Probing the impact of GFP tagging on Robo1-heparin interaction

Zhang, Fuming; Moniz, Heather; Walcott, Benjamin; Moremen, Kelley W.; Wang, Lianchun; Linhardt, Robert J.

doi:10.1007/s10719-014-9522-1

Cited by 11 publications

(6 citation statements)

References 43 publications

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“…16 An affinity resin was prepared by binding of a biotinylated fusion protein of human Robo1 and GFP to a streptavidin−agarose column. Recently, it was shown that GFP can bind HS, 17 and therefore an additional affinity resin for HS depletion was prepared containing biotinylated GFP alone. First, the natural HS mixture was passed through the GFP depletion column in a low-salt buffer (0.15 M NH 4 OAc, pH 7.4) to capture GFP-binding HS sequences, and the unbound flow-through and low-salt wash was collected.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1

Zong¹,

Huang²,

Condac³

et al. 2016

J. Am. Chem. Soc.

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An integrated methodology is described to establish ligand requirements for heparan sulfate (HS) binding proteins based on a workflow in which HS octasaccharides are produced by partial enzymatic degradation of natural HS followed by size exclusion purification, affinity enrichment using an immobilized HS-binding protein of interest, putative structure determination of isolated compounds by a hydrophilic interaction chromatography–high-resolution mass spectrometry platform, and chemical synthesis of well-defined HS oligosaccharides for structure–activity relationship studies. The methodology was used to establish the ligand requirements of human Roundabout receptor 1 (Robo1), which is involved in a number of developmental processes. Mass spectrometric analysis of the starting octasaccharide mixture and the Robo1-bound fraction indicated that Robo1 has a preference for a specific set of structures. Further analysis was performed by sequential permethylation, desulfation, and pertrideuteroacetylation followed by online separation and structural analysis by MS/MS. Sequences of tetrasaccharides could be deduced from the data, and by combining the compositional and sequence data, a putative octasaccharide ligand could be proposed (GlA-GlcNS6S-IdoA-GlcNS-IdoA2S-GlcNS6S-IdoA-GlcNAc6S). A modular synthetic approach was employed to prepare the target compound, and binding studies by surface plasmon resonance (SPR) confirmed it to be a high affinity ligand for Robo1. Further studies with a number of tetrasaccharides confirmed that sulfate esters at C-6 are critical for binding, whereas such functionalities at C-2 substantially reduce binding. High affinity ligands were able to reverse a reduction in endothelial cell migration induced by Slit2-Robo1 signaling.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1

Zong¹,

Huang²,

Condac³

et al. 2016

J. Am. Chem. Soc.

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“…An expression vector encoding the human roundabout homolog 1 precursor (Robo1, NP_002932, Uniprot Q9Y6N7) Ig domains 1 and 2 (residues 58–266) in the pGEn2 vector was previously described [36]. The vector encodes a fusion protein with an NH2-terminal signal sequence followed by an 8xHis tag, AviTag [37], “super-folder” GFP [38], the TEV protease recognition site [39], and the Ig1-2 domains of Robo1.…”

Section: Methodsmentioning

confidence: 99%

A Traveling Wave Ion Mobility Spectrometry (TWIMS) Study of the Robo1-Heparan Sulfate Interaction

Zhao

Yang

Thieker

et al. 2018

J. Am. Soc. Mass Spectrom.

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Roundabout 1 (Robo1) interacts with its receptor Slit to regulate axon guidance, axon branching, and dendritic development in the nervous system and to regulate morphogenesis and many cell functions in the nonneuronal tissues. This interaction is known to be critically regulated by heparan sulfate (HS). Previous studies suggest that HS is required to promote the binding of Robo1 to Slit to form the minimal signaling complex, but the molecular details and the structural requirements of HS for this interaction are still unclear. Here, we describe the application of traveling wave ion mobility spectrometry (TWIMS) to study the conformational details of the Robo1-HS interaction. The results suggest that Robo1 exists in two conformations that differ by their compactness and capability to interact with HS. The results also suggest that the highly flexible interdomain hinge region connecting the Ig1 and Ig2 domains of Robo1 plays an important functional role in promoting the Robo1-Slit interaction. Moreover, variations in the sulfation pattern and size of HS were found to affect its binding affinity and selectivity to interact with different conformations of Robo1. Both MS measurements and CIU experiments show that the Robo1-HS interaction requires the presence of a specific size and pattern of modification of HS. Furthermore, the effect of N-glycosylation on the conformation of Robo1 and its binding modes with HS is reported. Graphical Abstract ᅟ.

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“…However, seldom dissected are the plausible negative structural and functional ramifications of attaching the sizeable, ~ 27 kDa GFP moiety to the protein of interest. Indeed, in many instances, GFP-tagging has been shown to generate unstable fusion products 4 , 5 , cause aberrant protein localization 6 , promote aggregation 7 , prevent assembly 8 , and/or perturb protein function in more than subtle ways 9 – 12 . Such scrutiny has never been imposed on GFP-tagged dynamin-related protein 1 (Drp1), a long-studied yet controversial mechanoenzymatic GTPase recruited from the cytosol to the mitochondrial surface to mediate mitochondrial division 13 , 14 .…”

Section: Introductionmentioning

confidence: 99%

GFP fluorescence tagging alters dynamin-related protein 1 oligomerization dynamics and creates disassembly-refractory puncta to mediate mitochondrial fission

Montecinos-Franjola

Bauer

Mears

et al. 2020

Sci Rep

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Green fluorescent protein (GFP)-tagging is the prevalent strategy to monitor protein dynamics in living cells. However, the consequences of appending the bulky GFP moiety to the protein of interest are rarely investigated. Here, using a powerful combination of quantitative fluorescence spectroscopic and imaging techniques, we have examined the oligomerization dynamics of the GFP-tagged mitochondrial fission GTPase dynamin-related protein 1 (Drp1) both in vitro and in vivo. We find that GFP-tagged Drp1 exhibits impaired oligomerization equilibria in solution that corresponds to a greatly diminished cooperative GTPase activity in comparison to native Drp1. Consequently, GFP-tagged Drp1 constitutes aberrantly stable, GTP-resistant supramolecular assemblies both in vitro and in vivo, neither of which reflects a more dynamic native Drp1 oligomerization state. Indeed, GFP-tagged Drp1 is detected more frequently per unit length over mitochondria in Drp1-null mouse embryonic fibroblasts (MEFs) compared to wild-type (wt) MEFs, indicating that the drastically reduced GTP turnover restricts oligomer disassembly from the mitochondrial surface relative to mixed oligomers comprising native and GFP-tagged Drp1. Yet, GFP-tagged Drp1 retains the capacity to mediate membrane constriction in vitro and mitochondrial division in vivo. These findings suggest that instead of robust assembly-disassembly dynamics, persistent Drp1 higher-order oligomerization over membranes is sufficient for mitochondrial fission.

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Probing the impact of GFP tagging on Robo1-heparin interaction

Cited by 11 publications

References 43 publications

Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1

Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1

A Traveling Wave Ion Mobility Spectrometry (TWIMS) Study of the Robo1-Heparan Sulfate Interaction

GFP fluorescence tagging alters dynamin-related protein 1 oligomerization dynamics and creates disassembly-refractory puncta to mediate mitochondrial fission

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