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)     

香鱼补体成分C9基因的克隆、序列分析及表达

补体成分C9是构成膜攻击复合体引起靶细胞溶解破坏的重要组成成分。该文测定了香鱼C9(aC9)基因的cDNA全序列,序列全长2125个核苷酸,编码一个由592个氨基酸组成、相对分子质量为6.56×104的前体蛋白,N端22个氨基酸为信号肽序列。序列分析表明,aC9与虹鳟C9的氨基酸同源性最高,达56.8%,与其它鱼类C9的同源性介于40.9%~53.8%之间。aC9在健康香鱼肝、脾、肠、鳃和肌肉有表达,其中在肝内的表达量最高。实时荧光定量PCR的结果显示,鳗利斯顿氏菌侵染4h后,肝中aC9mRNA表达量显著上调,并随着时间的推移在16h时达到峰值。Westernblotting分析的结果显示,鳗利斯顿氏菌侵染后香鱼血清中的aC9蛋白随着时间的推移呈显著上调。以上结果表明,香鱼肝组织C9基因表达变化与鳗利斯顿氏菌的侵染密切相关,揭示了C9在鱼类抗细菌免疫反应中具有重要的作用。     

Complementary DNA sequence of lamprey fibrinogen beta chain

The cDNA sequence of the beta chain of lamprey fibrinogen has been determined. To that end, an oligonucleotide probe was synthesized that corresponded to an amino acid sequence from the carboxy-terminal region of the lamprey fibrinogen beta chain. The insert actually began with residue 3 of the fibrin beta chain; it ran through to a terminator codon following the carboxy-terminal residue at position 443 and then continued for an additional 606 nucleotides of noncoding sequence to its 3' end. The inferred amino acid sequence was verified by comparison with assorted cyanogen bromide fragments isolated from the beta-chain protein, including two carbohydrate-containing peptides that corresponded to segments containing the carbohydrate-attachment consensus sequence. Overall, the lamprey chain is 49% identical with the beta chain from human fibrinogen. This is the same degree of resemblance as was found for the lamprey and human gamma chains. Moreover, the principal regions of conservation are the same in both the beta and gamma chains. Differences and similarities in the physiological behavior of the two fibrinogens are assessed in terms of the observed amino acid replacements.     

The homology of complement factor C8 gamma chain and alpha-1-microglobulin

The sequence of the complement factor C8 gamma chain shares a remarkable degree of similarity with that of alpha-1-microglobulin, a member of the alpha-2u-globulin superfamily. This superfamily comprises a diverse group of distantly related animal proteins possessing characteristic structural features and similar functions. Comparison of the C8 gamma chain with these proteins supports its homology to them and suggests a possible functional role.     

The sequence and topology of human complement component C9.

A partial nucleotide sequence of human complement component C9 cDNA representing 94% of the coding region of the mature protein is presented. The amino acid sequence predicted from the open reading frame of this cDNA concurs with the amino acid sequence at the amino-terminal end of three proteolytic fragments of purified C9 protein. No long stretches of hydrophobic residues are present, even in the carboxy-terminal half of the molecule which reacts with lipid-soluble photoaffinity probes. Monoclonal antibody epitopes have been mapped by comparing overlapping fragments of C9 molecule to which the antibodies bind on Western blots. Several of these epitopes map to small regions containing other surface features (e.g., proteolytic cleavage sites and N-linked oligosaccharide). The amino-terminal half of C9 is rich in cysteine residues and contains a region with a high level of homology to the LDL receptor cysteine-rich domains. A model for C9 topology based on these findings is proposed.     

Functional studies of the MACPF domain of human complement protein C8alpha reveal sites for simultaneous binding of C8beta, C8gamma, and C9

Human C8 is one of five components of the membrane attack complex of complement (MAC). It contains three subunits (C8alpha, C8beta, C8gamma) arranged as a disulfide-linked C8alpha-gamma dimer that is noncovalently associated with C8beta. C8alpha, C8beta, and complement components C6, C7, and C9 form the MAC family of proteins. All contain N- and C-terminal modules and an intervening 40-kDa segment referred to as the membrane attack complex/perforin (MACPF) domain. During MAC formation, C8alpha binds and mediates the self-polymerization of C9 to form a pore-like structure on target cells. The C9 binding site was previously shown to reside within a 52-kDa segment composed of the C8alpha N-terminal modules and MACPF domain (alphaMACPF). In the present study, we examined the role of the MACPF domain in binding C9. Recombinant alphaMACPF and a disulfide-linked alphaMACPF-gamma dimer were successfully produced in Escherichia coli and purified. alphaMACPF was shown to simultaneously bind C8beta, C8gamma, and C9 and form a noncovalent alphaMACPF.C8beta.C8gamma.C9 complex. Similar results were obtained for the recombinant alphaMACPF-gamma dimer. This dimer bound C8beta and C9 to form a hemolytically active (alphaMACPF-gamma).C8beta.C9 complex. These results indicate that the principal binding site for C9 lies within the MACPF domain of C8alpha. They also suggest this site and the binding sites for C8beta and C8gamma are distinct. alphaMACPF is the first human MACPF domain to be produced recombinantly and in a functional form. Such a result suggests that this segment of C8alpha and corresponding segments of the other MAC family members are independently folded domains.     

Complementary deoxyribonucleic acid (cDNA) cloning and DNA sequence analysis of rat ovarian inhibins

Two forms of inhibin (A and B), gonadal polypeptide hormones that selectively suppress the secretion of FSH from the anterior pituitary, have been characterized from the porcine and human species, each being composed of a common alpha-chain and one of two distinct, but homologous beta-chains, i.e. alpha beta A and alpha beta B. Using cDNAs encoding the porcine inhibin subunits we have cloned and sequenced the cDNAs encoding the alpha, beta A, and beta B chains of rat ovarian inhibin. Northern analyses of rat testicular RNA with rat ovarian cDNA probes show the presence of mRNAs encoding alpha and beta B chains, but no detectable mRNA encoding the beta A chain under our experimental conditions. This suggests that there may be specific and distinct physiological roles for inhibins A and B. In addition, if there is no extratesticular source of beta A mRNA, then the male rat may be devoid of the stimulators of the secretion of FSH, i.e. activin (beta A beta B) and homoactivin A (beta A beta A), which are derived from the beta subunits of the two inhibins.     

Binding of human complement C8 to C9: role of the N-terminal modules in the C8 alpha subunit

Human C8 is one of five components of the membrane attack complex of complement (MAC). It is composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. The C8alpha and C8beta subunits contain a pair of N-terminal modules [thrombospondin type 1 (TSP1) + low-density lipoprotein receptor class A (LDLRA)] and a pair of C-terminal modules [epidermal growth factor (EGF) + TSP1]. The middle segment of each protein is referred to as the membrane attack complex/perforin domain (MACPF). During MAC formation, C8alpha mediates binding and self-polymerization of C9 to form a pore-like structure on the membrane of target cells. In this study, the portion of C8alpha involved in binding C9 was identified using recombinant C8alpha constructs in which the N- and/or C-terminal modules were either exchanged with those from C8beta or deleted. Those constructs containing the C8alpha N-terminal TSP1 or LDLRA module together with the C8alpha MACPF domain retained the ability to bind C9 and express C8 hemolytic activity. By contrast, those containing the C8alpha MACPF domain alone or the C8alpha MACPF domain and C8alpha C-terminal modules lost this ability. These results indicate that both N-terminal modules in C8alpha have a role in forming the principal binding site for C9 and that binding may be dependent on a cooperative interaction between these modules and the C8alpha MACPF domain.     

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)     

Molecular cloning and DNA homology of plasmid-mediated beta-lactamase genes

Summary Molecular cloning of DNA fragments between 1.5 and 8kb from BamHI, EcoRI, HindIII, SalI, or Sau3A digests permitted the isolation of structural genes coding for TEM-1, ROB-1, OXA-1, OXA-3, OXA-4, OXA-5, PSE-1, PSE-2, PSE-3, PSE-4, CARB-3, CARB-4, AER-1, and LCR-1 -lactamases. Ampicillin-resistant clones were selected and it was confirmed that they contained the respective -lactamase genes by isoelectric focusing. Detailed physical maps of 14 different recombinant plasmids were constructed using 8 restriction endonucleases. Plasmid deletions and lacZ fusions were used to localize the -lactamase structural genes. DNA probes were constructed for the TEM01, ROB-1, OXA-1, and PSE-1 genes. Under conditions of high stringency, hybridization was observed between the genes for TEM-1 and TEM-2 or TLE-1, OXA-1 and OXA-4, and PSE-1 and PSE-4 or CARB-3, while the ROB-1 gene probe showed no cross-hybridization. Such bla gene probes should facilitate studies of -lactamase molecular epidemiology.     

The protein sequence homology of gamma-crystallins among major vertebrate classes and their DNA sequence homology to heat-shock protein genes

A systematic characterization of lens crystallins from five major classes of vertebrates was carried out by exclusion gel filtration, cation-exchange chromatography and N-terminal sequence determination. All crystallin fractions except that of -crystallin were found to be N-terminally blocked. -Crystallin is present in major classes of vertebrates except the bird, showing none, or decreased amounts, of this protein in chicken and duck lenses, respectively. N-Terminal sequence analysis of the purified -crystallin polypeptides showed extensive homology between different classes of vertebrates, supporting the close relatedness of this family of crystallin even from the evolutionarily distant species. Comparison of nucleotide sequences and their predicted amino acid sequences between -crystallins of carp and rat lenses and heat-shock proteins demonstrated partial sequence homology of the encoded polypeptides and striking homology at the gene level. The unexpected strong homology of complementary DNA (cDNA) lies in the regions coding for 40 N-terminal residues of carp -II, rat 2-1, and the middle segments of 23,000- and 70,000-M _r heat-shock proteins. The optimal alignment of DNA sequences along these two segments shows about 50% homology. The percentage of protein sequence identity for the corresponding aligned segments is only 20%. The weak sequence homology at the protein level is also found between the invertebrate squid crystallin and rat -crystallin polypeptides. These results pointed to the possibility of unifying three major classes of vertebrate crystallins into one // superfamily and corroborated the previous supposition that the existing crystallins in the animal kingdom are probably mutually interrelated, sharing a common ancestry.     

Carbohydrate binding protein 35. Complementary DNA sequence reveals homology with proteins of the heterogeneous nuclear RNP

The complete nucleotide sequence of a cDNA clone for carbohydrate binding protein 35, a galactose-specific lectin identified in the nucleus of mouse 3T3 fibroblasts, has been determined. The deduced amino acid sequence suggests that the protein consists of two domains: (a) an amino-terminal portion that is homologous to certain regions of proteins of the heterogeneous nuclear ribonucleoprotein complex, and (b) a carboxyl-terminal portion that is homologous to beta-D-galactoside-specific lectins isolated from a number of animal tissues. This two-domain motif is reminiscent of several DNA- and RNA-binding proteins.     

Nucleotide sequence of complementary DNA and derived amino acid sequence of murine complement protein C3

The nucleotide sequences coding for murine complement component C3 have been determined from a cloned genomic DNA fragment and several overlapping cloned complementary DNA fragments. The amino acid sequence of the protein was deduced. The mature beta and alpha subunits contain 642 and 993 amino acids respectively. Including a 24 amino acid signal peptide and four arginines in the beta-alpha transition region, which are probably not contained in the mature protein, the unglycosylated single chain precursor protein preproC3 would have a molecular mass of 186 484 Da and consist of 1663 amino acid residues. The C3 messenger RNA would be composed of a 56 +/- 2 nucleotide long 5' non-translated region, 4992 nucleotides of coding sequence, and a 3' non-translated region of 39 nucleotides, excluding the poly A tail. The beta chain contains only three cysteine residues, the alpha chain 24, ten of which are clustered in the carboxy terminal stretch of 175 amino acids. Two potential carbohydrate attachment sites are predicted for the alpha chain, none for the beta chain. From a comparison with human C3 cDNA sequence (of which over 80% has been determined) an extensive overall sequence homology was observed. Human and murine preproC3 would be of very similar length and share several noteworthy properties: the same order of the subunits in the precursor, the same basic residue multiplet in the beta-alpha transition region, and a glutamine residue in the thioester region. The equivalent position of the known factor I cleavage sites in human C3 alpha could be located in the murine C3 alpha chain and the size and sequence of the resulting peptide were deduced. A comparison of the amino acid sequences of murine C3 and human alpha 2-macroglobulin is given. Several areas of strong sequence homology are observed, and we conclude that the two genes must have evolved from a common ancestor.     

Partial sequence homology of human myc oncogene protein to beta and gamma crystallins

The human cellular myc gene is one of about 20 cellular oncogenes which code for a variety of proteins including protein kinases and growth factors. The human gene is related to the avian myelocytomatosis leukaemia virus MC29 and produces a binding protein which may be involved in regulation of gene expression and cellular differentiation and proliferation. The crystallins are proteins in the eye lens synthesised at different stages of cell differentiation and proliferation, and whose short range order is necessary for lens transparency. Computer-based sequence comparisons show that beta Bp and gamma II crystallins, which show partial sequence homology and conservation of 'Greek Key' motives are also partially homologous to two regions on the human myc protein, though this protein probably does not conserve the 'Greek Key' structural motives.     

Interaction between the C8 alpha-gamma and C8 beta subunits of human complement C8: role of the C8 beta N-terminal thrombospondin type 1 module and membrane attack complex/perforin domain

Human C8 is one of five complement components (C5b, C6, C7, C8, and C9) that interact to form the cytolytic membrane attack complex (MAC). It is an oligomeric protein composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. C8alpha and C8beta are homologous; both contain an N-terminal thrombospondin type 1 (TSP1) module, a low-density lipoprotein receptor class A (LDLRA) module, an extended central segment referred to as the membrane attack/perforin (MACPF) domain, an epidermal growth factor (EGF) module, and a second TSP1 module at the C-terminus. In this study, the segment of C8beta that confers binding specificity toward C8alpha-gamma was identified using recombinant C8beta constructs in which the N- and/or C-terminal modules were deleted or exchanged with those from C8alpha. Constructs were tested for their ability to bind C8alpha-gamma in solution and express C8 hemolytic activity. Binding to C8alpha-gamma was found to be dependent on the TSP1 + LDLRA + MACPF segment of C8beta. Within this segment, the TSP1 module and MACPF domain are principally involved and act cooperatively to mediate binding. Results from activity assays suggest that residues within this segment also mediate binding and incorporation of C8 into the MAC.     

Complementary DNA cloning of poplar bark storage protein and control of its expression by photoperiod

Bark storage proteins accumulate in the bark of many woody plants during autumn and winter. In poplar (Populus deltoides Bartr. ex Marsh), the accumulation of the 32-kilodalton bark storage protein is controlled by photoperiod. We have isolated a full-length cDNA encoding for the poplar 32-kilodalton bark storage protein and determined its nucleotide sequence. The derived amino acid sequence shows that poplar bark storage protein is rich in serine, leucine, phenylalanine, and lysine. Poplar bark storage protein is similar to the poplar wound-induced cDNA clone 4 and clone 16 (TJ Parsons, HD Bradshaw, MP Gordon [1989] Proc Natl Acad Sci USA 86: 7895-7899). DNA gel blot analysis suggests that poplar bark storage protein is encoded by a multigene family of about five genes. Poplar plants grown in long days contained low levels of mRNA for the bark storage protein. Exposure to short days resulted in an increase in bark storage protein mRNA within 7 days. After 21 days of short day exposure, high levels of mRNA were detected. The accumulation of bark storage protein mRNA in response to short days was also observed in plants exposed to natural shortening daylengths. Our results indicate that the accumulation of poplar bark storage protein mRNA is controlled by photoperiod. This finding will provide a useful system for investigating photoperiodism in woody plants.     

Limited proteolysis of complement components C2 and factor B. Structural analogy and limited sequence homology.

A method is described for the simultaneous purification of milligram quantities of complement components C2 and Factor B. Both products are homogeneous by the criteria of polyacrylamide-gel electrophoresis and N-terminal sequence analysis. Component C2 is cleaved by serine proteinase C1s at an X-Lys bond to give fragment C2a (approx. mol.wt. 74000) and fragment C2b (approx. mol.wt. 34000). The two fragments can be separated by gel filtration without the need for reducing or denaturing agents. Fragment C2b represents the N-terminal end of the molecule. Similar results were seen on cleavage of Factor B by Factor D in the presence of component C3. Again two non-covalently linked fragments are formed. The smaller, fragment Ba (approx. mol.wt. 36,000),) has threonine as the N-terminal residue, as does Factor B; the larger, fragment Bb (approx. mol. wt. 58000), has lysine as the N-terminal residue. A similar cleavage pattern is obtained on limited proteolysis of Factor B by trypsin, suggesting an Arg-Lys-or Lys-Lys bond at the point of cleavage. Although component C2 and Factor B show no apparent N-terminal sequence homology, a limited degree of sequence homology is seen around the sites of proteolytic cleavage.