The functions of the ninth component of human complement are sustained by disulfide bonds with different susceptibilities to reduction.

Purified C9 with expected hemolytic and polymerizing activities was found to contain approximately 0.2 mol of sulfhydryl groups/mol of C9. By proteolysis of C9 with labeled SH groups, the SH residues on intact C9 were mapped to Cys-359 and Cys-384 which, presumably, form an intra-domain disulfide bond in the intact molecule. The blocking of these sulfhydryl residues by alkylation, however, had minimal influence on the functions of C9. On the other hand, reduction of C9 by 1 mM dithiothreitol (DTT) (6-fold molar excess over Cys residues) followed by alkylation resulted in a complete block of polymerization activity and a 50% loss of C9 hemolytic activity. In contrast, the ability of C9 to bind EAC1-8 remained largely unaffected. The loss of poly-C9 formation activity correlated with the alkylation of approx. 6 liberated sulfhydryl groups. Hemolytic activity was abolished by treatment with > 5 mM DTT which allowed the liberation of approximately 18 sulfhydryl groups. Most of the DTT-susceptible disulfides were within the C9a fragment (an N-terminal peptide derived by thrombin). Thus, three major functions of C9, EAC1-8 binding, polymerization, and hemolytic activity, are sustained by disulfide bond-dependent conformational motifs with different susceptibility to reducing reagents. The maintenance of the N-terminal C9a region is essential for polymerization, but not EAC1-8 binding activity of C9. Taken together, the results of the present study differentiate in molecular terms several of the functional portions of C9, and stress the significance of intra-chain disulfide linkages in maintaining the structural components necessary for the functions of C9.

Implication of membrane factors other than DAF and CD59 in complement-mediated lysis of paroxysmal nocturnal hemoglobinuria erythrocytes.

Erythrocytes from two patients (F.K. and A.M.) with paroxysmal nocturnal hemoglobinuria, which were almost completely deficient in decay-accelerating factor and CD59, were found to differ in their susceptibility to homologous complement. Whereas 50-70% of F.K. erythrocytes were lysed, almost 100% of the erythrocytes from A.M. were lysed. These observations were seen under both acidified and nonacidified conditions, and regardless of whether or not the normal human serum was adsorbed with normal erythrocytes to remove natural antibodies. Erythrocytes from two other patients, J.S. and Y.K., about 85% of which did not express CD59, showed lytic profiles similar to those of patient F.K. The differences between patients were not related to levels of natural antibody or other membrane regulatory proteins such as complement receptor type I or membrane cofactor protein. Erythrocytes from F.K. and A.M. differed in their reconstitution with decay accelerating factor and CD59. While erythrocytes from F.K. were reconstituted with CD59 in a unimodal pattern, erythrocytes from A.M. showed a bimodal pattern of reconstitution assessed by flow cytometry. After reconstitution with CD59, erythrocytes from F.K. and A.M. differed in their protection against homologous complement. It is concluded that erythrocyte membrane constituents other than the known inhibitors differ in these patients.

Genetic analysis of stunted growth by nuclear-cytoplasmic interaction in interspecific hybrids of Capsicum by using RAPD markers.

When eight cultivars of Capsicum annuum were used as female parents in interspecific crosses with two accessions of C. chinense, dwarfism occurred in hybrids originating from 10 out of 16 combinations, while hybrids of the remaining 6 combinations grew normally. In contrast, when C. chinense was used as female parent, all of the hybrids showed severely stunted growth as if affected by a virus. These results suggested that the stunted growth expressed in the cross of C. chinense x C. annuum is caused by an interaction between nuclear gene(s) from C. annuum and the cytoplasm of C. chinense. To examine the number of nuclear gene(s) which cause(s) the stunted growth, we backcrossed F1 hybrids of C. annuum x C. chinense to C. chinense. About one-quarter of the progeny in the backcrossed hybrids of C. chinense x (C. annuum x C. chinense) showed the same stunted growth shown by the f1 hybrids of C. chinense x C. annuum, suggesting that two complementary genes of C. annuum cause the stunted growth. However, the higher abortion rates of ovules and lower germination percentage of seeds in C. chinense x C. annuum than in the selfed C. chinense implied that the genetic ratio of the stunted type would have been higher than that observed in the C. chinense x (C. annuum x C. chinense) progeny. We then attempted a linkage analysis between the stunted growth and randomly amplified polymorphic DNA (RAPD) of C. chinense x (C. annuum x C. chinense) progeny. A RAPD marker that associated with 94% of the stunted plants but not with 94% of the normal one was identified. This confirmed that a single nuclear gene of C. annuum which is linked to the RAPD marker with a recombination value of 6% causes the stunted growth in an interaction with the cytoplasm of C. chinense.

IgA nephropathy in patients with congenital C9 deficiency.

The clinical, histologic, and immunopathological findings of three young Japanese males with congenital C9 deficiency and primary IgA nephropathy are reported. The C9 deficiency was discovered either through mass complement screening, or when low hemolytic activity for CH50 and normal C3 levels were detected in plasma. Hematuria and proteinuria were detected at the age of 8 or 9 years as a result of annual urinary screening tests for school children. Renal biopsy showed focal and segmental mesangial proliferation with small epithelial crescents in one patient, and mild, diffuse mesangial proliferation in two. IgA and C3 were deposited predominantly in the mesangial area, and staining for C9 was negative in these patients. Electron microscopy revealed electron dense deposits predominantly in mesangial and paramesangial zones. Immunohistochemical staining in renal biopsy tissues from two patients showed mesangial staining for C5, C8, and S-protein, but staining for C5b-9 neoantigen was completely negative. These results show that the formation of C5b-9 complex is not essential for the induction of human IgA nephropathy, and also for the proliferation of mesangial and even parietal epithelial cells.

Analysis of C5b-8 binding sites in the C9 molecule using monoclonal antibodies: participation of two separate epitopes of C9 in C5b-8 binding.

C5b-8 binding sites in C9 were examined using mAbs raised against C9. Among 16 mAbs, two, designated P40 and X197, blocked C9-mediated EAC1-8 lysis. C9 pretreated with the mAbs failed to bind to EAC1-8 at 4 degrees C. In addition, the mAbs became inaccessible to the C9 that had been incorporated into EAC1-8 at 4 degrees C. These findings suggest that C9 binding to EAC1-8, but not its membrane spanning or polymerization, is blocked by mAbs. By immunoblotting analysis using alpha-thrombin proteolytic fragments derived from C9 [a N-terminal fragment of mol. wt 25,000 (C9a) and a C-terminal one of mol. wt 37,000 (C9b)] and tryptic fragments of C9 (mol. wts 53,000 (C9a') and 20,000 (C9b')), the epitopes of P40 and X197 were mapped to the N-terminal and C-terminal regions of C9b, respectively. Both P40 and X197 bound to the C9 polymerized with Zn2+ in the fluid phase, whereas X197 but not P40 reacted with the membrane attack complex (MAC) formed on membranes. The results suggest that two distinct epitopes are involved in C9 binding to EAC1-8, and behave in a different manner for globular C9 bound to EAC1-8 at 4 degrees C, C9 assembled in MAC, or poly-C9 induced by Zn2+. These mAbs may be useful in clarifying the conformational states of C9 and in analyzing the molecular interaction between C9 and its inhibitors.

[A case of C5 deficiency with polyarthritis].

A 27-year-old female with polyarthritis was found to lack serum complement activity. Her serum CH50 was less than 1.9 U/ml. C5 protein in her serum was less than 2 mg/dl and its activity was not detected. The serum level of the other proteins of complement system examined were within the normal range. At 17 years old, she was diagnosed as rheumatic fever and was admitted to our hospital. She was treated with aminobenzylpenicillin and predonisolone for two months, and she was discharged from our hospital without any abnormalities. But she had no other episode of repeated infections. Family studies of this patient revealed that an elder sister of this patient was also homozygous deficiency of C5 and her parents were considered to be heterozygous deficiency of C5. From these results, the patient was considered to be inherited deficiency of C5.

Inherited deficiency of the ninth component of complement associated with streptococcal infection.

A 7 year old boy, who presented with streptococcal infection, was found to have a low serum complement level (CH50). The C9 component was undetectable. His CH50 rose to the normal value and remained normal for at least three weeks, but decreased to one-third of the normal level three months later. Family studies were consistent with a familial C9 deficiency, with autosomal co-dominant inheritance.

Gene responsible for deficient activity of the beta subunit of C8, the eighth component of complement, is located on mouse chromosome 4.

Sera from 73 strains of mice were tested for hemolytic activity through the classical and the alternative pathways (CP and AP) in a single radial hemolysis assay. Sera from 16 out of 45 laboratory inbred strains had no lytic activity, and Ouchterlony analysis with anti-C5 serum showed them to be C5-deficient. Sera from 2 out of 28 strains derived from wild mice also had no lytic activity, but the C5 molecule was detectable in both. The hemolytic activity of sera from these strains can be restored by the addition of partially purified human C8 or human serum deficient in C8 alpha-gamma, leading us to conclude that strains M.MOL-MSM (MSM) and Mae are deficient in the beta subunit of C8. Typing of (DBA/2J X MSM)F1 hybrids and of progeny of a backcross to MSM showed that this C8 deficiency is controlled by a single recessive gene, designated C8b; the allele with hemolytic activity is C8b1; and the allele with no activity C8b0. Because of synteny homologies in mouse and human, we looked for and found close linkage between C8b and Pgm-2. Typing of recombinant mice for Mup-1 mapped the C8b locus 2.3 centimorgans (cM) telomeric to Pgm-2 on mouse chromosome 4.

Paroxysmal nocturnal haemoglobinuria with coexisting deficiency of the ninth component of complement: lack of massive haemolytic attack.

A 47-year-old woman with paroxysmal nocturnal haemoglobinuria (PNH) was found to have an inherited deficiency in the ninth complement component (C9). In complement-sensitivity lysis tests, 80% of her erythrocytes were markedly complement-sensitive (PNH-III). Laser cytofluorimetry with a monoclonal antibody against decay-accelerating factor (DAF) revealed that 95% of her erythrocytes were DAF-negative. Surprisingly, she has suffered only mild haemolysis and has never experienced massive spontaneous haemolysis. Gross haemoglobinuria and jaundice occurred only after receiving postoperative transfusion of whole blood. In her serum, C9 was not detectable either by immunological or by functional assays. Both the Ham test and the sugar water test using normal human serum or plasma yielded marked haemolysis of the patient's erythrocytes. When the patient's serum or plasma was used, only a trace of lysis was detected. Addition of purified human C9 to her plasma fully restored haemolysis. These observations indicated that C9 may play a critical role in haemolytic attacks in patients with PNH and that characteristic haemolysis in PNH may be tempered by coexisting C9 deficiency.

A biotin-avidin sandwich ELISA for quantification of intact complement component C9. The sera from hereditary C9 deficient individuals completely lack C9.

A two-site sandwich ELISA method was developed for quantitating intact C9 protein using MoAb P40 (anti-C9b antibody). This antibody reacted with monomeric C9 but not with polymerized C9. MoAb P40 was used as a capture antibody and MoAb X195 (anti-C9a antibody) as a detection antibody. This method is highly sensitive and can detect approximately 0.5 ng/ml of native C9. No cross-reactivities of either C6, C7, or C8 were observed even at concentrations of 10 micrograms/ml per component. In addition, this method allows for measurement of only intact C9 molecules, eliminating the interference of polymerized C9 or inactivated C9. Using this assay, no C9 at all was detected in sera from inherited C9 deficient individuals, including both healthy blood donors and patients with meningococcal meningitis; although by hemolytic assay, C9 levels were reported to be less than 0.2% those of NHS. Therefore, this two-site sandwich ELISA method can replace the hemolytic assay, and is especially useful for measuring small amounts of C9 in serum.

Inherited deficiencies of the late-acting complement components other than C9 found among healthy blood donors.

Among sera from 145,640 healthy blood donors in Osaka, 16 were found to have abnormalities in late-acting complement components other than C9. It was found that of these 16 sera, 2 were deficient in C5, 4 in C6, 6 in C7 and 4 in C8 alpha-gamma-subunit. The incidence of deficiency of each component among the Osaka blood donors was calculated as follows: C5 deficiency, 0.0014%; C6 deficiency, 0.0027%; C7 deficiency, 0.0041%; C8 alpha-gamma-subunit deficiency, 0.0027%. We confirmed that 13 donors were healthy and 12 had no past history related to a complement component deficiency. From these results, not only C9 deficiency but also deficiencies of the other late-acting complement components were found among the healthy blood donors, but no early-acting component deficiencies were noted.

A high incidence of C9 deficiency among healthy blood donors in Osaka, Japan.

By the use of sucrose gelatin veronal buffer (SGVB), a simple screening test was developed by us to detect sera with low complement activity, including C9-deficient sera. Using this screening test, we were able to identify sera with low complement activity including C9-deficient sera among a large number of samples. Further examinations, estimation of the protein concentration of C9, C4, C3, etc., enabled classification of serum with low complement activity into C9-deficient serum, serum deficient in the other components, and serum with low complement activity caused by non-specific activation of complement through the classical pathway by low temperature in vitro. Among 145,640 sera from Osaka donors, 138 sera were found to be deficient in C9 by these methods. The whole complement activity (CH50) of the 138 sera was 13.1 +/- 3.0 U/ml. The C9 protein in these sera was undetectable, not only by the single radial immunodiffusion method, but also by the sensitive ELISA method. C9 activities in these sera were less than 0.1% of the level in pooled normal human serum. These findings and the family studies revealed that 138 blood donors unquestionably had a hereditary C9 deficiency. The incidence of C9 deficiency among Osaka donors was calculated to be 0.095%.

The role of the C9b domain in the binding of C9 molecules to EAC1-8 defined by monoclonal antibodies to C9.

Three mAb to human C9, X195, X197, and P40 were used to analyze the roles of the C9a and C9b domains in the reaction of the C9 molecule with sensitized sheep E bearing C1 to C8 (EAC1-8). X195 bound to NH2-terminal (C9a) fragments, and X197 bound to COOH-terminal (C9b) fragments obtained by cleavage of C9 with alpha-thrombin or trypsin. P40 recognized the epitope on the C9b fragment obtained by alpha-thrombin cleavage but did not react with the NH2-terminal or COOH-terminal fragment obtained by trypsin cleavage. In this respect, P40 differed from mAb to C9 reported previously. P40 almost completely inhibited the hemolytic activity of C9. X195 and X197 also inhibited C9 activity, but less effectively than P40. C9 molecules bound to P40 could not bind to EAC1-8 cells. C9 bound to X197 could not bind rapidly to EAC1-8, but prolonged incubation of the C9-X197 complex with EAC1-8 caused considerable lysis of the cells. C9 molecules bound to X195 could bind rapidly to EAC1-8, but their lytic activity was partially inhibited by the bound antibody. From these results, it is concluded that the C9b but not C9a domain contributes to the binding of C9 to EAC1-8 and that the epitope recognized by P40 or a closely adjacent site may be the binding site of C9 molecule to EAC1-8.

[Evaluation of immunosuppressive acid protein in genitourinary malignant diseases].

Clinical significance of serum immunosuppressive acid protein (IAP) was evaluated on the basis of experience on 55 patients with genitourinary malignant disease and 49 control patients. Although the measurement of serum level of IAP is not good enough to diagnose early stage of cancer, patients with 800 micrograms/ml or more of serum IAP can be suspected to have malignant diseases. With the exception of prostatic cancer, both mean serum level and positive rate of IAP were higher in patients with high stage of disease than in those with low stage. Furthermore, on an individual basis, serum level of IAP was elevated with the progress of malignant tumor and was reduced with effective treatment. Thus, serum IAP is considered as a valuable nonspecific tumor marker in monitoring clinical course of genitourinary malignant diseases except for cancer of the prostate. In patients with advanced prostatic cancer, no definite correlation was seen between serum IAP and stage of disease, histopathological finding or serum prostatic acid phosphatase.