SP-40,40, a newly identified normal human serum protein found in the SC5b-9 complex of complement and in the immune deposits in glomerulonephritis.

We report herein the isolation and initial characterization of a novel protein, termed SP-40,40, which is present at moderate levels (35-105 micrograms/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 SP-40,40 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.

Human seminal clusterin (SP-40,40). Isolation and characterization.

Molecular cloning of the human complement inhibitor SP-40,40, 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.

The apolipoprotein A-I binding protein of placenta and the SP-40,40 protein of human blood are different proteins which both bind to apolipoprotein A-I.

A complement-associated protein SP-40,40, which is a normal constituent of human blood, binds to the main apoprotein, apoA-I, of high density lipoprotein (HDL). This protein, which is identical to apolipoprotein J, was compared to another apoA-I binding protein purified from human placenta. Immunologically the two apoA-I binding proteins are different.

Cloning, expression and sequence analysis of the genes encoding the heterodimeric methylmalonyl-CoA mutase of Porphyromonas gingivalis W50.

Two genes that encode methylmalonyl-CoA mutase (MCM) have been characterised in Porphyromonas gingivalis W50 (Pg). The genes, designated mcmA and mcmB are transcribed in an operon and encode the MCM small subunit (SS, 68,626 Da) and the MCM large subunit (LS, 78,703 Da), respectively. A recombinant Escherichia coli (Ec) clone harbouring the Pg mcmA and mcmB genes expressed MCM activity 280-times higher than that of the Ec control. The C terminus of the MCM LS has sequence homology to domains of a variety of enzymes that consume or produce methylmalonyl-CoA, suggesting that the MCM LS C-terminal domain is involved in substrate binding. The MCM LS C-terminal region also exhibits homology to other enzymes that have cobalamin-containing cofactors. It is likely, therefore, that the C terminus of the MCM LS is an important MCM domain involved in both substrate and cofactor binding.

SP-40,40, a protein involved in the control of the complement pathway, possesses a unique array of disulphide bridges.

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. It contains 10 cysteine residues, the numbers and locations of which are conserved in several mammalian species. Evidence is presented that all the cysteine residues are involved in interchain (alpha-beta) disulphide bonds. There are no free cysteine residues. The disulphide bond motif established 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-40,40 and other multi-chain proteins containing disulphide bonds.

Complete amino acid sequence and comparative molecular modelling of HPr from Streptococcus mutans Ingbritt.

The heat-stable phosphocarrier protein (HPr) of Streptococcus mutans was extracted from whole cells using sodium lauroylsarcosinate/EDTA and purified to homogeneity by a single-step, ion-exchange chromatographic procedure. The complete amino acid sequence of the protein was determined from peptides generated by trypsin, alpha-chymotrypsin, endoproteinase Glu-C, and cyanogen bromide treatment. The HPr from S. mutans contains 86 or 87 amino acyl residues, depending on removal of the N-terminal Met and the protein shows high sequence homology with HPr from other Gram-positive bacteria. The predicted tertiary structure of the S. mutans HPr, from model building by homology, is an open-faced beta-sandwich consisting of two alpha-helices and a four-stranded antiparallel beta-sheet.

Complete nucleotide sequence of a gene prtR of Porphyromonas gingivalis W50 encoding a 132 kDa protein that contains an arginine-specific thiol endopeptidase domain and a haemagglutinin domain.

We have purified from Porphyromonas gingivalis W50 a 45 kDa arginine-specific, thiol-activated, EDTA-sensitive endopeptidase, designated prtR. Oligonucleotide probes based on the N-terminal amino acid sequence were used to isolate a genomic fragment containing an open reading frame (3654 bp) with the potential to encode a 132 kDa protein including the prtR N-terminus. Analysis of this prtR gene revealed that the predicted nascent product contains a protease domain followed by a haemagglutinin domain and is post-translationally processed by proteolytic (possibly autolytic) events to produce a 43-54 kDa arginine-specific, thiol protease and a 41-53 kDa haemagglutinin. Comparison of the prtR with the P. gingivalis prtH gene suggests that the prtH gene product also contains protease and haemagglutinin domains but in the reverse order to that in the prtR. An overlapping but shifted reading frame at the 3' end of the prtR encodes the 5' region of the prtH.

Regional localization of the gene for clusterin (SP-40,40; gene symbol CLI) to human chromosome 8p12-->p21.

The human clusterin (SP-40,40) gene, designated CLI (complement lysis inhibitor) by the Human Gene Nomenclature Committee, has previously been assigned to chromosome 8. In situ hybridization allowed us to map the locus at 8p12-->p21.

The alpha-chain of murine CD8 lacks an invariant Ig-like disulfide bond but contains a unique intrachain loop instead.

The CD8 Ag is a cell surface heterodimer which demarcates predominantly cytotoxic T cells which are restricted by class I MHC Ag. The disulfide bonds within the murine structure were assigned in this study and the alpha-beta-interchain bond involves one or more cysteine residues located in each chain proximal to the plasma membrane or included within it. The location of the intrachain disulfide loop within the CD8 beta-chain confirms its proposed structural homology to an IgV domain but no corresponding disulfide loop is present within the alpha-chain. The invariant IgV disulfide loop has been replaced by a unique, short loop involving an unusual cysteine which is conserved in the CD8 alpha-chains of man, mouse, and rat. Despite its lack of precedent in other Ig-related structures, this unusual disulfide loop can be parsimoniously accommodated into a modified domain which has retained the major features of the Ig structural motif.

Several vascular complement inhibitors are present on human sperm.

We have previously reported that the complement inhibitor SP-40,40 is present in human seminal plasma. We also speculated that other inhibitors of the vascular complement system may be present within semen for the purpose of providing protection for sperm against complement within the male and/or female genital tract. In this study, we examined human seminal plasma and spermatozoa for the presence of several major complement regulatory proteins. We detected the presence of decay-accelerating factor (DAF) and CD59 and have confirmed the presence of Membrane Cofactor Protein (MCP) and SP-40,40 on human sperm. As an approach to the possible functional significance of these inhibitors on sperm membranes, the presence of two key complement components, C3 and C9, in seminal plasma was used as a criterion for an active complement system. We failed to detect C9 in seminal plasma and showed that its concentration was less than 5% of the level detected in blood plasma. C3 was also undetectable in seminal plasma; as assessed by Western transfer, its level was less than 0.3% of that in blood plasma. The low level or indeed the absence of key components of the complement system in seminal plasma--together with the finding that human sperm possess an extensive array of the vascular complement inhibitors, some of known physiologic significance--strongly suggests that their role on sperm is to protect sperm from complement lysis in the female rather than the male genital tract.

Fractionation of detergent lysates of cells by ammonium sulphate-induced phase separation.

A procedure is described for fractionating detergent lysates of cells based on the ability of (NH4)2SO4 to induce phase separation of detergents such as Triton X-100, sodium deoxycholate, and sodium cholate, into detergent-rich and detergent-depleted phases. An analysis of six murine lymphocyte cell surface molecules revealed that the partitioning in Triton X-100 of each molecule was highly dependent upon the (NH4)2SO4 concentration, each antigen partitioning into the detergent-rich phase at a defined salt concentration. In contrast, none of the six molecules appeared in the detergent-rich phase of a Triton X-114 phase separation, even though two of the molecules, namely Ly-2/3 and L3T4, are well-characterized integral membrane proteins. It was also observed that (NH4)2SO4 resulted in the partitioning of many nonmembrane proteins into the detergent-rich phase, indicating that the procedure can be used to fractionate all cellular proteins. By judicious choice of (NH4)2SO4 concentrations, precipitation of cellular proteins at two different (NH4)2SO4 concentrations, and combining the method with subcellular fractionation prior to detergent solubilization, substantial enrichment and concentration of particular cellular proteins could be achieved.

Molecular cloning and characterization of the novel, human complement-associated protein, SP-40,40: a link between the complement and reproductive systems.

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.

The murine Fc receptor for immunoglobulin: purification, partial amino acid sequence, and isolation of cDNA clones.

The murine Fc receptor for IgG (Fc gamma R) was purified to homogeneity by immunoaffinity chromatography from detergent lysates of the macrophage cell line J774. Microsequencing of intact protein yielded a single amino-terminal sequence, which was confirmed and extended to 20 residues by the isolation of an overlapping peptide. The isolation of additional proteolytic fragments obtained by using Staphylococcus aureus V8 protease, cyanogen bromide, and lysine C proteinase, facilitated sequence analysis of a total of 119 amino acid residues. Codon usage charts were used to construct oligonucleotide probes based on the amino acid sequences of three nonoverlapping peptides. These probes were used to screen a cDNA library derived from the WEHI-3B myelomonocytic cell line, and a single cDNA clone (pFc24) to which all three probes hybridized was isolated. This clone, containing a 1.02-kilobase cDNA insert, has been characterized by restriction mapping and partial DNA sequencing, and it has been shown to encode the Fc gamma R. The sequence at the 5' end of the clone contained the coding information for the amino-terminal sequence of the Fc gamma R as well as a putative 13-amino acid signal sequence. The 3' end of the clone encoded a peptide identified in purified receptor preparations. Thus, the presence of coding information at the 5' and 3' ends of this clone suggests that full-length Fc receptor cDNA spans greater than 1 kilobase.

Molecular characterization of the murine cytotoxic T-cell membrane glycoprotein Ly-3 (CD8).

The murine Ly-2/3 glycoprotein is a surface marker of T cells restricted by class I major histocompatibility complex antigens. It is a disulfide-bonded heterodimer in which either the alpha or alpha' polypeptide chain encoded by Ly-2 is covalently linked to the beta polypeptide chain encoded by Ly-3. The nucleotide and predicted amino acid sequence of the murine Ly-3 cDNA, isolated by using the rat Ly-3 cDNA clone pX9.15, together with the amino acid sequence of Ly-3.1 peptides and the N terminus, are presented here. The alignment of peptide data from the Ly-3.1 antigen with that of the predicted amino acid sequence of the Ly-3.2 antigen confirmed that the putative Ly-3 cDNA clones do in fact encode the Ly-3 protein. The Ly-3.2 cDNA clones encode a protein of 213 amino acids, which includes a 21-residue leader sequence and structural features in common with immunoglobulin variable, joining, and hinge regions. Searches of protein data bases revealed that Ly-3 is a member of the immunoglobulin superfamily with significant homology to Ly-2, immunoglobulin variable region kappa and lambda light chains, and the beta chain of the T-cell receptor. A single N-linked glycosylation site was found at asparagine-13. The relative expression of two mRNA species (approximately 1.3 and 2.3 kilobases) varied according to the source of mRNA. A murine B1 repeat was located in the 3' untranslated region of Ly-3 cDNA clones.

SP-40,40 is an inhibitor of C5b-6-initiated haemolysis.

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.