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1.
Chembiochem ; 24(18): e202300454, 2023 09 15.
Article in English | MEDLINE | ID: mdl-37500587

ABSTRACT

Combinations of biological macromolecules can provide researchers with precise control and unique methods for regulating, studying, and manipulating cellular processes. For instance, combining the unmatched encodability afforded by nucleic acids with the diverse functionality of proteins has transformed our approach to solving several problems in chemical biology. Despite these benefits, there remains a need for new methods to site-specifically generate conjugates between different classes of biomolecules. Here we present a fully enzymatic strategy for combining nucleic acids and proteins using SNAP-tag and RNA-TAG (transglycosylation at guanosine) technologies via a bifunctional preQ1-benzylguanine small molecule probe. We demonstrate the robust ability of this technology to assemble site-specific SNAP-tag - RNA conjugates with RNAs of varying length and use our conjugation strategy to recruit an endonuclease to an RNA of interest for targeted degradation. We foresee that combining SNAP-tag and RNA-TAG will facilitate researchers to predictably engineer novel macromolecular complexes.


Subject(s)
Proteins , RNA , Proteins/chemistry , Chemical Phenomena
2.
J Am Chem Soc ; 145(14): 8099-8106, 2023 04 12.
Article in English | MEDLINE | ID: mdl-36988146

ABSTRACT

Bacterial tRNA guanine transglycosylases (TGTs) catalyze the exchange of guanine for the 7-deazaguanine queuine precursor, prequeuosine1 (preQ1). While the native nucleic acid substrate for bacterial TGTs is the anticodon loop of queuine-cognate tRNAs, the minimum recognition sequence for the enzyme is a structured hairpin containing the target G nucleobase in a "UGU" loop motif. Previous work has established an RNA modification system, RNA-TAG, in which Escherichia coli TGT exchanges the target G on an RNA of interest for chemically modified preQ1 substrates linked to a small-molecule reporter such as biotin or a fluorophore. While extending the substrate scope of RNA transglycosylases to include DNA would enable numerous applications, it has been previously reported that TGT is incapable of modifying native DNA. Here, we demonstrate that TGT can in fact recognize and label specific DNA substrates. Through iterative testing of rationally mutated DNA hairpin sequences, we determined the minimal sequence requirements for transglycosylation of unmodified DNA by E. coli TGT. Controlling steric constraint in the DNA hairpin dramatically affects labeling efficiency, and, when optimized, can lead to near-quantitative site-specific modification. We demonstrate the utility of our newly developed DNA-TAG system by rapidly synthesizing probes for fluorescent Northern blotting of spliceosomal U6 RNA and RNA FISH visualization of the long noncoding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). The ease and convenience of the DNA-TAG system will provide researchers with a tool for accessing a wide variety of versatile and affordable modified DNA substrates.


Subject(s)
Escherichia coli , RNA , RNA/chemistry , RNA, Transfer , DNA , Guanine
3.
bioRxiv ; 2023 Jan 23.
Article in English | MEDLINE | ID: mdl-36747847

ABSTRACT

Bacterial tRNA guanine transglycosylases (TGTs) catalyze the exchange of guanine for the 7-deazaguanine queuine precursor, prequeuosine1 (preQ1). While the native nucleic acid substrate for bacterial TGTs is the anticodon loop of queuine-cognate tRNAs, the minimum recognition sequence for the enzyme is a structured hairpin containing the target G nucleobase in a "UGU" loop motif. Previous work has established an RNA modification system, RNA-TAG, in which E. coli TGT exchanges the target G on an RNA of interest for chemically modified preQ1 substrates linked to a small molecule reporter such as biotin or a fluorophore. While extending the substrate scope of RNA transglycosylases to include DNA would enable numerous applications, it has been previously reported that TGT is incapable of modifying native DNA. Here we demonstrate that TGT can in fact recognize and label specific DNA substrates. Through iterative testing of rationally mutated DNA hairpin sequences, we determined the minimal sequence requirements for transglycosylation of unmodified DNA by E. coli TGT. Controlling steric constraint in the DNA hairpin dramatically affects labeling efficiency, and, when optimized, can lead to near quantitative site-specific modification. We demonstrate the utility of our newly developed DNA-TAG system by rapidly synthesizing probes for fluorescent Northern blotting of spliceosomal U6 RNA and RNA FISH visualization of the long noncoding RNA, MALAT1. The ease and convenience of the DNA-TAG system will provide researchers with a tool for accessing a wide variety of affordable modified DNA substrates.

4.
J Biol Chem ; 295(9): 2804-2821, 2020 02 28.
Article in English | MEDLINE | ID: mdl-31964714

ABSTRACT

Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate-protein interactions. The identification of membrane heparan sulfate-binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-type lectin 14a (CLEC14A), a member of the C-type lectin family that modulates angiogenesis. We found that the C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.


Subject(s)
Cell Adhesion Molecules/metabolism , Endothelium/chemistry , Heparitin Sulfate/metabolism , Lectins, C-Type/metabolism , Membrane Proteins/metabolism , Proteomics/methods , Animals , Binding Sites , Cells, Cultured , Endothelium/cytology , Humans , Mice , Protein Binding , U937 Cells
5.
ACS Chem Neurosci ; 9(12): 3086-3093, 2018 12 19.
Article in English | MEDLINE | ID: mdl-30095249

ABSTRACT

Achieving molecular control over the formation of synaptic contacts in the nervous system can provide important insights into their regulation and can offer means for creating well-defined in vitro systems to evaluate modes of therapeutic intervention. Agrin-induced clustering of acetylcholine receptors (AChRs) at postsynaptic sites is a hallmark of the formation of the neuromuscular junction, a synapse between motoneurons and muscle cells. In addition to the cognate agrin receptor LRP4 (low-density lipoprotein receptor related protein-4), muscle cell heparan sulfate (HS) glycosaminoglycans (GAGs) have also been proposed to contribute to AChR clustering by acting as agrin co-receptors. Here, we provide direct evidence for the role of HS GAGs in agrin recruitment to the surface of myotubes, as well as their functional contributions toward AChR clustering. We also demonstrate that engineering of the myotube glycocalyx using synthetic HS GAG polymers can replace native HS structures to gain control over agrin-mediated AChR clustering.


Subject(s)
Agrin/metabolism , Glycocalyx/metabolism , Heparan Sulfate Proteoglycans/metabolism , LDL-Receptor Related Proteins/metabolism , Muscle Fibers, Skeletal/metabolism , Neuromuscular Junction/metabolism , Receptors, Cholinergic/metabolism , Animals , Cell Engineering , Cell Line , Mice
6.
ACS Chem Biol ; 13(10): 2880-2887, 2018 10 19.
Article in English | MEDLINE | ID: mdl-30157624

ABSTRACT

Cell surface glycans, such as heparan sulfate (HS), are increasingly identified as co-regulators of growth factor signaling in early embryonic development; therefore, chemical tailoring of HS activity within the cellular glycocalyx of stem cells offers an opportunity to control their differentiation. The growth factors FGF2 and BMP4 are involved in mediating the exit of murine embryonic stem cells (mESCs) from their pluripotent state and their differentiation toward mesodermal cell types, respectively. Here, we report a method for remodeling the glycocalyx of mutant Ext1-/- mESCs with defective biosynthesis of HS to drive their mesodermal differentiation in an embryoid body culture. Lipid-functionalized synthetic HS-mimetic glycopolymers with affinity for both FGF2 and BMP4 were introduced into the plasma membrane of Ext1-/- mESCs, where they acted as functional co-receptors of these growth factors and facilitated signal transduction through associated MAPK and Smad signaling pathways. We demonstrate that these materials can be employed to remodel Ext1-/- mESCs within three-dimensional embryoid body structures, providing enhanced association of BMP4 at the cell surface and driving mesodermal differentiation. As a more complete understanding of the function of HS in regulating development continues to emerge, this simple glycocalyx engineering method is poised to enable precise control over growth factor signaling activity and outcomes of differentiation in stem cells.


Subject(s)
Biomimetic Materials/pharmacology , Bone Morphogenetic Protein 4/metabolism , Cell Differentiation/physiology , Embryonic Stem Cells/metabolism , Fibroblast Growth Factor 2/metabolism , Mesoderm/metabolism , Acrylic Resins/chemical synthesis , Acrylic Resins/metabolism , Acrylic Resins/pharmacology , Biomimetic Materials/chemical synthesis , Carbohydrate Sequence , Cell Engineering/methods , Disaccharides/chemical synthesis , Disaccharides/metabolism , Disaccharides/pharmacology , Glycocalyx/metabolism , Heparitin Sulfate/chemistry , Humans , N-Acetylglucosaminyltransferases/genetics , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Protein Binding , Signal Transduction/physiology
7.
Curr Protoc Chem Biol ; 10(2): e40, 2018 06.
Article in English | MEDLINE | ID: mdl-29927116

ABSTRACT

This article describes a protocol for remodeling cells with synthetic glycoprotein and glycolipid mimetics that are functionalized with lipid anchors, allowing for cell surface display of specific glycan structures in predefined nanoscale arrangements. The complex chemical heterogeneity of glycans found on the cell surface or the glycocalyx renders analysis of the individual contributions of glycans difficult. This technique allows for the precise study of individual glycans at different regions of the glycocalyx, and may be useful for interrogating glycan interactions in infection or immunity or in stem cell differentiation. CHO-Lec2 cells are prepared as adherent monolayers and, after reaching confluence, are incubated with the glycomaterials. Synthetic glycopolymers bearing α-2,3-sialyllactose glycans are used to decorate cellular surfaces in the form of 3D multivalent ligands projecting away from the cell surface, while α-2,6-sialyllactose glycolipid conjugates are used to anchor glycans in dynamic 2D arrays proximal to the cell membrane. Following washing, mimetic incorporation and glycan display can be analyzed using lectins with specificity for α-2,3- or α-2,6-linked sialic acids. Flow cytometry data reveals that cell surface remodeling with either glycoconjugate mimetic occurs efficiently in a dose-dependent manner. Combinations of glycoconjugates can also be employed simultaneously to generate a mixed glycocalyx with tunable composition and organization. © 2018 by John Wiley & Sons, Inc.


Subject(s)
Cell Membrane/metabolism , Glycocalyx/metabolism , Polysaccharides/metabolism , Animals , CHO Cells , Cell Membrane/chemistry , Cricetulus , Flow Cytometry , Glycocalyx/chemistry , Molecular Structure , Polysaccharides/chemistry , Surface Properties
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