We report herein the isolation and initial characterization of a novel protein, termed SP-40,40, which is present at moderate levels (35-105 gg/ml) in normal human serum. SP-40,40 is deposited in the renal glomeruli of patients with glomerulonephritis but is not found in normal glomeruli. The protein is a heterodimeric structure of relative molecular mass 80 kD, both chains of which are of a similar size (40 kD). The amino-terminal sequences of both chains are unrelated to one another and possess no significant homology to any known protein sequence.The tissue distribution of SP40,40 closely resembles that of the terminal complement components and its physicochemical properties are similar to, but distinct from, those of the S protein of complement. We have identified SP-40,40 in the SC5b-9 complex of complement and have demonstrated incorporation of labeled SP-40,40 into this complex. These data suggest that SP-40,40 is an additional component of SC5b-9.

The cDNA sequence encoding the human complement‐associated protein, SP‐40,40, is reported. The two chains of SP‐40,40 are coded in a single open reading frame on the same mRNA molecule, indicating the existence of a biosynthetic precursor protein which matures post‐synthetically by the proteolysis of at least one peptide bond. The precursor is preceded by a signal sequence for vectorial export and contains six N‐linked glycosylation sites distributed equally between the two chains of the structure. The sequence of the SP‐40,40 precursor bears a 77% identity to a rat sulphated glycoprotein‐2 (SGP‐2) which is the major secreted product of Sertoli cells. The presence of SP‐40,40 within human seminal plasma at levels comparable to those in serum was demonstrated, indicating that SP‐40,40 and SGP‐2 are serum and seminal forms of the same protein. A sequence of 23 amino acids within the beta‐chain of SP‐40,40 exhibited significant homology to corresponding segments located within complement components C7, C8 and C9. The short cysteine‐containing motif represented the only evidence of a possible vestigial relationship between SP‐40,40 and other complement components. The precise role of SP‐40,40 is not known in either blood or semen but the present findings document an intriguing link between the immune and the reproductive systems.

Molecular cloning of the human complement inhibitor SP40,40, has revealed strong homology to a major rat and ram Sertoli cell product, sulfated glycoprotein-2, known also as clusterin. This study reports the purification and characterization of human seminal clusterin. Two-dimensional gel electrophoresis revealed charge differences between clusterin purified from semen and the serum-derived material. Both preparations demonstrate comparable hemagglutination (clustering) activity and inhibition of C5b-6 initiated hemolysis.The average clusterin concentration in normal seminal plasma is considerably higher than that found in serum. Mean seminal plasma clusterin concentrations were significantly lower in azoospermia caused by obstruction or seminiferous tubule failure than with oligospermia or normospermia. Only men with vasal agenesis had undetectable seminal clusterin, suggesting that some of the seminal clusterin is produced by the seminal vesicles.Immunofluorescence of human spermatozoa revealed that clusterin was detected on 10% of spermatozoa, predominantly those that were immature or had abnormal morphology. A pilot study of 25 patients suggests that seminal clusterin concentration, together with sperm motility and morphology, is correlated with the fertilization rate in vitro. The function of seminal clusterin is unknown. Its extensive distribution in the male genital tract and its high concentration in seminal plasma suggests an important role in male fertility. (J. Clin. Invest.

This study examines the function of SP-40,40, a newly identified component of the SC5b-9 complement complex, in the regulation of the terminal complement pathway. Purified SP-40,40 was shown to inhibit, in a dose-dependent manner, C5b-6-initiated haemolysis. Apparently additive inhibition was also demonstrated in conjunction with complement S-protein, although SP-40,40 appears to be the more potent inhibitor on an equimolar basis. The data suggest that SP-40,40, like S-protein, probably combines with the nascent C5b-7 complex, forming a cytolytically inactive SC5b-7 - SP-40,40 complex. Preparations of S-protein, purified by an established technique, were shown to be contaminated with SP-40,40. Preparations of affinity-purified SP-40,40 were also shown to contain S-protein, suggesting that these proteins may be partially complexed in plasma.

SP-40,40 is a two-chain serum protein which acts in vitro as a potent inhibitor of the assembly of the membrane attack complex of human complement. I t contains I 0 cysteine resid ues, the numbers and locations of which are conserved in several mammalian species. Evidence is presented that all the cysteine residues are involved in interchain (~--~) disulphide bonds. There are no fr~ cystcine residues. The disulphide bond motif estal~lished in this study for SP.40,40 is unique and bears no obvious homology to those complement components whose disulphide bonds have been assigned, nor is there any homology apparent between SP-40040 and other multi-chain proteins containing disulphide bonds.