Involvement of transglutaminase 3 (TG3) in hair stiffness changing with age by capture of zinc.
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Presented by: Seiko Ueda
Introduction
We reported in 28th IFSCC Congress 2014 that zinc in the cuticle of hair contributes to hair stiffness, and the age-dependent decrease in zinc is one of the reasons for the decrease in hair stiffness . We also reported that zinc penetration into the aged hair restores hair stiffness. Furthermore, we analyzed the distribution and local structure of zinc by X-ray absorption fine structure (XAFS) analysis, and finally reported that zinc in the hair form a distorted tetrahedral structure with amino acids and the structure might contribute to hair stiffness.
Although it is not clear why zinc level in hair decreases with age, we speculated that there are possible zinc-binding proteins, which might also decrease with age in the cuticle of hair.
In this study, we tried to identify responsible proteins for binding zinc in the cuticle cell membrane complex (CMC). As one of the candidate proteins, we identified transglutaminase 3 (TG3), which is protein-crosslinking enzyme essential for hair formation. Then, we examined zinc-binding activity of TG3 and analyzed its distribution in hair and also age-dependent change of its expression level.
Methods
We used chemically untreated Chinese woman’s hair and treated it with formic acid for a short time to extract the proteins in the cuticle CMC, and identified proteins that bind zinc by a Q-Orbitrap mass spectrometric analysis. Next, to evaluate whether the identified proteins can bind to zinc reproducibly in vitro, we incubated the recombinant TG3 with zinc in solution. Then, free zinc was removed by a column for size-fractionation, and the amount of bound zinc in the eluted protein solution was analyzed by inductively-coupled plasma mass spectrometer.
As analysis at the cellular level, we made frozen sections of plucked hair and subjected to immunohistochemical staining to confirm the location of the TG3. Finally, we plucked several hairs from volunteers of each age group and extracted total RNA of the hair bulb cells, and analyzed the TG3 expression by quantitative-PCR (qPCR) to know whether it changes with age.
Results
Among several identified zinc-associating proteins by MS analysis, we selected TG3 as a candidate protein that bind zinc in cuticle CMC. Then, using recombinant protein, we confirmed that TG3 was capable to bind zinc notably, whereas negative control protein Enhanced Green Fluorescent Protein (EGFP) did not show any binding. Furthermore, we demonstrated the TG3 expression in hair root by immunohistochemical analysis. Finally, we showed that TG3 expression decreased with age evaluated by qPCR.
Conclusion
Based on these results, we assumed that the reduction of zinc in the hair with age is partly due to the decrease of TG3 in hair, in age-dependent manner. Our results firstly demonstrated TG3 is possible zinc-binding protein although the enzyme is known to bind calcium or magnesium. This is novel and additional role of TG3, because the enzyme plays a role for cross-linking of structural proteins in hair formation. As possible application in the future, the use of compounds that enhance the expression of TG3 will prevent from the decrease in zinc with age, thereby enables to contribute to the maintenance of hair stiffness.
We reported in 28th IFSCC Congress 2014 that zinc in the cuticle of hair contributes to hair stiffness, and the age-dependent decrease in zinc is one of the reasons for the decrease in hair stiffness . We also reported that zinc penetration into the aged hair restores hair stiffness. Furthermore, we analyzed the distribution and local structure of zinc by X-ray absorption fine structure (XAFS) analysis, and finally reported that zinc in the hair form a distorted tetrahedral structure with amino acids and the structure might contribute to hair stiffness.
Although it is not clear why zinc level in hair decreases with age, we speculated that there are possible zinc-binding proteins, which might also decrease with age in the cuticle of hair.
In this study, we tried to identify responsible proteins for binding zinc in the cuticle cell membrane complex (CMC). As one of the candidate proteins, we identified transglutaminase 3 (TG3), which is protein-crosslinking enzyme essential for hair formation. Then, we examined zinc-binding activity of TG3 and analyzed its distribution in hair and also age-dependent change of its expression level.
Methods
We used chemically untreated Chinese woman’s hair and treated it with formic acid for a short time to extract the proteins in the cuticle CMC, and identified proteins that bind zinc by a Q-Orbitrap mass spectrometric analysis. Next, to evaluate whether the identified proteins can bind to zinc reproducibly in vitro, we incubated the recombinant TG3 with zinc in solution. Then, free zinc was removed by a column for size-fractionation, and the amount of bound zinc in the eluted protein solution was analyzed by inductively-coupled plasma mass spectrometer.
As analysis at the cellular level, we made frozen sections of plucked hair and subjected to immunohistochemical staining to confirm the location of the TG3. Finally, we plucked several hairs from volunteers of each age group and extracted total RNA of the hair bulb cells, and analyzed the TG3 expression by quantitative-PCR (qPCR) to know whether it changes with age.
Results
Among several identified zinc-associating proteins by MS analysis, we selected TG3 as a candidate protein that bind zinc in cuticle CMC. Then, using recombinant protein, we confirmed that TG3 was capable to bind zinc notably, whereas negative control protein Enhanced Green Fluorescent Protein (EGFP) did not show any binding. Furthermore, we demonstrated the TG3 expression in hair root by immunohistochemical analysis. Finally, we showed that TG3 expression decreased with age evaluated by qPCR.
Conclusion
Based on these results, we assumed that the reduction of zinc in the hair with age is partly due to the decrease of TG3 in hair, in age-dependent manner. Our results firstly demonstrated TG3 is possible zinc-binding protein although the enzyme is known to bind calcium or magnesium. This is novel and additional role of TG3, because the enzyme plays a role for cross-linking of structural proteins in hair formation. As possible application in the future, the use of compounds that enhance the expression of TG3 will prevent from the decrease in zinc with age, thereby enables to contribute to the maintenance of hair stiffness.