| Abstract |
In their recent manuscript, Protective roles of highly conserved motif 1 in tardigrade cytosolic-abundant heat soluble protein in extreme environments, Kang et al. (2024) investigate the putative role of motif 1 of a cytoplasmic-abundant heat-soluble (CAHS) protein, PrCAHS 1, in desiccation protection. To do so, Kang et al. (2024) test the ability of different regions of PrCAHS 1 to confer protection to the desiccation-sensitive enzyme lactate dehydrogenase (LDH). In doing this, they compare how mixtures with different mass ratios of LDH and regions of PrCAHS 1 retain LDH activity after drying and rehydration. However, another way of comparing the protective capacity of protectants with different molecular weights is to compare their protective capacity at similar molar ratios. Since the 19-mer motif 1 of PrCAHS 1 has a molecular weight that is approximately 11 times lower than full-length PrCAHS 1, choices about how comparisons (weight vs. molar comparisons) are made could influence the outcome of the comparison. This led us to reanalyze the LDH protection data for PrCAHS 1 and motif 1 of PrCAHS 1 converting reported mass ratios to molar ratios, and performing a statistical analysis to compare LDH activity across different molar ratios (Figure 1a,b). This reanalysis confirmed the finding reported by Kang et al. that at similar molar ratios, motif 1 of PrCAHS 1 outperforms the full-length PrCAHS 1 protein in protecting LDH during drying. However, this result was surprising to us, since other full-length proteins from the CAHS family are reported to provide robust protection to LDH during drying (Biswas et al., 2024; Boothby et al., 2017; Hesgrove et al., 2021; Kc et al., 2024; Nguyen et al., 2022; Packebush et al., 2023; Piszkiewicz et al., 2019; Sanchez-Martinez et al., 2024) and do so more efficiently than their individual domains (Biswas et al., 2024; Hesgrove et al., 2021; Packebush et al., 2023). This observation raises the question of whether the protective role of CAHS motif 1 is a conserved feature among other CAHS proteins. To assess if the protective capacity of motif 1 from PrCAHS 1 is conserved among other motif 1s from different CAHS proteins, we performed a similar set of LDH protection assay to those of Kang et al. (2024), using an ortholog of PrCAHS 1, HeCAHS 8, and motif 1 derived from HeCAHS 8. Here we use HeCAHS 8 to test whether other CAHS proteins behave similarly to PrCAHS 1. We observed the opposite result obtained by Kang et al. (2024), where full-length HeCAHS 8 provided robust protection to LDH, whereas motif 1 derived from HeCAHS 8 did not (Figure 1c,d). From these results, one can infer that while motif 1 from some CAHS proteins may help retain LDH enzymatic activity during drying at a greater capacity than the full-length CAHS protein it is derived from, motif 1 sequences from some CAHS proteins do not. This observation suggests functional diversity among motif 1s from different CAHS proteins, rather than a conserved feature among all CAHS proteins. These results are in line with a recent study that found that while full-length LEA_4 proteins robustly protect LDH during drying, LEA_4 motifs do not (Kc et al., 2024; Nicholson et al., 2025). Why might we have obtained different results than were found in Kang et al (2024)? One possibility is that the motif 1s from distinct CAHS proteins, and full-length CAHS proteins in general, truly behave and function differently. In line with this possibility, is the fact that while the sequence of motif 1 from PrCAHS 1 and HeCAHS 8 are similar (Figure 1e), minor differences in sequence composition might account for the differential protection observed between these two motifs. Previous work has demonstrated that small changes in the sequence of CAHS proteins and CAHS-inspired peptides can lead to large changes in their structural ensemble (Biswas et al., 2024; Giubertoni et al., 2024), which in turn can affect the protective capacity of these mediators during desiccation (Biswas et al., 2024). Another possible source of this discrepancy is the use of different methods for measuring LDH activity. While Kang et al. (2024) use an indirect tetrazolium/formazan assay, we opted to use a method that directly assays for the presence of NADH (a primary product of LDH enzymatic activity), which is widely used in the desiccation tolerance field (Biswas et al., 2024; Boothby et al., 2017; Goyal et al., 2005; Kc et al., 2024; Nguyen et al., 2022; Piszkiewicz et al., 2019; Sanchez-Martinez et al., 2024). Although a similar methodology as Kang et al. has been utilized in a former study on LEA protein's ability to protect LDH activity during desiccation (Hatanaka et al., 2013), that does not necessarily make it the best approach for an LDH protection assay. Several issues exist with tetrazolium/formazan assays, including the propensity for formazan to precipitate at high concentration, thus causing major inconsistencies with quantification (Riss et al., 2016). The effect of such precipitation at high concentrations is that it can make a sample with high levels of LDH activity appear to actually have low levels of enzyme activity. Companies that sell these assays often mention that they include excipients to reduce precipitation; however, it is generally difficult to obtain precise information on what these excipients are and their effectiveness at preventing formazan precipitation. One possible explanation for why motif 1 of PrCAHS 1 might be more protective than full-length PrCAHS 1 suggested by Kang et al. (2024) is the ability of CAHS proteins, or their domains, to prevent the aggregation of LDH. To assess the anti-aggregation potential of CAHS proteins and their domains, Kang et al. (2024) measure the absorbance at 340 nm of solutions prepared by desiccating and then rehydrating LDH alone or with full-length or domains of PrCAHS 1. The authors first compare the absorbance of solutions of LDH alone to the absorbance of solutions of LDH + PrCAHS 1 (Figure 1f, replotted from Kang et al. 2024). They conclude that since the absorbance of the latter solution is greater than the absorbance of LDH alone, that PrCAHS 1 is causing the aggregation of LDH. The authors do mention that this increased absorbance might be due to aggregation of PrCAHS 1, but since they did not measure the absorbance of solutions of PrCAHS 1 by itself, the contribution of PrCAHS 1 aggregation to absorbance readings is impossible to assess based on their data. It is worth mentioning here that CAHS proteins are widely known to undergo a phase transition to form gels in a concentration-dependent manner, which is distinct from protein aggregation (Eicher et al., 2022; Malki et al., 2022; Sanchez-Martinez et al., 2024; Tanaka et al., 2022; Yagi-Utsumi et al., 2021). We suggest that the increase in absorbance observed by Kang et al. in their LDH + PrCAHS 1 sample is due to the gelation of PrCAHS 1 rather than aggregation of LDH or PrCAHS 1. To us, the conclusion that CAHS proteins cause LDH aggregation was surprising as CAHS proteins, as well as other desiccation-related IDPs, are thought to preserve enzyme function during drying through the prevention of aggregation, among other possible mechanisms (Chakrabortee et al., 2007; Hesgrove et al., 2021; Koubaa et al., 2019). To test whether CAHS proteins affect the aggregation of LDH, we conducted a similar set of experiments to those in Kang et al. (2024) utilizing HeCAHS 8. To assess LDH aggregation we measured the absorbance of UV-light (340 nm). We used this method since it is the same one used by Kang et al. (2024) making our results directly comparable to theirs. It is worth mentioning that size exclusion chromatography (SEC) is considered the gold standard method for studying protein aggregation (Hong et al., 2012). However, because CAHS proteins form gels (Eicher et al., 2022; Malki et al., 2022; Sanchez-Martinez et al., 2024; Tanaka et al., 2022; Yagi-Utsumi et al., 2021), SEC using these proteins is not feasible since they will clog the column in the absence of a strong denaturant, which might also perturb aggregation of LDH. We began by measuring the absorbance of solutions of LDH alone, HeCAHS 8 alone, and LDH mixed with HeCAHS 8, both before and after drying/rehydration. Dried/rehydrated samples of LDH alone showed a minimal but significant increase in absorbance after rehydration relative to hydrated samples. However, HeCAHS 8 alone and HeCAHS 8 mixed with LDH samples did not show any significant increase in absorbance upon drying-rehydration. This observation implies drying and rehydration of HeCAHS 8 does not lead to aggregation of the HeCAHS 8 itself. Moreover, the combination of HeCAHS 8 and LDH does not lead to more absorbance than HeCAHS 8 alone (Figure 1g). Finally, arithmetic addition of the absorbance value of LDH (alone) and HeCAHS 8 (alone) compared to the absorbance of HeCAHS 8 mixed with LDH (LDH + HeCAHS 8) shows a significant decrease in absorbance both in hydrated and dried state for the actual mixture (Figure 1h). If HeCAHS 8 was causing the aggregation of LDH during drying, we would expect to see a statistically similar or increased absorbance in the dried (LDH + HeCAHS 8) sample as compared to the arithmetic sum of the absorbance of LDH plus the absorbance of HeCAHS 8 alone. However, we observe a statistically significant decrease in the absorbance unit of LDH and HeCAHS 8 mixture compared to the arithmetic sum of the two individual components. This implies that, rather than causing aggregation, HeCAHS 8 helps reduce aggregation of LDH during drying (Figure 1h) and thus helps retain the enzymatic activity (Figure 1c). Using solutions composed of LDH + the motif 1 of PrCAHS 1, Kang et al. (2024) also report a decrease in absorbance relative to solutions consisting of just LDH. From this, they conclude that the motif 1 of PrCAHS 1 is preventing the aggregation of LDH. However, the authors provide no statistical analysis to assess whether this observed decrease in absorbance is significant. Using data provided by the authors, we conducted a one-way ANOVA analysis with Tukey's post-hoc test on their data (Figure 1f). This analysis revealed that there was not a statistically significant difference between the absorbance of LDH alone or LDH in the presence of motif 1. Furthermore, there was no significant difference between the absorbance of LDH with motif 1 or LDH with BSA (Figure 1f). This indicates that PrCAHS1 motif 1 is not preventing (or inducing) aggregation of LDH. This discrepancy in data interpretation emphasizes the importance of performing statistical tests to support conclusions drawn from an experiment, which unfortunately is lacking in the study by Kang et al. (2024). In conclusion, while portions of CAHS proteins, specifically the helical linker region, have previously been shown to be protective to enzymes during drying (Biswas et al., 2024; Hesgrove et al., 2021; Packebush et al., 2023), evidence for a conserved functional role of CAHS motif 1 in this process is lacking. We show that full-length HeCAHS 8 helps retain enzymatic activity of LDH, but in isolation motif 1 of HeCAHS 8 does not. Our data, alongside the observation by Kang et al. (2024), suggests a possible functional diversity in the protective role of conserved motif 1 from different proteins of CAHS family in mitigating drying-induced protein dysfunction. We would further like to caution that when analyzing IDP functionality, it is important to consider that extending functional relevance from observations on partial regions of an IDP to the full-length native protein context can be problematic. A partial fragment can take on a conformational state that may differ from its conformation in the context of a full-length native protein (Biswas et al., 2024; Das et al., 2015). This could be exacerbated under environmental conditions like desiccation which impart changes to the ensemble of IDPs (Kc et al., 2024). We acknowledge the work done by Kang et al. (2024) and hope this commentary can contribute to a fruitful discussion about the biology of tardigrades and their fascinating CAHS proteins. Sourav Biswas: Conceptualization; investigation; writing – original draft; methodology; visualization; writing – review and editing; formal analysis; data curation. Thomas C. Boothby: Conceptualization; writing – original draft; writing – review and editing. We would like to thank Dr. Chin-Ju Park (Gwangju Institute of Science and Technology) for providing raw data associated with the manuscript Kang et al. (2024) for reanalysis here. The authors declare no conflict of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. |
, Thomas C. Boothby 
