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In vitro functional characterization of bacterially expressed human fibroblast tropomyosin isoforms and their
R E Novy1, J R Sellers, L F Liu
1Department of Biological Sciences, University of Iowa, Iowa City 52242.
Abstract:
At least eight tropomyosin isoforms (hTM1, hTM2, hTM3, hTM4, hTM5, hTM5a, hTM5b, and hTMsm alpha) are expressed from four distinct genes in human fibroblasts. In order to elucidate isoform properties, we have subcloned hTM3 and hTM5 full-length cDNAs, as well as their chimeric cDNAs into the bacterial expression pET8C system. Bacterially expressed tropomyosin isoforms (called PEThTM3, PEThTM5, PEThTM5/3, and PEThTM3/5) were purified and characterized. Under optimal binding conditions, the binding of PEThTM5 isoform to F-actin was stronger than the PEThTM3 isoform. However, analysis of actin-binding by the McGhee and von Hippel equation revealed that PEThTM3 exhibits higher cooperativity in binding than PEThTM5 does. Furthermore, the chimera PEThTM5/3 which possessed the N-terminal fragment of hTM5 fused to the C-terminal fragment of hTM3 had even stronger actin binding ability. The reverse chimera PEThTM3/5 which possessed the N-terminal fragment of hTM3 fused to the C-terminal fragment of hTM5 demonstrated greatly reduced affinity to actin filaments. In addition, both chimeras had different KCl requirements for optimal binding to F-actin than their parental tropomyosins. A bacterially made C-terminal fragment of human fibroblast caldesmon (PETCaD39) and native chicken gizzard caldesmon were both able to enhance the actin-binding of these bacterially expressed tropomyosins. However, PETCaD39's enhancement of binding to F-actin was greater for PEThTM5 than PEThTM3. Under 30 mM KCl and 4 mM MgCl2, the low M(r) isoform PEThTM4 appeared to be able to amplify the actin-activated HMM ATPase activity by 4.7 fold, while the high M(r) isoform PEThTM3 stimulated the activity only 1.5 fold. The higher enhancement of ATPase activity by PEThTM5 than by PEThTM3 suggested that the low M(r) isoform hTM5 may be more involved in modulating nonmuscle cell motility than hTM3. These results further suggested that different isoforms of tropomyosin might have finite differences in their specific functions (e.g., cytoskeletal vs. motile) inside the cell.
Insights
Human tropomyosin isoforms (hTM) exhibit distinct actin-binding properties and functional roles. The hTM5 isoform shows stronger actin binding and higher ATPase activity, suggesting a greater role in cell motility compared to hTM3.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Human fibroblasts express at least eight tropomyosin isoforms (hTM1-hTM5, hTM5a, hTM5b, hTMsm alpha) from four genes.
- Understanding the specific properties and functions of these tropomyosin isoforms is crucial for elucidating their roles in cellular processes.
Purpose of the Study:
- To characterize bacterially expressed human tropomyosin isoforms (hTM3, hTM5) and their chimeras.
- To investigate the actin-binding properties and functional differences between hTM3 and hTM5 isoforms.
Main Methods:
- Subcloning of full-length hTM3 and hTM5 cDNAs and their chimeric versions into the pET8C bacterial expression system.
- Purification and characterization of bacterially expressed tropomyosin isoforms (PEThTM3, PEThTM5, PEThTM5/3, PEThTM3/5).
- Analysis of actin-binding affinity, cooperativity, and modulation by caldesmon, as well as impact on actin-activated HMM ATPase activity.
Main Results:
- PEThTM5 exhibited stronger F-actin binding than PEThTM3, while PEThTM3 showed higher binding cooperativity.
- Chimeric isoforms demonstrated altered actin-binding affinities and KCl requirements compared to parental isoforms.
- Caldesmon enhanced actin-binding, with greater effect on PEThTM5 than PEThTM3.
- The low molecular weight isoform PEThTM4 significantly amplified actin-activated HMM ATPase activity more than the high molecular weight PEThTM3.
Conclusions:
- Tropomyosin isoforms possess distinct functional properties, influencing their roles in cytoskeletal organization and cell motility.
- The hTM5 isoform appears more involved in modulating nonmuscle cell motility than hTM3.
- Differences in tropomyosin isoform function suggest specialized roles within the cell.