Killing of human melanoma cells by the membrane attack complex of human complement as a function of its molecular composition.

The efficiency of the membrane attack complex (MAC) in killing M21 melanoma cells was determined varying the molar ratio of cell-bound C9:C8. It was found that C5b-8 produced functional channels as evidenced by 86Rb release and propidium iodide uptake; cell killing occurred in the absence of C9 with greater than 5 X 10(5) C5b-8/cell; the maximal molar ratio of C9:C8 was 6.6:1; using nonlytic numbers of C5b-8 (4.7 X 10(5)/cell), greater than 90% killing ensued at a C9:C8 molar ratio of 2.8:1 at which approximately 9,000 poly C9/cell were formed, and 50% killing at a ratio of 1:1; (e) when the MAC was assembled on cells at 0 degree C, consisting of C5b-8(1)9(1), and unbound C9 was removed before incubation at 37 degrees C, killing was similar to that observed when poly C9 formation was allowed to occur. Thus, MAC lytic efficiency toward M21 cells may be enhanced by but does not depend on poly C9 formation.

Molecular mechanisms of cytotoxicity: comparison of complement and killer lymphocytes.

The membrane attack complex of complement is an amphiphilic fusion product of 5 glycoproteins, C5, C6, C7, C8 and C9. The membrane attack complex forms transmembrane channels that vary in size depending on the number of C9 molecules incorporated into the complex. The C5b-8 complex forms small channels and at high multiplicity can kill nucleated cells. At least 12 C9 molecules are required to form tubular poly C9 which evokes the ultrastructural image of the classical membrane lesion produced by complement. The membranes of erythrocytes and other blood cells contain a 70,000 dalton protein that can inhibit channel formation by the membrane attack complex. This protein is species specific and has been called homologous restriction factor. A cytotoxic protein immunochemically related to C9 was isolated from cytotoxic human large granular lymphocytes and from OKT3 activated human peripheral blood mononuclear cells. In the presence of Ca++, isolated C9 related protein (C9RP) formed circular structures that resembled poly C9. C9RP efficiently killed K562 cells, human melanoma cells, Raji cells and human large granular lymphocytes. The results suggest that the channel forming protein of cytotoxic lymphocytes and C9 of complement have a common evolutionary ancestry.

Catabolites of the third component of complement in urines of hereditary nephritis patients.

Hereditary nephritis protein (HNP), an unusual urine protein from patients with hereditary nephritis (Alport Syndrome), was purified 120-fold to homogeneity. A slightly larger protein, pro-HNP, was similarly purified and was found to be a precursor of HNP. Both pro-HNP and HNP showed immunological identity to the third component of human complement, C3, and to its catabolite C3c. Pro-HNP had a molecular weight of 143,000 and, in equimolar ratio, polypeptide chains or fragments of molecular weights 75,000, 40,000, and 28,000. The largest and smallest chains contained carbohydrate. HNP had a molecular weight of 141,000 and fragments of molecular weights 60,000, 38,000, 26,000, and 17,000 in equimolar ratio; the two smallest fragments contained carbohydrate. Plasmin digestion of pro-HNP showed that the 75,000-Da chain, identical with the intact beta-chain of C3, broke down to the 60,000- and 17,000-Da fragments of HNP. In both pro-HNP and HNP, the polypeptide chains were linked by disulfide bonds, with the exception of the 17,000-Da fragment of HNP. This fragment was readily dissociated from the rest of the HNP molecule in the presence of sodium dodecyl sulfate. Amino acid analyses showed that both pro-HNP and HNP contained approximately 22 half-cystine residues per molecule. Extinction coefficients, epsilon 1% 1cm, at 280 nm were calculated to be 8.5 and 8.8 for pro-HNP and HNP, respectively.

Polypeptide fragments of the third component of complement in urine of hereditary nephritis patients.

Pro-HNP, a urine protein isolated from hereditary nephritis patients, is derived from C3 and resembles the C3c domain. It contains disulfide-linked polypeptides of beta 75, alpha 40, and alpha 28. Plasmin degraded pro-HNP in vitro to HNP, which was also isolated from the urine of patients and which contained disulfide-linked polypeptides of beta 60, alpha 38, and alpha 26, and noncovalently bound polypeptide of beta 17. Amino terminal sequence analyses and amino acid compositions of the seven polypeptides isolated from pro-HNP and HNP show that beta 75 degrades to beta 60 and beta 17 (beta 17 locates at the amino end of beta 75), alpha 40 degrades to alpha 38 (both locate at the carboxyl end of the alpha-chain of C3), and alpha 28 degrades to alpha 26 (both are from the amino end of the alpha'-chain of C3b). These results confirm the enzymatic specificity of plasmin on pro-HNP. In HNP, the half-cystine contents of beta 60, alpha 38, alpha 26, and beta 17 were approximately 3, 12, 3, and 4, respectively. Partial reduction readily released alpha 40 from pro-HNP and alpha 38 from HNP. There were about five intra-chain disulfide bonds in alpha 40 or alpha 38; stepwise reduction of these intra-polypeptide bonds apparently accounted for multiple conformations of alpha 40 or alpha 38.

Mechanism of cytotoxicity of human large granular lymphocytes: relationship of the cytotoxic lymphocyte protein to the ninth component (C9) of human complement.

A Mr 70,000 protein was isolated from cytotoxic human large granular lymphocytes and shown to have cytotoxic activity. The protein was demonstrated to be immunochemically related to the ninth component (C9) of complement and was therefore designated C9-related protein (C9RP). This finding suggests that C9RP and C9 share homology in primary structure and have a common evolutionary ancestry. C9RP was isolated, by affinity chromatography employing anti-human C9-Sepharose, from either purified cytoplasmic granules or whole-cell lysates of cultured human large granular lymphocytes. The cells were isolated from healthy blood donors and maintained in interleukin-2-dependent cultures. The immunochemical crossreactivity of C9 with C9RP was 3-4%, using a murine anti-C9RP antiserum. Certain murine monoclonal antibodies to C9RP and to C9 inhibited killing of K562 cells by human large granular lymphocytes. Killed target cells, identified by propidium iodide staining and isolated by fluorescence-activated cell-sorting, exhibited clusters of circular membrane lesions that resembled poly(C9) in appearance. Polymerization of isolated C9RP in the presence of Ca2+ resulted in the formation of two different circular structures, one having an inner diameter of approximately equal to 60 A, and the other, of 125 A. Polymerized C9RP could be incorporated into liposomes and, as such, gave rise to channels of two different sizes. The smaller channel had a functional diameter of 50-90 A, and the bigger channel, a diameter greater than 102 A.