Functional Characterization of the Venus Flytrap Domain of the Human TAS1R2 Sweet Taste Receptor

Round 1

Reviewer 1 Report

Review of ijms-1813053

Functional characterization of the Venus Flytrap domain of the human TAS1R2 sweet taste receptor by Laffitte et al.,

Sweet taste receptor binding assays have been difficult to develop, mainly due to the poor yields in producing purified and functional homomers, heterodimer or functional fragments, and the typically low affinity of ligands. Nevertheless, there have been a few manuscripts, over the last 17 years, reporting the measurement of sweet taste receptor ligand affinity using purified whole receptor, or VFT subunits, and showing a relatively good agreement with sweetness potency (Nie et al. 2005; Maitrepierre et al. 2012; Belloir et al. 2021), while other binding assays showed only partial or poor correlation between assay potency and sweetness potency (Assadi-Porter, 2018; Jeong et al. 2022). The present study is clearly of interest to scientists following the field. The expression and purification approach reported in the manuscript produce a functional protein with relatively high yields and it is the most complete pharmacological characterization of binding properties of sweet taste receptor ligands on a purified VFT domain with affinity results that are, overall, trending with sweetness potency. In addition, mutations known to impair sweet taste receptor function in cell-based assays were evaluated and their effects reproduced with the purified hT1R2-VFD. This is a significant advancement for developing higher throughput binding assays for the sweet taste receptor or to solve the structure of the VFD bound to different ligands.

My comments require only minor revisions.

1)     The authors provide an overview of similar studies performed over the years (lines 49 to 57) but what were the limitations of these other approaches? Why are their new approach superior?

2)     Introduction should end with some sort of a concluding statement.

3)     Line156: “allowed us to confirm…” should read “allowed us to determine…”

4)     Table 1 should be modified as shown below (as an example) with increasing level of sweetness potency. It is easier to assess correlation between assay and sweetness potency

5)     Include a correlation plot?

6)     Figures 5A and 5B dose response on WT are reused from Figures 4 dose-responses. Same issue with figure 6A. This should be mentioned in the figure caption.

 

 

 

 

 

 

 

 

 

Sweetener	Kd (uM)	Assay Potency Relative to Sucrose	Sweetness Potency Relative to Sucrose
Lactose	70,000	0.1	0.3
Glucose	1,500	2.8	0.6
Fructose	900	4.7	0.7
Sucrose	4200	1.0	1
Cyclamate	n.b.		30-50
Aspartame	17	257	160-220
AceK	0.9	4667	200
Saccharin	0.06	70000	300
Sucralose	7	600	600
Alitame	0.15	28000	2000
Perrilartine	n.b.		2000
Neotame	0.09	46667	11000

 

Author Response

1) The authors provide an overview of similar studies performed over the years (lines 49 to 57) but what were the limitations of these other approaches? Why are their new approach superior?

Our answer: According to referee’s remark, we added a short sentence (line 51-55) introducing the difficulties in the production of the sweet taste receptor and explaining why sweet receptor was only study by part and not on the whole receptor complex.

Our approach is close to that previously described by our laboratory for the hTAS1R3-VFT (Maitrepierre et al, 2012) with slight modifications, so we can’t tell it if this new approach is superior. However, the methodology described here for hTAS1R2 provides new opportunities to study VFT domain of the sweet taste receptor and ligand binding. We also have added this objective at the end of our introduction (line 81-83).

 

2) Introduction should end with some sort of a concluding statement.

Our answer: As requested also by reviewer 2, we added a short sentence (line 81-83) in order to clarify the contribution made by this methodology for the study of the sweet taste receptor.

3) Line 156: “allowed us to confirm…” should read “allowed us to determine…”

Our answer: As requested by referee, the sentence has been modified (line 165).

4) Table 1 should be modified as shown below (as an example) with increasing level of sweetness potency. It is easier to assess correlation between assay and sweetness potency.

Our answer: Table 1 has been changed as suggested by referee with increasing level of sweetness potency. It also includes modification request by reviewer 2 about sucrose equivalence for sweetness potency.

5) Include a correlation plot?

Our answer: We have done this plot using log of sweetness potency and log of Kd values, the correlation was about 0.86 but we think that the development of a linear adjustment model with a predictive vocation would require further studies.

6) Figures 5A and 5B dose response on WT are reused from Figures 4 dose-responses. Same issue with figure 6A. This should be mentioned in the figure caption.

Our answer: This information has been added in the figure caption of figures 5 and 6.

Author Response File: Author Response.pdf

Reviewer 2 Report

The study by Laffitte et al. describes the functional characterization of the VFT-domain of TAS1R2 heterologously expressed in E.coli.

In general the study is novel, has originality and is highly relevant for the field. The methods are mostly precisely described, and the conclusions are supported by the data provided. The experiments are suitable for addressing the research question and a clearly presented. Overall, the manuscript is well-written and structured, and relatively easy to follow.

I only have a couple of minor remarks that should be addressed before publishing:

 

1.       Introduction and Abstract: There is no clear aim of the study and a clear hypothesis stated. The last paragraph of the introduction ends with a brief summary / description of the study without any hypothesis.

2.       Introduction, l.41. This statement is not precise enough (“[…] where sugars and non-caloric sweeteners interact”). Not all sugars and non-caloric sweeteners interact exclusively with hTAS12-VFT. Especially in the case of glucose and sucrose, it has been previously shown that both compounds target TAS1R2 and TAS1R3, with different binding affinities. See also: Nie, Y., Vigues, S., Hobbs, J. R., Conn, G. L., & Munger, S. D. (2005). Distinct contributions of T1R2 and T1R3 taste receptor subunits to the detection of sweet stimuli. Current Biology, 15(21), 1948-1952.

3.       Results: l.70-77. This whole paragraph reads like another introduction, as far as I understood it correctly, this is only referring to a previous work of the authors without showing new results. If this is regarded as necessary background information, it should be provided in the introduction section.

4.       Table 1: The sweetness potency values are taken from literature. This is of course acceptable; however, it remains unclear how the sweetness potency was calculated and to what concentration of sucrose the sweetness potency is referring to. Results from different sensory studies are usually difficult to compare. The authors should provide the information to what concentration of sucrose the potency was calculated or use a sensory study that tested more compounds in one batch. Where were the sweetness potencies of perillartine and cyclamate taken from?

5.       Figure legends 5 and 6: Data are shown +/- SEM. In that case it is required to give the exact number of n for each measurement, because the SEM depends on the number of replicates and they are therefore necessary to judge the error bars. Otherwise, just simply use SD, which would also probably be more appropriate for the statement that the authors want make.

6.       The purities of the chemicals used need to be provided, as well as the ordering number of the antibodies used. Also the software version of SigmaPlot needs to be provided.

7.       The data availability statement does not seem sufficient. The raw data should be available – at least upon request.

Author Response

I only have a couple of minor remarks that should be addressed before publishing:

1. Introduction and Abstract: There is no clear aim of the study and a clear hypothesis stated. The last paragraph of the introduction ends with a brief summary / description of the study without any hypothesis.

Our answer: Following the referee’s remark, Abstract (line 17-18, 23-25) and end of the introduction (line 83-85) have been modified in order to clarify the contribution made by this methodology for the study of the sweet taste receptor.

 

2. Introduction, l.41. This statement is not precise enough (“[…] where sugars and non-caloric sweeteners interact”). Not all sugars and non-caloric sweeteners interact exclusively with hTAS12-VFT. Especially in the case of glucose and sucrose, it has been previously shown that both compounds target TAS1R2 and TAS1R3, with different binding affinities. See also: Nie, Y., Vigues, S., Hobbs, J. R., Conn, G. L., & Munger, S. D. (2005). Distinct contributions of T1R2 and T1R3 taste receptor subunits to the detection of sweet stimuli. Current Biology, 15(21), 1948-1952.

Our answer: As suggested by the referee, we have specified the contribution of TAS1R2 and TAS1R3 to the detection of sweet stimuli especially in the case of glucose and sucrose. The introduction part (line 47 to 49) has been modified and the citation Nie et al. 2005 has been cited. To be more precise, we added “glucose and sucrose” for natural sugars able to bind TAS1R3-VFT (line 47).

 

3. Results: l.70-77. This whole paragraph reads like another introduction, as far as I understood it correctly, this is only referring to a previous work of the authors without showing new results. If this is regarded as necessary background information, it should be provided in the introduction section.

Our answer: We agree with referee’s remark. This part has been moved to the introduction section (between line 66 and 73).

4. Table 1: The sweetness potency values are taken from literature. This is of course acceptable; however, it remains unclear how the sweetness potency was calculated and to what concentration of sucrose the sweetness potency is referring to. Results from different sensory studies are usually difficult to compare. The authors should provide the information to what concentration of sucrose the potency was calculated or use a sensory study that tested more compounds in one batch. Where were the sweetness potencies of perillartine and cyclamate taken from?

Our answer: According to the referee’s remark, we added an asterisk to Table 1 to specify that sweetness potency is described on molar basis. To clarify this point, we took most of the information on sweetness potency from the same reference (Schiffman and Galtin, 1993). This study is based on water equivalent to 2% sucrose (fructose, cyclamate, aspartame, AceK, saccharin, sucralose, alitame). Accordingly, some data were modified. Sucrose equivalence was added in table legend.

5. Figure legends 5 and 6: Data are shown +/- SEM. In that case it is required to give the exact number of n for each measurement, because the SEM depends on the number of replicates and they are therefore necessary to judge the error bars. Otherwise, just simply use SD, which would also probably be more appropriate for the statement that the authors want make.

Our answer: The experimental data calculation of mean +/- SEM and graphic presentation were proceeded as previously published by our team for cTAS1R1 or hTAS1R2 or by Nie et al., 2005. In this study, the replicates were already described in figure caption. Following the referee’s remark, we specified n=12 in the legends of Figures 5 and 6.

 

6. The purities of the chemicals used need to be provided, as well as the ordering number of the antibodies used. Also the software version of SigmaPlot needs to be provided.

Our answer: According to the referee’s remark, the precision about the purities of chemicals was specified by adding the following sentence: “with a purity greater than or equal to 98%” in paragraph 4.1 (line 394). Ordering number and manufacturer for primary and secondary antibody was added in paragraph 4.4 (line 477 and 478). Software version and description was added in paragraph 4.7 (line 521).

7. The data availability statement does not seem sufficient. The raw data should be available – at least upon request.

Our answer: According to the referee’s remark, a sentence was added in the data Availability Statement (line 545): “The datasets generated and analysed during the present study are available from the corresponding author on request.”

 

Author Response File: Author Response.pdf