Referee 1's review (Marie-Christine Rio):

Review of the original submission:
In their manuscript Shu Bai Liu and collaborators report the identification and characterisation of a new amphibian protein complex including non-lens bg-crystallin and TFF sub-units. Their data clearly indicate that the resulting protein, bg-CAT, plays a key role in mammalian erythrocyte hemolysis and K+ efflux via membrane pore formation. Moreover, using human endothelial HUVEC cells, they show that bg-CAT favors cell migration or death depending on the concentration used. The technics used are correctly done and appropriate to the study. Several points need to be clarified.

Major comments:

-The number of assays and/or statistical data are missing for almost all experiments. Several times it is written that the results are significant (ie : pages 9 lines 19 and 23, page 10 line 1...) but without giving p values. Only one p value is indicated Figure 7. Does it mean that all the other results are not statistically significant ? p values should be added to each experiment. All experiments should be done at least in triplicate (not the case Figure 6)
In the material section add a paragraph concerning the statistical analysis of the results.

-The identified b-sub-unit corresponds to a new TFF. Indeed, to date, no TFF containing 3 TFF domains has been identified. This point should be highlighted and discussed. Possible structure of the protein ? relative organization of the 3 domains ? The primary sequence of the b-sub-unit signal peptide should be added to the figure 3. Is the b-sub-unit synthesized somewhere else in the body ? ie : gastrointestinal tract ?

- What could be the structural organization of the bg-CAT ? How the 1 a- and 2 b- sub-units might be associated ?

- Cellular function : the effect on cell migration of the a-sub-unit alone should also be tested?

-page 6 : How has been calculated the relative amounts of the 2 sub-units. This data is not clear.

-page 9, line 12 : « Qian unpublished observations » Qian is not a co-author of the present paper. Does he or she agree to mention this data?

-Since K+ efflux could be induced without complete hemolytic activity, has bg-CAT a potential interest in therapeutics ? discuss this point.

-About the oligomers. It cannot be excluded that this band results from a SDS-stable interaction between a-sub-unit and another protein (ie : a membrane receptor ?). Figure 5, lane 7 : in this control, several bands corresponding to polymerisation are seen, but they disappear when cells are treated to give only 1 band. How this can be explained ?

-Why erythrocytes from other species are resistant to bg-CAT ?

-How could be explained the drastic difference of function depending on protein concentration ? similar effects already reported for other proteins ?

-Finally, the last part concerning endocytosis is not correct. It should be further studied: cells ? Huvec ? concentration used? PicoM or nanoM ? same observations at the 2 concentrations ? co-staining with endocytic proteins ? The conclusions should be discussed taking into account the previous results showing that apoptosis might be induced. Figure 8 control ? is it after or before treatment ? legend gives data different from those included in the text concerning the localization.

Minor comments :

-page 6, line 4 : indicate the size of the purified single band
-page 8, line 7 : give informations concerning antisera : done in the laboratory, polyclonal rabbit antibodies, types, antigene targetted
-page 16 : reducing conditions ? Figure 2 : what means : native, reducing and non-reducing conditions ? to be clearly indicated in material section.
-Add addresses of Compagnies providing material : Invitrogen, Promega...
-AgNO3 technique ?
-few references are missing : ex : MTT method
-Figure 5 : indicate that the lower band in C 12-15 is an artefact.
-Figure 7 C : indicate the scales
-Figure 6 : the effect is dependent of collagen. Discuss this point. Moreover the legend is not clear. COL ? Tran ? Explain better
- There are errors in the Reference section : ie ref 10 « Hauster » at the place of « Hauser », « P-damain » for « P-domain ».....

Review of the first revised manuscript:
The manuscript entitled "A novel Non-lens betagamma-Trefoil Factor Complex from amphibian skin and its functional implications", from Shu Bai Liu and collaborators has been satisfactory revised.

Few points need to be clarified for a better understanding :
-Page 2, replace « absent in melanoma » by « Absent In Melanoma »
-Result section, page 5, Characterization of bg-CAT : add Da at all molecular weights : ex : replace « 38000 » by « 38000 Da ». Same in the discussion p11 line 1.

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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.