Related Experiment Videos
Domain structure, stability, and interactions of human complement C1s-: characterization of a derivative lacking most
1Biochemistry Laboratory, American Red Cross Biomedical Research and Development, Rockville, Maryland 20855.
Insights
This study identifies the A chain of C1-s as crucial for calcium-dependent assembly and interaction with other complement components. The findings clarify the structural domains of C1-s, enhancing our understanding of the complement system
Area of Science:
- Immunology
- Biochemistry
- Complement System
Background:
- Understanding the structure and function of the C1 complex is essential for elucidating the complement system's role in immunity.
- Calcium ions (Ca2+) play a critical role in the assembly and function of complement component 1 (C1).
- The subcomponent C1-s contains distinct regions (domains) responsible for Ca2+-dependent interactions.
Purpose of the Study:
- To identify the specific regions of C1-s involved in Ca2+-dependent assembly.
- To investigate the functional and structural independence of C1-s domains.
- To determine the location of Ca2+ binding sites and interaction sites within C1-s.
Main Methods:
- Limited proteolysis of C1-s with trypsin in the presence of Ca2+ to generate a functional fragment (C1-s-A).
- Fast exclusion chromatography to analyze the properties of C1-s-A.
- Differential scanning calorimetry to study thermal transitions (LTT and HTT) of C1-s and C1-s-A in the presence and absence of Ca2+.
- Reconstitution assays to assess the functional activity of C1-s-A.
Main Results:
- A 56-kDa fragment (C1-s-A), comprising the A chain and a small piece of the B chain, retained Ca2+-dependent dimerization, tetramer formation with C1-r, and association with C1q.
- C1-s-A competitively inhibited C1-s hemolytic activity, indicating its interaction domain is on the A chain.
- Thermal analysis revealed that the low-temperature transition (LTT) of C1-s is Ca2+-dependent and primarily associated with the A chain, while the high-temperature transition (HTT) is linked to the catalytic domain.
Conclusions:
- The A chain of C1-s contains the Ca2+ binding sites and is responsible for the Ca2+-dependent interactions with C1-r and C1q.
- The catalytic and interaction domains of C1-s are structurally and functionally independent.
- These findings support current models of C1 complex assembly and function within the complement cascade.
Abstract:
A better understanding of the structure and function of C1 requires knowledge of the regions (domains) of the subcomponents that are responsible for Ca2+-dependent assembly. Toward this end, C1-s was digested with trypsin in the presence of Ca2+, a treatment that rapidly degraded the B chain, leaving a 56-kDa fragment comprised of a complete A chain disulfide linked to a small (less than 4-kDa) residual piece of the B chain. The purified fragment, referred to as C1-s-A, was shown by fast exclusion chromatography to be similar to C1-s in its ability to (1) reversibly dimerize in the presence of Ca2+, (2) substitute for C1-s in the formation of C1-r2-s2 tetramers, and (3) associate with C1-r and C1q to form macromolecular C1. Although C1-s-A was itself catalytically and hemolytically inactive, it competitively inhibited the expression of the hemolytic activity of C1-s in a reconstitution assay. When heated in the absence of Ca2+, C1-s exhibited a low-temperature transition (LTT) near 31 degrees C and a high-temperature transition (HTT) near 51 degrees C, similar to those previously observed in the homologous protein C1-r [Busby, T. F., & Ingham, K. C. (1987) Biochemistry 26, 5564-5571]. The midpoint of the LTT was shifted to 58 degrees C in 5 mM Ca2+ whereas the HTT was unaffected by Ca2+. C1-s-A exhibited only a LTT whose midpoint and Ca2+ dependence were similar to those of the LTT in C1-s. The HTT, which was accompanied by a loss of esterolytic activity, was reproduced in a plasmin-derived fragment representing the catalytic domain. These results provide strong support for the structural and functional independence of the catalytic and interaction domains of C1-s and strengthen current models regarding the role of these domains in various interactions. They also provide direct proof for the occurrence of Ca2+ binding sites on the A chain and demonstrate that all or most of the sites on C1-s that are responsible for its interaction with C1-r and C1q are located on the A chain.