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J M Sodetz

Publications and source records attributed to J M Sodetz.

At least 37 records · Page 2Linked to original sources
PubMed1989

DNA polymorphism of the human complement C8 beta gene: formal genetics and intragenic localization.

The eighth component of human complement consists of three subunits of different molecular mass, which are coded for by three separate genetic loci. Polymorphisms have been described at the protein level for the alpha and beta subunits by means of sodium dodecyl sulfate gel electrophoresis and isoelectric focusing. Using a full-length human C8 beta cDNA probe, we have studied more than 100 individuals by Southern blot analysis to detect DNA polymorphisms. We have found two restriction fragment length polymorphisms (RFLPs) with the enzymes Taq I and Bam HI. The Taq I polymorphism is defined by two alleles, i.e., a single 4.9 kb fragment or two 2.8/2.1 kb fragments. The allele frequencies are 0.68 and 0.32, respectively. The second RFLP with Bam HI is correlated with the Taq I variants: 3 kb Bam HI; 4.9 kb Taq I and 3.3 kb Bam HI; 2.8/2.1 kb Taq I. Both RFLPs could be mapped to the 3' portion of the C8 beta gene. Based on the size of genomic restriction fragments, the C8 beta gene can be estimated to have a size of 32-36 kb. Because of the even frequency distribution, the C8 beta DNA polymorphisms may be useful in gene mapping and disease association studies.

Alleles↗
PubMed1989

Chromosomal assignment of genes encoding the alpha, beta, and gamma subunits of human complement protein C8: identification of a close physical linkage between the alpha and the beta loci.

The eighth component of human complement (C8) is a serum protein containing three nonidentical subunits (alpha, beta, gamma) that are arranged as a disulfide-linked alpha-gamma dimer and a noncovalently associated beta chain. In earlier genetic studies, electrophoretic analysis of C8 protein polymorphisms revealed several allelic variants of alpha-gamma and beta. These were governed by separate loci designated C8A and C8B for alpha-gamma and beta, respectively. Genetic linkage analyses indicated that these loci were linked to each other and to chromosome 1 marker loci PGM1 and Rh, but it was unclear at the time if C8A was a single locus coding for a single-chain precursor form of alpha-gamma or if separate loci existed for alpha and gamma. Since evidence now indicates that alpha, beta, and gamma are encoded by separate genes, cDNA probes corresponding to each subunit were used to make direct assignments of the individual loci. Analysis of somatic cell hybrids revealed that only the alpha and beta loci are located on chromosome 1. Parallel analysis of genomic DNA digests using 5' and 3'-specific cDNA probes showed they are physically linked (less than 2.5 kb) and oriented 5' alpha-beta 3'. Further probing of the hybrid panel revealed that gamma is located on chromosome 9q. Thus, the observed genetic linkage of alpha-gamma to beta must be determined solely by alpha. In accordance with these findings, the C8 loci should now be designated C8A, C8B, and C8G for alpha, beta and gamma, respectively.

Animals↗
PubMed1987

Biosynthesis of C8 by hepatocytes. Differential expression and intracellular association of the alpha-gamma- and beta-subunits.

The subunit structure of C8 is unusual in that it consists of a disulfide-linked alpha-gamma-dimer and a non-covalently associated beta-chain. To gain insight into the biosynthesis and assembly of C8, we examined its production by short term cultures of rat hepatocytes. Synthesis was detected by functional assays as well as immunologically with antisera against C8, alpha-gamma or beta. Control experiments showed that all three antisera were capable of precipitating C8 from rat serum. When antiserum against C8 was used to analyze hepatocyte cell lysates, precursor as well as mature forms of alpha-gamma and beta were detected. Precursor-product relationships were confirmed by pulse-chase experiments, which also revealed a significantly faster rate of synthesis for alpha-gamma. Importantly, no single chain form of alpha-gamma was detected in the intracellular pool. Evidence that C8 is assembled before secretion was provided by analyzing cell lysates with antiserum specific for alpha-gamma or beta. Precipitation of both subunits with either antiserum confirmed they were associated. Parallel analyses of the extracellular pool revealed that alpha-gamma and beta remain associated in the media but that excess alpha-gamma is secreted as a consequence of a faster synthesis rate. These results provide evidence for differential expression of alpha-gamma and beta and are consistent with the existence of different loci for these subunits. Furthermore, they indicate that association of alpha-gamma with beta is a presecretory event in normal C8 synthesis.

Animals↗
PubMed1987

Evidence that C5b recognizes and mediates C8 incorporation into the cytolytic complex of complement.

The aim of this study was to identify constituents of the intermediate C5b-7 complex of human complement that mediate binding of C8 and formation of C5b-8. Analysis of interactions between purified C8 and C5, C6, or C7 indicate that C5 and C8 associate to form a dimer in solution. This interaction is specific and involves a single C5 binding site located on the beta-subunit of C8. Simultaneous interaction of C8 with C5 and C9 in solution suggests that during assembly of the cytolytic C5b-9 complex on membranes, C8 binds to C5b-7 through association of beta with C5b, after which C9 associates through interaction with the previously identified C9-specific site on the alpha-subunit. Other evidence of interaction with C5b was provided by the fact that C8 can bind purified C5b6. Also, in situ cross-linking experiments showed that within C5b-8, the beta-subunit is in close proximity to C5b. These results indicate that C8 binding to C5b-7 is mediated by a specific C5b recognition site on beta, thus explaining the requirement for this subunit in C5b-8 formation. They also reveal that C5b contains a specific site for interaction with beta.

Binding Sites↗
PubMed1987

The eighth component of human complement: evidence that it is an oligomeric serum protein assembled from products of three different genes.

The eighth component of human complement (C8) consists of three nonidentical subunits arranged asymmetrically as a disulfide-linked alpha-gamma dimer and a noncovalently associated beta chain. Genetic studies of C8 polymorphisms established that alpha-gamma and beta are encoded at different loci. Implicit in this finding was the existence of two different genes and the likelihood that alpha-gamma would be synthesized in single-chain precursor form. However, recent characterization of cDNA clones revealed separate mRNAs for human alpha and beta but no evidence of a single-chain precursor for alpha-gamma. A cDNA clone containing the entire coding region for human gamma has now been characterized, and its sequence supports the existence of a separate gamma mRNA. Included are a consensus translation initiation sequence, an apparent initiation methionine, and a signal peptide. By use of cDNA probes specific for human alpha, beta, or gamma, analysis of poly(A) RNA from normal baboon liver revealed separate mRNAs of 2.5, 2.6, and 1.0 kilobases (kb), respectively. Parallel analysis of poly(A) RNA from rat liver identified mRNAs of 3.4, 2.3, and 0.9 kb. These results argue against the possibility that C8 is assembled from products of two different genes (alpha-gamma and beta) and suggest it is comprised of three different gene products (alpha, beta, and gamma) that undergo both covalent and noncovalent association to yield the mature protein.

Amino Acid Sequence↗
PubMed1987

Complementary DNA and derived amino acid sequence of the alpha subunit of human complement protein C8: evidence for the existence of a separate alpha subunit messenger RNA.

The entire amino acid sequence of the alpha subunit (Mr 64,000) of the eighth component of complement (C8) was determined by characterizing cDNA clones isolated from a human liver cDNA library. Two clones with overlapping inserts of net length 2.44 kilobases (kb) were isolated and found to contain the entire alpha coding region [1659 base pairs (bp)]. The 5' end consists of an untranslated region and a leader sequence of 30 amino acids. This sequence contains an apparent initiation Met, signal peptide, and propeptide which ends with an arginine-rich sequence that is characteristic of proteolytic processing sites found in the pro form of protein precursors. The 3' untranslated region contains two polyadenylation signals and a poly(A) sequence. RNA blot analysis of total cellular RNA from the human hepatoma cell line HepG2 revealed a message size of approximately 2.5 kb. Features of the 5' and 3' sequences and the message size suggest that a separate mRNA codes for alpha and argues against the occurrence of a single-chain precursor form of the disulfide-linked alpha-gamma subunit found in mature C8. Analysis of the derived amino acid sequence revealed several membrane surface seeking domains and a possible transmembrane domain. These occur in a cysteine-free region of the subunit and may constitute the structural basis for alpha interaction with target membranes. Analysis of the carbohydrate composition indicates 1 or 2 asparagine-linked but no O-linked oligosaccharide chains, a result consistent with predictions from the amino acid sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Complementary DNA and derived amino acid sequence of the beta subunit of human complement protein C8: identification of a close structural and ancestral relationship to the alpha subunit and C9.

A cDNA clone encoding the beta subunit (Mr 64,000) of the eighth component of complement (C8) has been isolated from a human liver cDNA library. This clone has a cDNA insert of 1.95 kilobases (kb) and contains the entire beta sequence [1608 base pairs (bp)]. Analysis of total cellular RNA isolated from the hepatoma cell line HepG2 revealed the mRNA for beta to be approximately 2.5 kb. This is similar to the message size for the alpha subunit of C8 and confirms the existence of different mRNAs for alpha and beta. This finding supports genetic evidence that alpha and beta are encoded at different loci. Analysis of the derived amino acid sequence revealed several membrane surface seeking segments that may facilitate beta interaction with target membranes during complement-mediated cytolysis. Determination of the carbohydrate composition indicated 1 or 2 asparagine-linked but no O-linked oligosaccharide chains. Comparison of the beta sequence to that reported for alpha in the preceding paper [Rao, A. G., Howard, O. M. Z., Ng, S. C., Whitehead, A. S., Colten, H. R. & Sodetz, J. M. (1987) Biochemistry (preceding paper in this issue)] and to that of human C9 revealed a striking homology between all three proteins. For beta and alpha, the overall homology is 33% on the basis of identity and 53% when conserved substitutions are allowed. For beta and C9, the values are 26% and 47%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

Analysis of the specific association of the eighth and ninth components of human complement: identification of a direct role for the alpha subunit of C8.

The basis for the physical association between C8 and C9 in solution was examined by isolating the noncovalently associated alpha-gamma and beta subunits of C8 and determining their respective affinities for C9. Results indicate that only alpha-gamma associates with C9 and this association, though reversible, is complete at near equimolar ratios of each component. Further experiments using purified alpha or gamma revealed that only alpha was capable of forming a stable complex with C9. Although the strength of this interaction was dependent on salt concentration, association was observed in buffer of physiological ionic strength and in human serum. These results establish that the domain on C8 responsible for interaction with C9 is located entirely within alpha. In related experiments, addition of beta to performed dimers of either (alpha-gamma + C9) or (alpha + C9) resulted in complete association of this subunit. These particular results indicate that there are two physically distinct sites on alpha that separately mediate association of alpha with beta and with C9. Furthermore, occupation of one site does not impair interaction at the other.

Complement C8↗
PubMed1985

Functional domains of the alpha subunit of the eighth component of human complement: identification and characterization of a distinct binding site for the gamma chain.

The purified gamma subunit of the eighth component of human complement (C8) was used to characterize its site of interaction within C8 and to probe the ultrastructure of membrane-bound C5b-8 and C5b-9 complexes. Purification of gamma was accomplished by separating the disulfide-linked alpha-gamma subunit from the noncovalently associated beta chain and subjecting the former to limited reduction, alkylation, and ion-exchange chromatography. Upon mixing, purified alpha and gamma exhibited a high affinity for each other, as evidenced by their ability to form a noncovalent, equimolar complex at dilute concentrations and in the presence of excess serum albumin. Purified gamma also exhibited an affinity for C8', a previously described derivative that is functionally similar to C8 although it is composed of only alpha and beta. These results indicate that alpha possesses a specific site for interaction with gamma and that this site is preserved in the isolated subunit. Furthermore, this site remains accessible when alpha is associated with beta. In related experiments, gamma was found to specifically associate with membrane-bound C5b-8' and C5b-(8')9 complexes. These results indicate that the site for gamma interaction remains accessible on alpha in C5b-8' and is not shielded by C9 within C5b-(8')9. It is concluded that the gamma subunit of C8 is located on the surface of membrane-bound C5b-8 and C5b-9.

Binding Sites↗
PubMed1984

Measurement of the ratio of the eighth and ninth components of human complement on complement-lysed membranes.

The mole ratio of the eighth (C8) and ninth (C9) components of human complement on membranes carrying the cytolytic C5b-9 complex was measured by direct binding assays. Erythrocytes from two different species were used as the membrane system. Antibody-treated sheep erythrocytes carrying a relatively small number of precursive membrane-bound C5b-7 complexes were prepared by exposure to human C8-depleted serum. These complexes were subsequently converted to C5b-8 by addition of saturating amounts of C8. Parallel binding assays using 125I-C8 were used to determine the exact amount bound and thus the number of C5b-8 complexes per cell. These cells were subsequently incubated with excess 125I-C9 and the amount bound relative to C8 on the membrane was measured. Results indicated the C8:C9 ratio remained constant at approximately 1:4 as the number of complexes varied from 40 to 310 per cell. Similar results were obtained regardless of whether C8 and C9 were added sequentially or simultaneously to cells bearing C5b-7. For comparison, experiments were also performed using membranes that contained a high number of complexes. Here, rabbit erythrocytes which carried approximately 25 000 C5b-7 per cell were incubated with limited amounts of C8 to form C5b-8 complexes on the membrane surface, the exact number of which was measured by 125I-C8 binding assays. When erythrocytes prepared in this manner were incubated with excess 125I-C9, the ratio of C8:C9 on the membrane was found to be essentially constant at approximately 1:3 as the number of these complexes varied from 50 to 4000 per cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Function of subunits within the eighth component of human complement: selective removal of the gamma chain reveals it has no direct role in cytolysis.

The eighth component of human complement (C8) consists of a disulfide-linked alpha-gamma dimer that is noncovalently associated with beta. Previous results from this laboratory established that two of these subunits have distinct roles in the cytolytic function of C8. Binding of C8 to the precursive C5b-7 complex is mediated strictly through beta while interaction between C8 and the lipid bilayer of target membranes occurs primarily through alpha. In the present study, we examined the importance of the gamma subunit in cytolysis by characterizing functional properties of C8', a derivative of C8 that lacks gamma. Preparation of this derivative was accomplished by limited cleavage of disulfide bonds in purified alpha-gamma and separation of alpha from gamma. When mixed, purified alpha and beta combined to form C8'. When tested for functional similarity to normal C8, the following results were obtained. (1) Specific and saturable binding of C8' to the C8 binding site on C5b-7 could be achieved. Significantly, the resulting C5b-8' supported subsequent C9 binding and cell lysis, which was equivalent to that observed with C5b-8. (2) Complement activation of (C8 + C9)-depleted serum containing C8' resulted in formation of SC5b-8'. Inclusion of C9 in the serum resulted in further conversion to SC5b-(8')9. These observations indicate that C8' is functionally similar to C8. Furthermore, they indicate that unlike alpha and beta, the gamma subunit has no direct role in facilitating C8 interaction with the nascent cytolytic complex or in mediating C9 binding and membrane lysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Purification of functional subunits of the eighth component of human complement (C8) under nondenaturing conditions.

A simple procedure is described for purifying the noncovalently associated alpha-gamma and beta subunits of human C8 in the absence of denaturants and in yields significantly higher than previously obtainable. The procedure utilizes either conventional or high-pressure molecular sieve chromatography in the presence of 1.5 M NaCl. This ionic strength promotes dissociation of alpha-gamma from beta and facilitates their physical separation under mild conditions. Yields are typically 60-75% and, as judged by several criteria, each subunit is functionally identical to those isolated by earlier methods using sodium dodecyl sulfate. This new procedure eliminates the need for this detergent and thereby avoids the major solubility and yield problems encountered during its removal.

Chromatography, Gel↗
PubMed1983

Studies of the association of the eighth and ninth components of human complement within the membrane-bound cytolytic complex.

The association of the eighth (C8) and ninth (C9) components of human complement within membrane-bound C5b-9 was investigated using the photosensitive cross-linking reagent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. Reaction of this reagent with either the purified alpha-gamma or beta subunit of C8 resulted in the introduction of 6-8 mol/mol of photosensitive 6-(4'-azido-2'-nitrophenylamino)hexanoate (ANH) as an intrinsic ligand on each protein. The resulting ANH-(alpha-gamma) or ANH-(beta) was capable of recombining with equimolar amounts of beta or alpha-gamma, respectively, to yield ANH-C8. Parallel modifications of purified C9 resulted in incorporation of 3-4 mol/mol of ANH-ligand. Both ANH-C8 and ANH-C9 retained their ability to incorporate into C5b-9. Two approaches were used to determine the proximity of C8 subunits to C9 within C5b-9. In one, the complex was assembled on erythrocytes by incubating EAC1-7 cells separately with each form of ANH-C8 and subsequently saturating with 125I-C9. After lysis, membranes were irradiated, solubilized, and analyzed by gel electrophoresis. Cross-linking was assessed by a shift in electrophoretic mobility of 125I-C9 to a higher molecular weight. Results using either form of ANH-C8 in C5b-9 showed that, although at least 30% was involved in cross-linking, none was cross-linked to C9. Similar results were obtained using a second approach in which cross-linker and radiolabel were transposed between C8 and C9. Here, EAC1-7 cells were incubated first with 125I-C8 containing either 125I-(alpha-gamma) or 125I-(beta) and subsequently with ANH-C9. Although at least 48% of ANH-C9 in C5b-9 was involved in cross-linking in these experiments, no cross-linking to either subunit of C8 was detected. These results suggest that C8 is not in close physical association with C9 within membrane-bound C5b-9.

Azides↗
PubMed1983

Evidence of direct insertion of terminal complement proteins into cell membrane bilayers during cytolysis. Labeling by a photosensitive membrane probe reveals a major role for the eighth and ninth components.

Radioiodinated hexanoyldiiodo-N-(4-azido-2-nitrophenyl)tyramine (HNT) was employed as a photosensitive membrane-restricted probe to establish whether terminal complement proteins insert into membrane bilayers during cytolysis. The system studied consisted of natural membranes carrying intermediate (C5b-8) or fully assembled (C5b-9) cytolytic complexes of human complement prepared by two different methods. In one method, C5b-8 and C5b-9 were assembled on membranes de novo by incubating rabbit erythrocytes with C9-depleted or whole human serum, respectively. The probe was partitioned into lipid bilayers of the resulting MC5b-8 and MC5b-9 membranes by post-addition of HNT. Membranes were irradiated and photolabeled C5b-8 and C5b-9 were extracted, purified, and analyzed on polyacrylamide gels. Those constituents labeled by HNT within each complex were identified by autoradiography. The second method involved pre-addition of HNT to membranes carrying the precursive C5b-7 complex and subsequent conversion to MC5b-8 and MC5b-9 by addition of exogenous C8 and C9. After irradiation, C5b-8 and C5b-9 were again purified and analyzed for the presence of photolabel. Results from both methods were similar and indicated all constituents of each complex are labeled to a measurable extent. However, the C8 alpha subunit was predominantly labeled in C5b-8 and both C8 alpha and C9 were predominantly labeled in C5b-9. Because labeling by HNT is specific for intramembrane structural domains of proteins, these results provide direct evidence that constituents of terminal complement complexes insert into the lipid bilayer of cell membranes during lysis. Further, we conclude that in these complexes, C8 alpha and C9 are the primary contributors of inserted peptide domains.

Animals↗
PubMed1981

Role of the beta subunit in interaction of the eighth component of human complement with the membrane-bound cytolytic complex.

This study is concerned with the subunits of the eighth component of human complement (C8) and their role in facilitating specific incorporation of C8 into the membrane-bound cytolytic complex of complement. The noncovalently associated alpha-gamma and beta subunits of C8 were purified and examined for their ability to interact independently with the C8 binding site on C5b-7, the membrane-bound precursor of the cytolytic complex. Using erythrocyte-bound C5b-7 (EAC1-7), it was observed that native C8 and its isolated beta subunit have similar high affinities for this complex. Binding of beta to EAC1-7 was specific for the C8 binding site as evidenced by the fact that 1) nearly identical molar amounts of either C8 or beta were required to saturate C8 binding sites on EAC1-7; 2) pretreatment of EAC1-7 with saturating amounts of beta prevented binding of C8; and 3) pretreatment of EAC1-7 with saturating amounts of C8 prevented binding of beta. In related experiments, alpha-gamma alone had no affinity for EAC1-7, however, binding comparable to that exhibited by native C8 occurred if alpha-gamma was first incubated with an equimolar amount of beta. Other experiments showed that if EAC1-7 was first saturated with beta to produce EAC1-7(beta), alpha-gamma also bound to yield functional C8 on the cell surface. These results provide conclusive evidence that structural features of C8 which are essential for specific recognition by membrane-bound C5b-7 are contained in the beta subunit.

Cell Membrane↗
PubMed1980

The eighth component of human complement. Purification and physicochemical characterization of its unusual subunit structure.

The eighth component of human complement (C8) has been purified in high yield from Cohn Fraction III and characterized with regard to its physicochemical properties and subunit structure. The purified product was found to be similar to functional C8 isolated from plasma or serum. Human C8 possesses a molecular weight of 151,000 and is composed of a 1:1:1 ratio of three nonidentical subunits: alpha (Mr = 64,000), beta (Mr = 64,000), and gamma (Mr = 22,000). These subunits occur as a covalently linked alpha-gamma dimer which is noncovalently associated with beta. After purification and characterization of alpha, beta, and gamma, each was found to possess different amino acid compositions and NH2-terminal sequences. Both alpha and beta subunits contain similar but exceptionally high percentages of hydrophobic aromatic amino acids. As measured by circular dichroism, the secondary structure of C8 contains 12% alpha-helix, 24% beta structure, and 64% unordered structure, values typical of globular proteins. Complete secondary structure, as well as hemolytic activity, can be recovered after exposure to 6 M guanidinium hydrochloride or 8 M urea. The alpha-gamma and beta subunits were dissociated and isolated in the presence of sodium dodecyl sulfate and after removal of detergent, neither was found to possess independent hemolytic activity. Significantly, activity equivalent to that of native C8 was generated when alpha-gamma and beta were recombined in an equimolar ratio. These results indicate that C8 is an atypical serum protein with regard to both its subunit structure and denaturation characteristics.

Amino Acids↗