James M. Sodetz

Structure of Human C8 Protein Provides Mechanistic Insight into Membrane Pore Formation by Complement

C8 is one of five complement proteins that assemble on bacterial membranes to form the lethal pore-like “membrane attack complex” (MAC) of complement. The MAC consists of one C5b, C6, C7, and C8 and 12–18 molecules of C9. C8 is composed of three genetically distinct subunits, C8α, C8β, and C8γ. The C6, C7, C8α, C8β, and C9 proteins are homologous and together comprise the MAC family of proteins. All contain N- and C-terminal modules and a central 40-kDa membrane attack complex perforin (MACPF) domain that has a key role in forming the MAC pore. Here, we report the 2.5 Å resolution crystal structure of human C8 purified from blood. This is the first structure of a MAC family member and of a human MACPF-containing protein. The structure shows the modules in C8α and C8β are located on the periphery of C8 and not likely to interact with the target membrane. The C8γ subunit, a member of the lipocalin family of proteins that bind and transport small lipophilic molecules, shows no occupancy of its putative ligand-binding site. C8α and C8β are related by a rotation of ∼22° with only a small translational component along the rotation axis. Evolutionary arguments suggest the geometry of binding between these two subunits is similar to the arrangement of C9 molecules within the MAC pore. This leads to a model of the MAC that explains how C8-C9 and C9-C9 interactions could facilitate refolding and insertion of putative MACPF transmembrane β-hairpins to form a circular pore.

The effect of ϵ-amino caproic acid on the gross conformation of plasminogen and plasmin☆

Abstract Previous studies from several laboratories have demonstrated that the native form of circulating plasminogen, Pga, undergoes a gross conformational alteration upon binding the amino acid, ϵ-amino caproic acid (ϵ-ACA), and its analogues. Sedimentation velocity measurements show that plasminogen b Pgb), prepared by removal of a peptide of Mr 6000–8000 from the amino terminus of Pga as a consequence of its activation to plasmin, possesses a strikingly dissimilar gross conformation when compared to Pga. However, the overall conformation of Pgb is very similar to the Pga· ϵ-ACA complex. Although Pgb retains its full capacity for binding ϵ-ACA, no additional gross conformational alteration results as a consequence of saturating the binding site of Pgb with this amino acid. Similar measurements show that plasmin a (Pga), which is a direct activation product of Pga, displays a native gross conformation which is very similar to native Pga. Pma binds ϵ-ACA in an identical manner to Pga and also undergoes a similar conformational alteration as Pga, as a consequence of this binding. On the other hand, plasmin b (Pga), which is a direct activation product of Pgb, and also results from removal of the above mentioned peptide from Pma, possesses an overall gross conformation similar to Pgb, the Pga·ϵ-ACA complex and the Pma·ϵ-ACA complex. Although Pmb also retains full capabilities for binding ϵ-ACA, there is no further gross conformational alteration as a result of this interaction. These studies illustrate the importance of the amino terminal region of the original Pga in controlling the overall conformation of the molecule as well as controlling the conformation achieved upon saturation of the molecule by ϵ-ACA like molecules.

[23] Rabbit plasminogen and plasmin isozymes

Publisher Summary Assays of plasminogen first require its conversion to the enzyme plasmin by common activators. The enzyme is then assayed on the basis of its esterolytic activity toward N-a-toluenesulfonyl-L-arginine methylester (TosArgOMe, TAME) utilizing a recording pH stat to titrate the amount of acid, Na-tosyl-L-arginine, liberated with time. In this chapter the Colorimetric method is described. This method is used if the equipment to perform esterolytic activity is not available. Colorimetric method is very sensitive and is used for analysis of the concentration of TosArgOMe remaining after incubation of plasmin with this substrate for a given period of time. This method is based upon the quantitative reaction of esters, such as TosArgOMe, with hydroxylamine, yielding the hydroxamic acid derivative. Upon addition of ferric chloride, a ferric ion-hydroxamic acid complex is formed, possessing spectral properties different from ferric chloride. The chapter also explains the Potentiometric Method. The purification procedure of plasminogen, chemical, physical and kinetic properties of rabbit plasminogen are also discussed.

Structure and function of C8 in the membrane attack sequence of complement

Complement-mediated cell lysis occurs as a result of interactions between complement proteins C5b, C6, C7, C8, and C9 to produce the membrane attack complex C5b-9 (Muller-Eberhard 1986):

Role of the human C8 subunits in complement-mediated bacterial killing: evidence that C8γ is not essential

C5b\;\xrightarrow{{C6}}\;C5b - 6\;\xrightarrow{{C7}}\;C5b - 7\xrightarrow{{C8}}\;C5b - 8\;\xrightarrow{{nC9}}\;C5b - 9

[45] Membrane attack complex proteins C5b-6, C7, C8, and C9 of human complement

Assembly of C5b-9 begins with proteolytic conversion of C5 to C5b by the C5 convertases formed as a consequence of complement activation. Development of a transient binding site for C6 leads to formation of a stable C5b-6 dimer. Subsequent binding of C7 and formation of C5b-7 coincides with the expression of a high-affinity lipid-binding site that mediates a strong but noncovalent interaction between the nascent complex and target membranes. Binding of C8 yields the tetramolecular C5b-8 complex. Although capable of slowly lysing erythrocytes and some nucleated cells, C5b-8 functions primarily as a receptor for C9 and thereby mediates formation of the more lytically effective C5b-9 complex. The number of C9 molecules per complex differs depending on C9 input and conditions of formation. The ultrastructure varies accordingly from what are functional lesions with one or a few C9s to highly organized porelike structures formed by polymerization of as many as 16 C9s per C5b-8. Facts and controversies about the function of C9 and the stoichiometry, structure, and mechanism of action of C5b-9 are summarized in other reports (Podack 1986; Muller-Eberhard 1986; Esser 1987; Stanley, this volume).

Regional chromosomal assignment of genes encoding the α and β subunits of human complement protein C8 to 1p32

Two major forms of plasminogen exist in the plasma of many animal species and are distinguished by their affinities for certain antifibrinolytic amino acids. Quantitative end group analysis demonstrated that each isolated form of rabbit plasminogen possessed a single amino terminal residue of glutamic acid. Amino acid sequence analysis indicated that at least the first twelve amino terminal amino acids were identical in the two forms. The unique amino terminal sequence obtained for each form was NH2-glu-pro-leu-asp-asp-tyr-val-asn-thr-gln-gly-ala-. Analysis of the carbohydrate content of each major plasminogen form revealed some striking differences. The first major form of rabbit plasminogen isolated from affinity chromatography columns contained 1.5–1.7 percent neutral carbohydrate and 3.0–3.3 moles of sialic acid per mole of protein. The second major form of rabbit plasminogen isolated from affinity chromatography columns contained 0.6–0.8 percent neutral carbohydrate and 1.8–2.2 moles of sialic acid per mole of protein.

Genomic organization of human complement protein C8α and further examination of its linkage to C8β

Human C8 is one of five complement components (C5b, C6, C7, C8 and C9) that interact to form the cytolytic membrane attack complex (MAC) on bacterial cell membranes. It is an oligomeric protein composed of a disulfide-linked C8 alpha-gamma heterodimer and a non-covalently associated C8 beta chain. Previous studies revealed that C8 alpha and C8 beta have distinct roles in the formation of the MAC on simple cells such as erythrocytes and that both subunits are essential for cell lysis. These studies also determined that C8 gamma is not required for expression of MAC hemolytic activity. To determine if these conclusions are applicable to more biologically relevant systems, the C8 subunits were examined for their ability to support complement-mediated killing of Gram-negative bacteria. Results indicate: (1) C8 alpha-gamma, C8 alpha, C8 beta and C8 gamma have no independent bactericidal activity; (2) bacterial killing requires C8 beta and either C8 alpha-gamma or C8 alpha; (3) C8 alpha is an effective substitute for C8 alpha-gamma in bacterial killing; and (4) C8 gamma enhances, but is not required for C8 bactericidal activity. Together, these data suggest that C8 alpha and C8 beta have correspondingly similar roles in MAC-mediated lysis of erythrocytes and bacterial killing. Furthermore, they provide the first direct evidence that C8 gamma is not required for complement-mediated killing of Gram-negative bacteria.