ABSTRACT
The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is the standard method for bioorthogonal conjugation. However, current Cu(I) catalyst formulations are toxic, hindering their use in living systems. Here we report that BTTES, a tris(triazolylmethyl)amine-based ligand for Cu(I), promotes the cycloaddition reaction rapidly in living systems without apparent toxicity. This catalyst allows, for the first time, noninvasive imaging of fucosylated glycans during zebrafish early embryogenesis. We microinjected embryos with alkyne-bearing GDP-fucose at the one-cell stage and detected the metabolically incorporated unnatural sugars using the biocompatible click chemistry. Labeled glycans could be imaged in the enveloping layer of zebrafish embryos between blastula and early larval stages. This new method paves the way for rapid, noninvasive imaging of biomolecules in living organisms.
Subject(s)
Biocompatible Materials/chemistry , Copper/chemistry , Molecular Imaging/methods , Polysaccharides/metabolism , Alkynes/chemistry , Animals , Azides/chemistry , Biocompatible Materials/toxicity , Blastula/metabolism , Catalysis , Cell Line , Cell Survival , Click Chemistry , Embryonic Development , Fucose/metabolism , Humans , Microinjections , Time Factors , Triazoles/chemistry , Zebrafish/embryologyABSTRACT
Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells by a low-pH-triggered membrane fusion reaction mediated by the viral E1 protein. E1 inserts into target membranes and refolds to a hairpin-like homotrimer containing a central core trimer and an outer layer composed of domain III and the juxtamembrane stem region. The key residues involved in mediating E1 trimerization are not well understood. We recently showed that aspartate 188 in the interface of the core trimer plays a critical role. Substitution with lysine (D188K) blocks formation of the core trimer and E1 trimerization and strongly inhibits virus fusion and infection. Here, we have isolated and characterized revertants that rescued the fusion and growth defects of D188K. These revertants included pseudorevertants containing acidic or polar neutral residues at E1 position 188 and a second-site revertant containing an E1 K176T mutation. Computational analysis using multiconformation continuum electrostatics revealed an important interaction bridging D188 of one chain with K176 of the adjacent chain in the core trimer. E1 K176 is completely conserved among the alphaviruses, and mutations of K176 to threonine (K176T) or isoleucine (K176I) produced similar fusion phenotypes as D188 mutants. Together, our data support a model in which a ring of three salt bridges formed by D188 and K176 stabilize the core trimer, a key intermediate of the alphavirus fusion protein.
Subject(s)
Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Mutation, Missense , Protein Multimerization , Semliki forest virus/physiology , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Virus Internalization , Amino Acid Sequence , Amino Acid Substitution , Animals , Cricetinae , Membrane Fusion , Membrane Glycoproteins/metabolism , Molecular Sequence Data , Semliki forest virus/chemistry , Semliki forest virus/genetics , Semliki forest virus/metabolism , Viral Envelope Proteins/metabolism , Viral Fusion Proteins/chemistry , Viral Fusion Proteins/genetics , Viral Fusion Proteins/metabolismABSTRACT
A combination of recombinant FKP and alpha-(1-->3)-fucosyltransferase allows the facile synthesis of the sialyl Lewis X tetrasaccharide glycan and its derivatives in excellent yield. In this system, the universal fucosyl donor, guanidine 5'-diphosphate-beta-L-fucose (GDP-fucose), or its analogues can be generated in situ by cofactor recycling using pyruvate kinase.
