, the major type of layer 4 (L4) neurons, in the mouse primary somatosensory cortex (barrel cortex), where mature dendrites display a strong orientation bias toward the barrel center. Longitudinal imaging at 8-h intervals revealed the long-term dynamics by which SS neurons acquire this unique dendritic pattern. However, the spatiotemporal resolution was insufficient to detect the more rapid changes in SS neuron dendrite morphology during the critical neonatal period. In the current study, we imaged neonatal L4 neurons hourly for 8 h and improved the spatial resolution by uniform cell-surface labeling. The improved spatiotemporal resolution allowed detection of precise changes in dendrite morphology and revealed aspects of short-term dendritic dynamics unique to the neonatal period. Basal dendrites of barrel cortex L4 neurons were highly dynamic. In particular, both barrel-inner and barrel-outer dendrites (trees and branches) emerged/elongated and disappeared/retracted at similarly high frequencies, suggesting that SS neurons acquire biased dendrite patterns through rapid trial-and-error emergence, elongation, elimination, and retraction of dendritic trees and branches. We also found correlations between morphology and behavior (elongation/retraction) of dendritic tips. Thus, the current study revealed short-term dynamics and related features of cortical neuron dendrites during refinement.Significance StatementThe formation of proper dendritic patterns during early postnatal development is essential for normal neuronal circuit function in adulthood. To elucidate the mechanisms responsible for this refinement, in vivo imaging with high spatiotemporal resolution is useful. Our previous long-term in vivo imaging studies have clarified aspects of dendritic refinement mechanism; however, due to the long intervals (8-h) between image acquisitions, rapid changes in dendritic morphology were missed. Here hourly in vivo time-lapse imaging of neonatal mouse barrel cortex over 8 h revealed the rapid changes in the dendrite morphology of layer 4 neurons, thereby providing a more comprehensive record of dendritic refinement during postnatal development for mechanistic analysis.

Click-electrochemistry for the rapid labeling of virus, bacteria and cell surfaces

Methods for direct covalent ligation of microorganism surfaces remain poorly reported, and mostly based on metabolic engineering for bacteria and cells functionalization. While effective, a faster method avoiding the bio-incorporation step would be highly complementary. Here, we used N-methylluminol (NML), a fully tyrosine-selective protein anchoring group after one-electron oxidation, to label the surface of viruses, living bacteria and cells. The functionalization was performed electrochemically and in situ by applying an electric potential to aqueous buffered solutions of tagged NML containing the viruses, bacteria or cells. The broad applicability of the click-electrochemistry method was explored on recombinant adeno-associated viruses (rAAV2), Escherichia coli (Gram-) and Staphyloccocus epidermidis (Gram + ) bacterial strains, and HEK293 and HeLa eukaryotic cell lines. Surface electro-conjugation was achieved in minutes to yield functionalized rAAV2 that conserved both structural integrity and infectivity properties, and living bacteria and cell lines that were still alive and able to divide.

Chemically programmable bacterial probes for the recognition of cell surface proteins.

Common methods to label cell surface proteins (CSPs) involve the use of fluorescently modified antibodies (Abs) or small-molecule-based ligands. However, optimizing the labeling efficiency of such systems, for example, by modifying them with additional fluorophores or recognition elements, is challenging. Herein we show that effective labeling of CSPs overexpressed in cancer cells and tissues can be obtained with fluorescent probes based on chemically modified bacteria. The bacterial probes (B-probes) are generated by non-covalently linking a bacterial membrane protein to DNA duplexes appended with fluorophores and small-molecule binders of CSPs overexpressed in cancer cells. We show that B-probes are exceptionally simple to prepare and modify because they are generated from self-assembled and easily synthesized components, such as self-replicating bacterial scaffolds and DNA constructs that can be readily appended, at well-defined positions, with various types of dyes and CSP binders. This structural programmability enabled us to create B-probes that can label different types of cancer cells with distinct colors, as well as generate very bright B-probes in which the multiple dyes are spatially separated along the DNA scaffold to avoid self-quenching. This enhancement in the emission signal enabled us to label the cancer cells with greater sensitivity and follow the internalization of the B-probes into these cells. The potential to apply the design principles underlying B-probes in therapy or inhibitor screening is also discussed here.

On the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation.

Peptide asparaginyl ligases (PALs) are precision tools for peptide cyclization, cell-surface labelling, protein semisynthesis and protein conjugation. PALs are expressed as inactive proenzymes requiring low pH activation. During activation, a large portion of the cap domain of the proenzyme that covers the substrate binding site is proteolytically removed, exposing the active site to solvent and releasing a population of heterogenous active enzymes. The availability of a readily active ligase not requiring acid activation and subsequent purification of active forms would facilitate manufacturing and streamline applications. Here, we engineered the OaAEP1b-C247A hyperactive ligase via serial truncations along the linker connecting the cap and core domain of the proenzyme. The recombinant expression of the truncated constructs was carried out in Escherichia coli. Following a solubilization/refolding protocol, one truncated construct termed 'OaAEP1b-C247A-∆351' could be overexpressed in the insoluble fraction, purified, and displayed a level of ligase activity comparable to the acid-activated OaAEP1b-C247A enzyme. This constitutively active protein can be stored for up to 2 years at -80 °C and readily used for peptide cyclization and protein conjugation. We were able to express and purify a stable constitutively active asparaginyl ligase that can be stored for months without significant activity loss. The removal of the low pH proenzyme activation step eliminates the heterogeneity introduced by this procedure. The yield of purified recombinant active ligase that can be routinely obtained per 100 mL of E.coli cell culture is about 0.9 mg. This recombinant active ligase can be used to carry out protein conjugation.

"Cell Surface Capture" Workflow for Label-Free Quantification of the Cell Surface Proteome.

Over the past decade, mass spectrometry-based proteomics has enabled an in-depth characterization of biological systems across a broad array of applications. The cell surface proteome ("surfaceome") in human disease is of significant interest, as plasma membrane proteins are the primary target of most clinically approved therapeutics, as well as a key feature by which to diagnostically distinguish diseased cells from healthy tissues. However, focused characterization of membrane and surface proteins of the cell has remained challenging, primarily due to the complexity of cellular lysates, which mask proteins of interest by other high-abundance proteins. To overcome this technical barrier and accurately define the cell surface proteome of various cell types using mass spectrometry proteomics, it is necessary to enrich the cell lysate for cell surface proteins prior to analysis on the mass spectrometer. This paper presents a detailed workflow for labeling cell surface proteins from cancer cells, enriching these proteins out of the cell lysate, and subsequent sample preparation for mass spectrometry analysis.

Labeling cell surface glycosylphosphatidylinositol-anchored proteins through metabolic engineering using an azide-modified phosphatidylinositol

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.

Fluorescent dendrimer-based probes for cell membrane imaging: Zebrafish epidermal labeling-based toxicity evaluation

Visualizing the plasma membrane of living mammalian cells both in vitro and in vivo is crucial for tracking their cellular activities. However, due to the complex and dynamic nature of the plasma membrane, most commercial dyes for membrane staining can only realize very limited imaging performance. Thus, precise and stable plasma membrane imaging remains technically challenging. Here, by taking advantage of the small, well-defined, and amine-rich dendrimers, we prepared poly(ethylene glycol)-cholesterol (PEG-Chol)-conjugated and cyanine dye (e.g., cyanine2, cyanine3, and cyanine5)-labeled dendrimer nanoprobes (termed DPC-Cy2, DPC-Cy3, and DPC-Cy5 NPs). It was revealed that these probes enabled universal, wash-free, long-term (at least 8 h), and multicolor (green, yellow, and red) plasma membrane labeling of a variety of live mammalian cells. Further, we confirmed that the nanoprobes (using DPC-Cy5 as a representative) could achieve high-quality, wash-free, and stable cell surface labeling of live zebrafish embryos. More importantly, we demonstrated that our probes could act as biosensors to visualize the toxicity of metal–organic frameworks (MOFs) toward the epidermal cells of zebrafish embryos, and thus they hold great potential for identifying the toxic effect of drugs/materials at the single-cell scale or in live animals. The present work highlights the advantages of utilizing dendrimers for constructing functional imaging materials, and it is also believed that the fluorescent dendrimer nanoprobes developed in this work may find wide applications like cell imaging, drug toxicity evaluation, and cellular state monitoring.

Preparation of CD52-targeted chimeric antigen receptor-modified T cells and their anti-leukemia effects

目的构建一种靶向CD52的嵌合抗原受体T细胞（CD52 CAR-T），探索其对CD52+白血病的治疗效果。方法应用分子克隆技术构建以CD52为抗原结合区的CD52 scFv、4-1BB为共刺激分子的二代CAR-T细胞。慢病毒载体包装后感染T细胞，制备CD52 CAR-T细胞，通过流式细胞术检测CD52 CAR-T细胞CD4、CD8细胞亚群及其分化状态。通过体外杀伤实验、脱颗粒实验及细胞因子的释放等功能实验，观察CD52 CAR-T细胞对CD52+白血病细胞的特异性细胞毒作用。结果①成功构建了pCDH-CD52 scFv-CD8α-4-1BB-CD3ζ-GFP表达载体，感染人T细胞，获得靶向CD52的CAR-T细胞。②感染CD52 CAR表达载体的第6天，T细胞中表达CD52的细胞可被清除［CD52 CAR-T组CD52+ T细胞为（4.48±4.99）％，Vector-T组为（56.58±19.8）％，P＝0.011］。③CD52 CAR-T的自我杀伤不会影响T细胞的增殖，培养后期CD52 CAR-T组的增殖速度高于Vector-T组。④T细胞表型检测发现CD52 CAR可以促进CAR-T细胞向中心记忆T细胞及效应记忆T细胞分化，减少CAR-T细胞向终末效应细胞分化。⑤CD52 CAR-T细胞与HuT78-19t及MOLT4-19t靶细胞以效靶比1∶1共培养24 h，HuT78-19t细胞可被清除，而对MOLT4-19t细胞无明显杀伤作用［（2.66±1.60）％对（56.66％±5.74）％，P<0.001］。⑥脱颗粒实验显示，HuT78-19t细胞对CD52 CAR-T细胞的激活作用显著高于MOLT4-19t细胞［（57.34±11.25）％对（13.06±4.23）％，P<0.001］。⑦CD52 CAR-T与HuT78-19t细胞共培养48 h上清中细胞因子分泌水平［IFN-γ（3706±226）pg/ml、TNF-α（1732±560）pg/ml］远高于MOLT4-19t细胞组［IFN-γ（1577±846）pg/ml、TNF-α（74±12）pg/ml］（P值均<0.01）。结论该研究成功制备了具有抗白血病作用的CD52 CAR-T细胞，为进一步的靶向CD52的免疫治疗奠定了基础。

Highly selective enrichment of surface proteins from living cells by photo-crosslinking probe enabled in-depth analysis of surfaceome

Cell surface-exposed proteins (CSPs), termed the surfaceome, play a key role in many cellular processes. In-depth CSP analysis is significant for screening candidate biomarkers and drug targets. Highly selective enrichment of CSPs in physiological cellular environments is attractive but remains technically challenging. Here, we present a photocrosslinking-assisted cell surface protein enrichment (PCSPE) strategy. In this strategy, CSP labeling would be achieved within 2 min of UV irradiation by developing a new photocrosslinking probe (SDB) followed by one-step enrichment. The enrichment selectivity of CSPs reached 70.5%, and we identified up to 1017 CSPs from living HEK-293T cells, attributed to the high photocrosslinking reactivity and inherent impermeability of SDB, as well as the high cell viability maintained after rapid cell surface labeling to decrease the interference of intracellular proteins. Finally, this strategy was successfully applied to sorafenib-resistance cells for quantitative surfaceome analysis. All results demonstrated that our developed PCSPE method might provide a valuable toolkit for in-depth surfaceome profiling and comprehensive functional analysis.

Labeling Cell Surface Receptors with Ligand.BirA* Bispecifics.

BirA*, a mutant form of the biotinylating enzyme BirA, can nonspecifically biotinylate ε-amino groups on lysines of proteins. Based on the promiscuous labeling nature of BirA*, plasmids expressing fusion constructs of BirA* to a given ligand have been used to transfect eukaryotic cells, leading to the biotinylation of intracellular proteins interacting or in close proximity to such Ligand.BirA* constructs. Mass spectrometry performed on the recovered biotinylated partners allows one to map intracellular protein interactors, a technique known as BioID. In contrast, the expression and purification of recombinant Ligand.BirA* constructs could serve as a powerful tool for labeling and detecting cell surface receptors. Here, we report the design and expression of recombinant Affibody.BirA* constructs, ZEGFR:1907.BirA* and ZHER2:243.BirA*, as protein bispecifics able to biotinylate their respective receptors EGFR and HER2 on the surface of MDA-MB-231 (EGFR+, EpCaM+, and CD44+) and SK-OV-3 (HER2++, EGFR+, EpCaM+, and CD44+) cancer cells. These Affibody.BirA* constructs retain both their BirA* enzymatic activity as well as their receptor-binding function. Importantly, MDA-MB-231 and SK-OV-3 cells biotinylated with Affibody.BirA* constructs did label their receptors EGFR and HER2 but did not biotinylate irrelevant antigens such as EpCaM or CD44 present on the surface of both cell lines. Ligand.BirA* bispecifics may represent a promising class of agents to identify unknown receptors on cell surfaces.

Astrocyte Cell Surface Antigen 2 and Other Potential Cell Surface Markers of Enteric glia in the Mouse Colon.

Enteric glia regulate gut functions in health and disease through diverse interactions with neurons and immune cells. Intracellular localization of traditional markers of enteric glia such as GFAP, s100b, and Sox10 makes them incompatible for studies that require antigen localization at the cell surface. Thus, new tools are needed for probing the heterogeneous roles of enteric glia at the protein, cell, and functional levels. Here we selected several cell surface antigens including Astrocyte Cell Surface Marker 2 (ACSA2), Cluster of differentiation 9 (CD9), lysophosphatidic acid receptor 1 (LPAR1), and Proteolipid protein 1 (PLP1) as potential markers of enteric glia. We tested their specificity for enteric glia using published single-cell/-nuclei and glia-specific translating mRNA enriched transcriptome datasets, immunolabeling, and flow cytometry. The data show that ACSA2 is a specific marker of mucosal and myenteric glia while other markers are suitable for identifying all subpopulations of enteric glia (LPAR1), glia and immune cells (CD9), or are not suitable for cell-surface labeling (PLP1). These new tools will be useful for future work focused on understanding specific glial functions in health and disease. Summary Statement This study identifies astrocyte cell surface antigen 2 as a novel marker of myenteric glia in the intestine. This, in combination with other markers identified in this study, could be used for selective targeting of enteric glia.

Cell-surface Labeling via Bioorthogonal Host-Guest Chemistry.

The widespread adoption of the bioorthogonal chemical reporter strategy revolutionized chemical biology. However, its translation to living mammals has been challenging, due to the size/stability properties of the chemical reporter group and/or the reaction kinetics of the labeling step. While developing new bioorthogonal reactions has been the traditional approach to optimizing the bioorthogonal chemical reporter strategy, here we present a different avenue, leveraging intermolecular interactions, to create bioorthogonal host-guest pairs. This approach, deemed "bioorthogonal complexation, does not rely on activated functional groups or second-order rate constants. We utilize the cucurbit[7]uril (CB[7]) scaffold to showcase bioorthogonal complexation and determine that medium-affinity (Ka ≈ 108-109 M-1) guests efficiently label cell surfaces and outperform the strain-promoted azide-alkyne cycloaddition. Finally, we implement bioorthogonal complexation in the chemical reporter strategy through the metabolic incorporation of ortho-carborane into cell-surface glycans and detection with a CB[7]-fluorescein conjugate.

Sortase A: A chemoenzymatic approach for the labeling of cell surfaces.

Sortase A, a transpeptidase enzyme is present in many Gram-positive bacteria and helps in the recruitment of the cell surface proteins. Over the last two decades, Sortase A has become an attractive tool for performing in vivo and in vitro ligations. Sortase A-mediated ligation has continuously been used for its specificity, robustness, and highly efficient nature. These properties make it a popular choice among protein engineers as well as researchers from different fields. In this review, we give an overview of Sortase A-mediated ligation of various molecules on the cell surfaces, which can have diverse applications in interdisciplinary fields.