RESUMEN
Mammalian cells orchestrate signalling through interaction events on their surfaces. Proteoglycans are an intricate part of these interactions, carrying large glycosaminoglycan polysaccharides that recruit signalling molecules. Despite their importance in development, cancer and neurobiology, a relatively small number of proteoglycans have been identified. In addition to the complexity of glycan extension, biosynthetic redundancy in the first protein glycosylation step by two xylosyltransferase isoenzymes XT1 and XT2 complicates annotation of proteoglycans. Here, we develop a chemical genetic strategy that manipulates the glycan attachment site of cellular proteoglycans. By employing a tactic termed bump- and-hole engineering, we engineer the two isoenzymes XT1 and XT2 to specifically transfer a chemically modified xylose analogue to target proteins. The chemical modification contains a bioorthogonal tag, allowing the ability to visualise and profile target proteins modified by both transferases in mammalian cells. The versatility of our approach allows pinpointing glycosylation sites by tandem mass spectrometry, and exploiting the chemical handle to manufacture proteoglycans with defined GAG chains for cellular applications. Engineered XT enzymes permit a view into proteoglycan biology that is orthogonal to conventional techniques in biochemistry.
RESUMEN
This paper investigates upper-limb kinematic reaching responses during a mechanical perturbation to understand interjoint arm coordination used towards powered prosthesis control development. Common prosthesis arm controllers use electromyography sensors with data-driven models to decode muscle activation signals in controlling prosthesis joint movements. However, these control approaches produce non-natural, discrete movements with no guarantee the controller can react to unexpected disturbances during continuous task motion. Determining a continuous phase-dependent variable for measuring a human's progression during reaching can derive a time-invariant kinematic function to control the prosthesis joint in a natural, continuous manner. A perturbation experimental study was conducted across three participants in evaluating the shoulder and elbow joint kinematics to examine the existence of a phase shift during reaching. Experimental results demonstrated the effects of arm proximal-distal interjoint coordination that validated the proposed mechanical phase variable of the shoulder used in parameterizing elbow joint kinematic for reaching. This could allow for a continuous phase-based control strategy that can handle disturbances to achieve arm reaching in prosthesis control.
Asunto(s)
Miembros Artificiales , Articulación del Codo , Humanos , Brazo/fisiología , Hombro/fisiología , Articulación del Codo/fisiología , Movimiento/fisiologíaRESUMEN
We report an operationally facile protocol to prepare photoactivatable probes of the bioactive mammalian disaccharide, Man(ß1,4)GlcNAc. Using conformationally restricted mannosyl hemi-acetal donors in a one-pot chlorination, iodination and glycosylation sequence, ß-mannosides were generated in excellent diastereoselectivities and yields. Upon accessing the disaccharide, we generated the corresponding photoactivatable probes by appending a diazirine-alkyne equipped linker via a condensation reaction between a diazirine-containing linker and C-1 and C-2 derivatized mannosylamines to furnish the desired C-1 and C-2 modified Man(ß1,4)GlcNAc-based probes. This new synthetic protocol greatly simplifies the preparation of this important bioactive disaccharide to enable future work to identify its protein binding partners in cells.
RESUMEN
The presence of glycan modifications at the cell surface and other locales positions them as key regulators of cell recognition and function. However, due to the complexity of glycosylation, the annotation of which proteins bear glycan modifications, which glycan patterns are present, and which proteins are capable of binding glycans is incomplete. Inspired by activity-based protein profiling to enrich for proteins in cells based on select characteristics, these endeavors have been greatly advanced by the development of appropriate glycan-binding and glycan-based probes. Here, we provide context for these three problems and describe how the capability of molecules to interact with glycans has enabled the assignment of proteins with specific glycan modifications or of proteins that bind glycans. Furthermore, we discuss how the integration of these probes with high resolution mass spectrometry-based technologies has greatly advanced glycoscience.
RESUMEN
The surface proteome or "surfaceome" is a critical mediator of cellular biology, facilitating cell-to-cell interactions and communication with extracellular biomolecules. Constituents of the surfaceome can serve as biomarkers for changing cell states and as targets for pharmacological intervention. While some pathways of cell surface trafficking are well characterized to allow prediction of surface localization, some non-canonical trafficking mechanisms do not. Basigin (Bsg), a cell surface glycoprotein, has been shown to chaperone protein clients to the cell surface. However, understanding which proteins are served by Bsg is not always straightforward. To accelerate such identification, we applied a surfaceome proximity labeling method that is integrated with quantitative mass spectrometry-based proteomics to discern changes in the surfaceome of hepatic stellate cells that occur in response to the genetic loss of Bsg. Using this strategy, we observed that the loss of Bsg leads to corresponding reductions in the cell surface expression of monocarboxylate transporters MCT1 and MCT4. We also found that these relationships were unique to Bsg and not found in neuroplastin (Nptn), a related family member. These results establish the utility of the surfaceome proximity labeling method to determine clients of cell surface chaperone proteins.
Asunto(s)
Basigina , Glicoproteínas de Membrana , Humanos , Basigina/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Membrana Celular/metabolismo , Chaperonas Moleculares/metabolismoRESUMEN
Glycosylation is a ubiquitous post-translational modification read by glycan-binding proteins (GBP) to encode important functions, but a robust understanding of these interactions and their consequences can be challenging to uncover. Glycan-GBP interactions are transient and weak, making them difficult to capture, and glycosylation is dynamic and heterogenous, necessitating study in native cellular environments to identify endogenous ligands. Proximity labeling, an experimental innovation that labels biomolecules close to a protein of interest, has recently emerged as a powerful strategy to overcome these limitations, allowing interactors to be tagged in cells for subsequent enrichment and identification by mass spectrometry-based proteomics. We will describe this nascent technique and discuss its applications in the last five years with different GBP classes, including Siglecs, galectins, and non-human lectins.
Asunto(s)
Galectinas , Procesamiento Proteico-Postraduccional , Galectinas/química , Galectinas/metabolismo , Glicosilación , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Polisacáridos/químicaRESUMEN
Cell surface proteins (CSPs) are vital molecular mediators for cells and their extracellular environment. Thus, understanding which CSPs are displayed on cells, especially in different cell states, remains an important endeavor in cell biology. Here, we describe the integration of cell surface engineering with radical-mediated protein biotinylation to profile CSPs. This method relies on the prefunctionalization of cells with cholesterol lipid groups, followed by sortase-catalyzed conjugation with an APEX2 ascorbate peroxidase enzyme. In the presence of biotin-phenol and H2O2, APEX2 catalyzes the formation of highly reactive biotinyl radicals that covalently tag electron-rich residues within CSPs for subsequent streptavidin-based enrichment and analysis by quantitative mass spectrometry. While APEX2 is traditionally used to capture proximity-based interactomes, we envisioned using it in a "baitless" manner on cell surfaces to capture CSPs. We evaluate this strategy in light of another CSP labeling method that relies on the presence of cell surface sialic acid. Using the APEX2 strategy, we describe the CSPs found in three mammalian cell lines and compare CSPs in adherent versus three-dimensional pancreatic adenocarcinoma cells.
Asunto(s)
Adenocarcinoma , Membrana Celular , Proteínas de la Membrana , Proteómica , Animales , Humanos , Adenocarcinoma/metabolismo , Biotinilación/métodos , Membrana Celular/química , Membrana Celular/metabolismo , Peróxido de Hidrógeno/metabolismo , Mamíferos/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Neoplasias Pancreáticas/metabolismo , Estreptavidina/metabolismo , Proteómica/métodosRESUMEN
Glycoproteins bearing mucin domains serve important biological functions, yet they are understudied due to their dense glycosylation. Malaker et al. describe a new tool that will advance the capture, identification, and prediction of new members of the 'mucinome'.
Asunto(s)
Glicoproteínas , Mucinas , Mucinas/metabolismo , GlicosilaciónRESUMEN
Human milk oligosaccharides (HMOs) are a family of unconjugated soluble glycans found in human breast milk that exhibit a myriad of biological activity. While recent studies have uncovered numerous biological functions for HMOs (antimicrobial, anti-inflammatory & probiotic properties), the receptors and protein binding partners involved in these processes are not well characterized. This can be attributed largely in part to the low affinity and transient nature of soluble glycan-protein interactions, precluding the use of traditional characterization techniques to survey binding partners in live cells. Here, we present the use of synthetic photoactivatable HMO probes to capture, enrich and identify HMO protein targets in live cells using mass spectrometry-based chemoproteomics. Following initial validation studies using purified lectins, we profiled the targets of HMO probes in live mouse macrophages. Using this strategy, we mapped hundreds of HMO binding partners across multiple cellular compartments, including many known glycan-binding proteins as well as numerous proteins previously not known to bind glycans. We expect our findings to inform future investigations of the diverse roles of how HMOs may regulate protein function.
RESUMEN
Open-access databases can facilitate data sharing among researchers and provide normative data for objective clinical assessment development, robotic design, and biomechanical modeling. However, most existing databases focus on gait, balance, and hand gestures without providing elbow and shoulder kinematics that are required in activities of daily living. Furthermore, the few existing upper limb datasets include small sample sizes without consistent data collection protocols, which hinder robotic engineers' ability to design robotic devices that accommodate the general population. To address the literature gap, an open-access upper limb kinematic database was proposed. Due to the impact of COVID-19 on human research, only data from 16 participants were collected. Clinical Relevance-This provides baseline kinematics for developing objective clinical assessments and rehabilitation robots.
Asunto(s)
COVID-19 , Robótica , Actividades Cotidianas , Fenómenos Biomecánicos , Humanos , Robótica/métodos , Extremidad SuperiorRESUMEN
Proteoglycans are now well regarded as key facilitators of cell biology. Although a majority of their interactions and functions are attributed to the decorating glycosaminoglycan chains, there is a growing appreciation for the roles of the proteoglycan core protein and for considering proteoglycans as replete protein-glycan conjugates. This appreciation, seeded by early work in proteoglycan biology, is now being advanced and exalted by modern approaches in chemical glycobiology. In this review, we discuss up-and-coming methods to unearth the fine-scale architecture of proteoglycans that modulate their functions and interactions. Crucial to these efforts is the production of chemically defined materials, including semisynthetic proteoglycans and the in situ capture of interacting proteins. Together, the integration of chemical biology approaches promises to expedite the dissection of the structural heterogeneity of proteoglycans and deliver refined insight into their functions.
Asunto(s)
Glicosaminoglicanos , Proteoglicanos , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Proteoglicanos/química , Relación Estructura-ActividadRESUMEN
Proteoglycans are heterogeneous macromolecular glycoconjugates that orchestrate many important cellular processes. While much attention has focused on the poly-sulfated glycosaminoglycan chains that decorate proteoglycans, other important elements of their architecture, such as core proteins and membrane localization, have garnered less emphasis. Hence, comprehensive structure-function relationships that consider the replete proteoglycan architecture as glycoconjugates are limited. Here we present an extensive approach to study proteoglycan structure and biology by fabricating defined semisynthetic modular proteoglycans that can be tailored for cell surface display. The expression of proteoglycan core proteins with unnatural amino acids permits bioorthogonal click chemistry with functionalized glycosaminoglycans for methodical dissection of the parameters required for optimal binding and function of various proteoglycan-binding proteins. We demonstrate that these sophisticated materials can recapitulate the functions of native proteoglycan ectodomains in mouse embryonic stem cell differentiation and cancer cell spreading while permitting the analysis of the contributing architectural elements toward function.
Asunto(s)
Proteoglicanos , Animales , Membrana Celular/metabolismo , Ratones , Proteoglicanos/análisis , Proteoglicanos/metabolismoRESUMEN
Post-translational modifications (PTMs) immensely expand the diversity of the proteome. Glycosylation, among the most ubiquitous PTMs, is a dynamic and multifarious modification of proteins and lipids that generates an omnipresent foliage on the cell surface. The resulting protein glycoconjugates can serve important functions in biology. However, their vast complexity complicates the study of their structures, interactions, and functions. There is now a growing appreciation of the need to study glycans and proteins together as complete entities, as the sum of these two components can exhibit unique functions. In this review, we discuss the growing forestry toolbox to characterize the structure, interactions, and biological functions of protein glycoconjugates, as well as the potential payouts of understanding and controlling these enigmatic biomolecules.
Asunto(s)
Proteoma , Proteómica , Glicoconjugados , Glicosilación , Procesamiento Proteico-Postraduccional , Proteómica/métodosAsunto(s)
Glicómica , Polisacáridos/química , Humanos , Polisacáridos/metabolismo , Proteínas/metabolismoRESUMEN
Myogenic differentiation, the irreversible developmental process where precursor myoblast muscle stem cells become contractile myotubes, is heavily regulated by glycosylation and glycan-protein interactions at the cell surface and the extracellular matrix. The glycan-binding protein galectin-1 has been found to be a potent activator of myogenic differentiation. While it is being explored as a potential therapeutic for muscle repair, a precise understanding of its glycoprotein interactors is lacking. These gaps are due in part to the difficulties of capturing glycan-protein interactions in live cells. Here, we demonstrate the use of a proximity tagging strategy coupled with quantitative mass-spectrometry-based proteomics to capture, enrich, and identify the glycan-mediated glycoprotein interactors of galectin-1 in cultured live mouse myoblasts. Our interactome dataset can serve as a resource to aid the determination of mechanisms through which galectin-1 promotes myogenic differentiation. Moreover, it can also facilitate the determination of the physiological glycoprotein counter-receptors of galectin-1. Indeed, we identify several known and novel glycan-mediated ligands of galectin-1 as well as validate that galectin-1 binds the native CD44 glycoprotein in a glycan-mediated manner.
Asunto(s)
Galectina 1/metabolismo , Glicoproteínas/metabolismo , Animales , Biotina/análogos & derivados , Biotinilación , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Endonucleasas/química , Endonucleasas/metabolismo , Galectina 1/química , Glicómica , Glicoproteínas/química , Humanos , Ligandos , Ratones , Sondas Moleculares/química , Enzimas Multifuncionales/química , Enzimas Multifuncionales/metabolismo , Mioblastos , Fenoles/química , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Interactions between glycans and glycan-binding proteins (GBPs) consist of weak, noncovalent, and transient binding events, making them difficult to study in live cells void of a static, isolated system. Furthermore, the glycans are often presented as protein glycoconjugates, but there are limited efforts to identify these proteins. Proximity labeling permits covalent tagging of the glycoprotein interactors to query GBP in live cells. Coupled with high-resolution mass spectrometry, it facilitates determination of the proteins bearing the interacting glycans. In this method, fusion protein constructs of a GBP of interest with a peroxidase enzyme allows for in situ spatiotemporal radical-mediated tagging of interacting glycoproteins in living cells that can be enriched for identification. Using this method, the capture and study of glycan-GBP interactions no longer relies on weak, transient interactions, and results in robust capture and identification of the interactome of a GBP while preserving the native cellular environment. This protocol focuses on (1) expression and characterization of a recombinant fusion protein consisting of a peroxidase and the GBP galectin-3, (2) corresponding in situ labeling and visualization of interactors, (3) and proteomic workflow and analysis of captured proteins for robust identification using mass spectrometry. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Expression, purification, and characterization of recombinant fusion protein Alternate Protocol 1: Manual Ni-NTA purification of recombinant fusion protein Basic Protocol 2: In situ proximity labeling and evaluation by fluorescence microscopy Alternate Protocol 2: Western blot analysis of in situ proximity labeling Basic Protocol 3: Proximity labeling of cells for quantitative MS-based proteomics with tandem mass tags.
Asunto(s)
Proteínas Portadoras , Proteómica , Glicoproteínas , Espectrometría de Masas , PolisacáridosRESUMEN
At the surface of many cells is a compendium of glycoconjugates that form an interface between the cell and its surroundings; the glycocalyx. The glycocalyx serves several functions that have captivated the interest of many groups. Given its privileged residence, this meshwork of sugar-rich biomolecules is poised to transmit signals across the cellular membrane, facilitating communication with the extracellular matrix and mediating important signalling cascades. As a product of the glycan biosynthetic machinery, the glycocalyx can serve as a partial mirror that reports on the cell's glycosylation status. The glycocalyx can also serve as an information-rich barrier, withholding the entry of pathogens into the underlying plasma membrane through glycan-rich molecular messages. In this review, we provide an overview of the different approaches devised to engineer glycans at the cell surface, highlighting considerations of each, as well as illuminating the grand challenges that face the next era of 'glyco-engineers'. While we have learned much from these techniques, it is evident that much is left to be unearthed.
Asunto(s)
Ingeniería Genética/métodos , Glicocálix/fisiología , Glicoconjugados/química , Animales , Sistemas CRISPR-Cas , Química Clic , Técnicas de Inactivación de Genes , Glicocálix/química , Glicoconjugados/síntesis química , Glicoproteínas/metabolismo , Glicosilación , Glicosiltransferasas/genética , Humanos , Monosacáridos/química , Mucinas/metabolismo , Oligosacáridos/química , Polisacáridos/metabolismo , Ingeniería de Proteínas/métodos , Procesamiento Proteico-Postraduccional , ARN Interferente Pequeño/genética , Proteínas Recombinantes/metabolismo , Propiedades de SuperficieRESUMEN
Galectin-3 is a glycan-binding protein (GBP) that binds ß-galactoside glycan structures to orchestrate a variety of important biological events, including the activation of hepatic stellate cells and regulation of immune responses. While the requisite glycan epitopes needed to bind galectin-3 have long been elucidated, the cellular glycoproteins that bear these glycan signatures remain unknown. Given the importance of the three-dimensional (3D) arrangement of glycans in dictating GBP interactions, strategies that allow the identification of GBP receptors in live cells, where the native glycan presentation and glycoprotein expression are preserved, have significant advantages over static and artificial systems. Here we describe the integration of a proximity labeling method and quantitative mass spectrometry to map the glycan and glycoprotein interactors for galectin-3 in live human hepatic stellate cells and peripheral blood mononuclear cells. Understanding the identity of the glycoproteins and defining the structures of the glycans will empower efforts to design and develop selective therapeutics to mitigate galectin-3-mediated biological events.
Asunto(s)
Galectina 3/metabolismo , Polisacáridos/metabolismo , Técnicas de Cultivo de Célula , Galectina 3/fisiología , Galectinas/química , Glicoproteínas/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Polisacáridos/fisiología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/fisiología , Transducción de SeñalRESUMEN
The 2019 Faraday Discussion on the Nanolithography of Biointerfaces brought together a diverse set of interdisciplinary scientists involved in the seemingly disparate fields of materials science, nanolithography and glycoscience. The setting and format of this meeting renders the experience unique, and anyone in the audience is instantly engaged in the debate. This Faraday Discussion attracted about sixty delegates, ranging from graduate students and early career researchers to full professors. The meeting was a reflection on how far lithography techniques, tissue engineering and glycoscience have come, with the aid of scientists working at the realm of the nanoscale. True to its name, this gathering was also a discussion on what the outstanding questions in glycobiology are and how nanolithography can be appropriately applied to answer them. In this report, we will give an overview of the topics and discussions covered during the meeting and highlight the content of each session.