Stable Production of a Tethered Recombinant Eel Luteinizing Hormone Analog with High Potency in CHO DG44 Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Summary

This manuscript reports the production of recombinant eel luteinizing hormone (LH) analog in Chinese hamster ovary (CHO) cells. The system worked well, and the produced LH successfully activated cAMP signaling, resulting in ERK phosphorylation. The authors also produced the LH mutant (LH-M) with an equine chorionic gonadotropin β-subunit carboxyl-terminal peptide (eCTPβ) region to enhance LH function. However, the obvious difference between LH-wt and LH-M was not shown and explained in the manuscript, as I pointed out below. The authors need to provide some evidence for the advantage of LH-M.

 

Comments

1.          Figures 5 and 6: In these Western blotting images, there are multiple bands per lane. The bands should be labeled with arrows or arrowheads to make it clear which band is described in the main text. For example, in Lines 275 and 292-295.

2.          Table 1: Are there statistical differences between LH-wt and LH-M in “Basal” and “Rmax” cAMP levels?

3.          Figure 8A: There is no label in the images or description in the legend that the images are Western blotting for pERK1/2.

4.          The Introduction describes “LH-M exhibiting a more potent biological activity” (Line 85), but there was no difference between LH-wt and LH-M on ERK phosphorylation (Figure 8). Although Figure 7 shows the difference between LH-wt and LH-M on cAMP responses, there was no description of the statistical difference. The conclusion of this study is still unclear, whether LH-M is more effective than LH-wt? In order to clarify the significance of the present study clear, the authors undoubtedly need to present the advances of LH-M.

Author Response

Reviewer 1

This manuscript reports the production of recombinant eel luteinizing hormone (LH) analog in Chinese hamster ovary (CHO) cells. The system worked well, and the produced LH successfully activated cAMP signaling, resulting in ERK phosphorylation. The authors also produced the LH mutant (LH-M) with an equine chorionic gonadotropin β-subunit carboxyl-terminal peptide (eCTPβ) region to enhance LH function. However, the obvious difference between LH-wt and LH-M was not shown and explained in the manuscript, as I pointed out below. The authors need to provide some evidence for the advantage of LH-M.

Comments

1. Figures 5 and 6: In these Western blotting images, there are multiple bands per lane. The bands should be labeled with arrows or arrowheads to make it clear which band is described in the main text. For example, in Lines 275 and 292-295.

→We inserted “the MW marker” in the left.

 

2. Table 1: Are there statistical differences between LH-wt and LH-M in “Basal” and “Rmax” cAMP levels?

→We inserted “the different superscript” in the Table 1.

 

3. Figure 8A: There is no label in the images or description in the legend that the images are Western blotting for pERK1/2.

→The description was in the legend content. Thus, we inserted in the title.

 

4. The Introduction describes “LH-M exhibiting a more potent biological activity” (Line 85), but there was no difference between LH-wt and LH-M on ERK phosphorylation (Figure 8). Although Figure 7 shows the difference between LH-wt and LH-M on cAMP responses, there was no description of the statistical difference. The conclusion of this study is still unclear, whether LH-M is more effective than LH-wt? In order to clarify the significance of the present study clear, the authors undoubtedly need to present the advances of LH-M.

→As reviewer’s comments, cAMP responsiveness is more potent activity than that of eel LH-wt. However, pERK1/2 activation did not any differ between eel LH-wt and eel LH-M. Thus, we suggest that in the Abstract section “had more potent activity in cAMP response” than the tethered eel LH-wt in vitro.

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

 

The present paper by M. Byambaragchaa et al. deals with the stable production in CHO cells and partial characterization of single-chain eel LH analogs encompassing or not an equine β-CTP sequence as a linker between its β and α subunits.

General comment :
The production of active eel LH in sufficient quantity is of interest in view of the management of eel reproduction. Although this paper presents interesting preliminary data, some approximations and the absence of any in vivo bioactivity measurement weaken its conclusions and claims.

Specific comments :

·       Title: it should be mentioned that this recombinant eel LH is a single-chain analog.

·       Line 18: eLH/CG instead of eCG as the same gene encodes both eLHβ and eCGβ.

·       Figure 1 and elsewhere: The denomination LH-wt is misleading as LH-wt would be heterodimeric and not single-chain.

·       Does M in LH-M stand for Monocatenary? It is also the case for LH-wt. They are both single-chain eel LH analogs encompassing or not an equine β-CTP sequence as a linker.

·       SDS-PAGEs of the two sc-eel LHs should be presented to check for their purity.

·       Figures 3, 5, and 6: a MW standard protein scale would be helpful.

·       Figures 5 and 6: What is the meaning of the two bands for the different eel LH-M?

·       The glycanase F hydrolysis seems to exert a lesser effect on LH-M than on LH-wt. It is unclear whether glycanase was used in disulfide bridge-reducing conditions.
If not, can the CTP hinder the enzyme’s access to the N-saccharides?
If yes, could the presence of the CTP (and hence O-glycosylations) disturb N-glycans’ maturation during the hormone’s synthesis in CHO cells?

·       Figure 7: it should be mentioned under the abscissa that hormone concentrations derive from ELISA measurements.

·       The last experiments dealing with ERK1/2 activation appear somewhat marginal, and their significance is not obvious.

·       The main interest of inserting the eLH/CG CTP sequence in eel LH is to increase its half-life and provide it with a longer half-life in vivo. Such an in-vivo experiment is necessary.

Author Response

Reviewer 2

The present paper by M. Byambaragchaa et al. deals with the stable production in CHO cells and partial characterization of single-chain eel LH analogs encompassing or not an equine β-CTP sequence as a linker between its β and α subunits.

General comment:
The production of active eel LH in sufficient quantity is of interest in view of the management of eel reproduction. Although this paper presents interesting preliminary data, some approximations and the absence of any in vivo bioactivity measurement weaken its conclusions and claims.

Specific comments:

• Title: it should be mentioned that this recombinant eel LH is a single-chain analog.

→We inserted “Tethered” in the title as reviewer’s comment.

• Line 18: eLH/CG instead of eCG as the same gene encodes both eLHβ and eCGβ.

→We changed “eCG” to “eLH/CG” in all contents as reviewer’s comment.

• Figure 1 and elsewhere: The denomination LH-wt is misleading as LH-wt would be heterodimeric and not single-chain.

→We changed “LH-wt” to “tethered LH-wt” in all contents as reviewer’s comment. However, we presented the meaning of the LH-wt in the Abstract in the Line20.

• Does M in LH-M stand for Monocatenary? It is also the case for LH-wt. They are both single-chain eel LH analogs encompassing or not an equine β-CTP sequence as a linker.

→We suggested that “M” in the eel LH-M means insertion of eLH/CG CTP β-subunit regions in the middle between β-subunit and a-subunit.

• SDS-PAGEs of the two sc-eel LHs should be presented to check for their purity.

→We analyzed tethered rec-LH-wt and LH-M proteins by western blotting method. The supernatant sample (20 uL) was subjected to 12% SDS-PAGE. It didn’t go through any refining process purification. Therefore, we couldn’t confirm the purity at the presented point..  

• Figures 3, 5, and 6: a MW standard protein scale would be helpful.

→We inserted “MW marker” in the Left of Fig. 3. 5. and 6.

• Figures 5 and 6: What is the meaning of the two bands for the different eel LH-M?

→We inserted “Two bands with different molecular weights are presumed to be due to the modification of the oligosaccharide chains.” in the Line 284-285.

• The glycanase F hydrolysis seems to exert a lesser effect on LH-M than on LH-wt. It is unclear whether glycanase was used in disulfide bridge-reducing conditions.
  If not, can the CTP hinder the enzyme’s access to the N-saccharides?
  If yes, could the presence of the CTP (and hence O-glycosylations) disturb N-glycans’ maturation during the hormone’s synthesis in CHO cells?

→We suggested that the molecular weight of the tethered eel LH-wt and LH-M were decreased to approximately 8 kDa and 6-8 kDa by PNGase treatment, respectively. The eLH/CG CTP β-subunit linker has only 34 amino acids. However, the increased molecular weight of LH-M indicates broadly 38-44 kDa. Thus, we insisted that the MW in the LH-M increase approximately 6-9 kDa than those of observed in LH-wt. Therefore, we suggest that LH-M partially modified an additional oligosaccharide in the O-linked glycosylation sites. In conclusion, we insist that the addition of eLH/CG CTP β-subunit linker hinder the PGNase F enzyme access.

• Figure 7: it should be mentioned under the abscissa that hormone concentrations derive from ELISA measurements.

→We don’t understand meaning of “abscissa”.

→Thus, we inserted “by increasing concentrations (0-1,500 ng/mL)“ in the Figure 7. Legend.

• The last experiments dealing with ERK1/2 activation appear somewhat marginal, and their significance is not obvious.

→Although pERK activation is not clearly differed between eel LH-wt and LH-M, we confirmed that tethered eel LH-wt and LH-M have an influence on pERK1/2 activation by eel LH/CGR-mediated signal (PKA pathway). Therefore, next we are going to determine which signal pathway factors (β-arrestin 1, 2 and GRKs) have a decisive impaction on the pERK1/2. 

• The main interest of inserting the eLH/CG CTP sequence in eel LH is to increase its half-life and provide it with a longer half-life in vivo. Such an in-vivo experiment is necessary.

→We think that the test of longer half-life in vivo should have been presented. Now, we are conducting an in vivo experiment for sex-maturation induction in male eel. 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors revised the manuscript according to the reviewer's comments.

Author Response

Reviewer 1’s comments

Open Review

Comments and Suggestions for Authors

The authors revised the manuscript according to the reviewer's comments.

→As the reviewer’s comments, we rechecked all sentences by native English expert.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have adequately answered the points I raised except for my request for an in vivo assay to check that the presence of the CTP-linker indeed enhanced the hormone's half-life and, consequently, its in-vivo potency.

I recommend that the authors point out this important issue at the end of the discussion and in their conclusion.

 

Author Response

Reviewer’s comments

 

Comments and Suggestions for Authors

The authors have adequately answered the points I raised except for my request for an in vivo assay to check that the presence of the CTP-linker indeed enhanced the hormone's half-life and, consequently, its in-vivo potency.

I recommend that the authors point out this important issue at the end of the discussion and in their conclusion.

→We inserted “Although we didn’t present in vivo results in this experiment, the addition of eLH/CG β-subunit CTP linker will probably enhance the hormone’s half-life by post-translation modification of the CTP region. In general, eel males need to inject hormones (SPE and hCG) once a week for a long time for 7-8 weeks to induce maturity. Therefore, in vivo experiments are the most important to utilize these recombinants for the induction of sex-maturation in eel males. Nevertheless, we suggest that the eCTP linker attachment contributes to the biological activity and long-acting functions of proteins in vivo for the increased molecular weight.”  In the Line 397-403.

 

→We inserted “In addition, an in vivo study on the effect of inducing sexual maturity must be presented for the tethered rec-eel LH-M. Currently, in vivo research to induce male maturity is being conducted in collaboration with a specialized research institute.” In the Line 446-449.