Heparan sulfate dependent phase separation of CCL5 and its chemotactic activity
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eLife assessment
How the triplicate interaction between chemokines with both GAGs and G protein-coupled receptors (GPCR) works and how gradients are created and potentially maintained in vivo are poorly understood. The authors provide solid evidence to show phase separation can drive chemotactic gradient formation. The paper is a useful advance in the field of chemokine biology.
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Abstract
Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid–liquid phase separation (LLPS) and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin binding sequence) or by addition of heparin to wild type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.
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eLife assessment
How the triplicate interaction between chemokines with both GAGs and G protein-coupled receptors (GPCR) works and how gradients are created and potentially maintained in vivo are poorly understood. The authors provide solid evidence to show phase separation can drive chemotactic gradient formation. The paper is a useful advance in the field of chemokine biology.
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Reviewer #1 (Public Review):
Summary:
In their manuscript, Yu et al. describe the chemotactic gradient formation for CCL5 bound to - i.e. released from - glycosaminoglycans. The authors provide evidence for phase separation as the driving mechanism behind chemotactic gradient formation. A conclusion towards a general principle behind the finding cannot be drawn since the work focuses on one chemokine only, which is particularly prone to glycan-induced oligomerisation.
Strengths:
The principle of phase separation as a driving force behind and thus as an analytical tool for investigating protein interactions with strongly charged biomolecules was originally introduced for protein-nucleic acid interactions. Yu et al. have applied this in their work for the first time for chemokine-heparan sulfate interactions. This opens a novel way to …
Reviewer #1 (Public Review):
Summary:
In their manuscript, Yu et al. describe the chemotactic gradient formation for CCL5 bound to - i.e. released from - glycosaminoglycans. The authors provide evidence for phase separation as the driving mechanism behind chemotactic gradient formation. A conclusion towards a general principle behind the finding cannot be drawn since the work focuses on one chemokine only, which is particularly prone to glycan-induced oligomerisation.
Strengths:
The principle of phase separation as a driving force behind and thus as an analytical tool for investigating protein interactions with strongly charged biomolecules was originally introduced for protein-nucleic acid interactions. Yu et al. have applied this in their work for the first time for chemokine-heparan sulfate interactions. This opens a novel way to investigate chemokine-glycosaminoglycan interactions in general.
Weaknesses:
As mentioned above, one of the weaknesses of the current work is the exemplification of the phase separation principle by applying it only to CCL5-heparan sulfate interactions. CCL5 is known to form higher oligomers/aggregates in the presence of glycosaminoglycans, much more than other chemokines. It would therefore have been very interesting to see, if similar results in vitro, in situ, and in vivo could have been obtained by other chemokines of the same class (e.g. CCL2) or another class (like CXCL8).
In addition, the authors have used variously labelled CCL5 (like with the organic dye Cy3 or with EGFP) for various reasons (detection and immobilisation). In the view of this reviewer, it would have been necessary to show that all the labelled chemokines yield identical/similar molecular characteristics as the unlabelled wildtype chemokine (such as heparan sulfate binding and chemotaxis). It is well known that labelling proteins either by chemical tags or by fusion to GFPs can lead to manifestly different molecular and functional characteristics.
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Reviewer #2 (Public Review):
Although the study by Xiaolin Yu et al is largely limited to in vitro data, the results of this study convincingly improve our current understanding of leukocyte migration.
(1) The conclusions of the paper are mostly supported by the data although some clarification is warranted concerning the exact CCL5 forms (without or with a fluorescent label or His-tag) and amounts/concentrations that were used in the individual experiments. This is important since it is known that modification of CCL5 at the N-terminus affects the interactions of CCL5 with the GPCRs CCR1, CCR3, and CCR5 and random labeling using monosuccinimidyl esters (as done by the authors with Cy-3) is targeting lysines. Since lysines are important for the GAG-binding properties of CCL5, knowledge of the number and location of the Cy-3 labels on …
Reviewer #2 (Public Review):
Although the study by Xiaolin Yu et al is largely limited to in vitro data, the results of this study convincingly improve our current understanding of leukocyte migration.
(1) The conclusions of the paper are mostly supported by the data although some clarification is warranted concerning the exact CCL5 forms (without or with a fluorescent label or His-tag) and amounts/concentrations that were used in the individual experiments. This is important since it is known that modification of CCL5 at the N-terminus affects the interactions of CCL5 with the GPCRs CCR1, CCR3, and CCR5 and random labeling using monosuccinimidyl esters (as done by the authors with Cy-3) is targeting lysines. Since lysines are important for the GAG-binding properties of CCL5, knowledge of the number and location of the Cy-3 labels on CCL5 is important information for the interpretation of the experimental results with the fluorescently labeled CCL5. Was the His-tag attached to the N- or C-terminus of CCL5? Indicate this for each individual experiment and consider/discuss also potential effects of the modifications on CCL5 in the results and discussion sections.
(2) In general, the authors appear to use high concentrations of CCL5 in their experiments. The reason for this is not clear. Is it because of the effects of the labels on the activity of the protein? In most biological tests (e.g. chemotaxis assays), unmodified CCL5 is active already at low nM concentrations.
(3) For the statistical analyses of the results, the authors use t-tests. Was it confirmed that data follow a normal distribution prior to using the t-test? If not a non-parametric test should be used and it may affect the conclusions of some experiments.
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