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PubMed2003

Optimized protocol for linear RNA amplification and application to gene expression profiling of human renal biopsies.

Gene expression analysis using high-density cDNA or oligonucleotide arrays is a rapidly emerging tool for transcriptomics, the analysis of the transcriptional state of a cell or organ. One of the limitations of current methodologies is the requirement of a relatively large amount of total or polyadenylated RNA as starting material. Standard array hybridization protocols require 5-15 micrograms labeled RNA. To obtain these quantities from small amounts of starting RNA material, RNA can be amplified in a linear fashion. Here we introduce an optimized protocol for rapid and easy-to-use amplification of as little as 1 ng total RNA. Our analysis shows that this method is linear and highly reproducible and that it preserves similarities as well as dissimilarities between normal and disease-related samples. We applied this technique to the RNA expression profiling of human renal allograft biopsies with normal histology and compared them to the profiles of renal biopsies with histological evidence of chronic transplant nephropathy or chronic rejection. Among others, complement component C1r was found to be significantly up-regulated in chronic rejection and chronic transplant nephropathy biopsies compared to normal samples, while fructose-1,6-biphosphatase showed lower-than-normal expression.

Gene Expression Profiling↗
PubMed1988

Selective synthesis of mRNA and proteins by human peripheral blood neutrophils.

Human peripheral blood polymorphonuclear neutrophils (PMN) have been considered to be capable of little if any protein biosynthesis. We evaluated the ability of PMN to synthesize both mRNA and proteins. Using in vitro [35S]methionine pulse-chase labeling of purified PMN, followed by immunoprecipitation of cell lysates with immobilized mAb and analysis by gel electrophoresis, PMN were shown to synthesize CR1, FcR, CR3 alpha-chain, MHC class I, and actin. In contrast, incorporation of [35S]methionine into either CR3 beta-chain or the secondary granule protein lactoferrin was not detected. Purification of mRNA from PMN and analysis by Northern blots demonstrated the presence in PMN of CR1, actin, and MHC class I transcripts. However, despite the apparent lack of CR3 beta-chain biosynthesis, specific beta-chain message was detectable in PMN RNA. Inhibition of mRNA synthesis in PMN with actinomycin D resulted in decreased synthesis of nascent CR1, FcR, MHC class I, and actin compared with control cells. Thus, PMN continue to transcribe and translate the genes for certain membrane and cytoskeletal proteins. In contrast, the lack of detectable synthesis of either lactoferrin or CR3 beta-chain suggested that biosynthesis in circulating PMN is selective.

Blood Proteins↗
PubMed1981

[Isolation and crystallization of the Clr subunit of the first component of human complement (author's transl)].

Isolation of Clr from Cohn-Fraction I of human plasma is accomplished by a series of steps which include affinity chromatography on IgG-p-azobenzyloxyethylsulfonylethoxy-Sepharose CL-4B, ion exchange chromatography and preparative zone electrophoresis. The protein is easily soluble in 0.1 M Tris/HCl buffer, pH 10.0 and is crystallized readily by means of dialysis against 0.15 M saline, pH 7.0 in the form of hexagonal dipyramides. The structural elucidation using X-ray analysis is now possible.

Chromatography, Affinity↗
PubMed1996

Impaired production of both normal and mutant C1 inhibitor proteins in type I hereditary angioedema with a duplication in exon 8.

In the autosomal dominant disorder type I hereditary angioedema, reduced levels of C1 inhibitor may be due in part to increased turnover and decreased synthesis of normal C1 inhibitor protein. A type I hereditary angioedema patient was recently described in whom the C1 inhibitor mutation consisted of a 20-bp duplication of nucleotides 1414 to 1433 in exon 8 that introduced a frame shift predicting the loss of a normal stop codon and the translation of a protein 52 amino acids longer than normal. In this study, we analyzed the expression of C1 inhibitor in fibroblasts obtained from a skin biopsy of this patient. Two proteins of approximately 78 and 94 kDa were found intracellularly, corresponding to the products of normal and mutated alleles, respectively. Pulse-chase analysis showed a complete lack of secretion of the mutated form. In addition, there was decreased extracellular production of the normal C1 inhibitor, suggesting either decreased secretion or increased intracellular catabolism of the normal protein because of the presence of the mutant allele. The production of other complement proteins was normal. This study provides a model for further analysis of autosomal dominant genetic disorders in which production of the functional protein may be affected by the product of the mutated allele.

Alleles↗
PubMed1984

Structure and activity of C1r and C1s.

During activation of the first component of the classical complement pathway the two zymogen subcomponents, C1r and C1s are converted to active proteolytic enzymes. Activated C1r cleaves C1s which then becomes the activator of C4 and C2. Amino acid sequence studies of the proteolytic chains of C1r and C1s, carried out in Oxford and Aberdeen respectively, have shown that they belong to the serine proteinase family. Modelling of these sequences to the three-dimensional coordinates of chymotrypsin (Birktoft & Blow 1972) reveals that both molecules have a conserved structural core, and that most of the differences lie in the external loops. Catalytically functional residues (Ile-16, His-57, Asp-102, Ser-195) are conserved, and residue 189 is aspartic acid, consistent with the known trypsin-like specificity of cleavage. Examination of the amino acid sequences of C4a, and comparison with those of the homologous molecules C3a and C5a, shows that there is a marked difference in the distribution of basic residues near the C-terminal arginine residue which is the site of action of C1s. When these amino acid sequences are modelled to the coordinates of C3a (Huber et al. 1980) and docked to the active site of C1s, the basic residues of C4a appear to interact with two glutamate residues peculiar to C1s, suggesting that this interaction may contribute to the ability of C1s to discriminate C4 from C3 and C5.

Amino Acid Sequence↗
PubMed1980

Assembly of subcomponents C1r and C1s of first component of complement: electron microscopic and ultracentrifugal studies.

Monomeric C1s (Mr, 85,000; s20,w, 4.3S), a subcomponent of first component of complement (C1), the dimer (Mr, 170,000; s20,w, 6.7 S) of C1r, another subcomponent, and the tetrameric complex (C1r,C1s)2 (Mr, 340,000; s20,w, 8.7 S) are elongated molecules. Hydrodynamic equivalents of cylindrical shape have a diameter of 3.3 nm and lengths of 20 nm for C1s, 36 nm for (C1r)2, and 64 nm for (C1r,C1s)2. In electron micrographs the C1r,C1s complex appears as a chain composed of six to eight globular domains with a contour length of 51 nm. A structure is proposed in which (C1r)2 forms a core to which C1s protomers are associated at both ends. The C1 complex (s20,w, 16.3 S) reconstituted from C1q, C1r, and C1s dissociates under the conditions used for electron microscopy. Some features of the C1 complex are revealed in the dissociation products.

Centrifugation, Density Gradient↗
PubMed2000

A novel PCR-based technique using expressed sequence tags and gene homology for murine genetic mapping: localization of the complement genes.

The complement system is a cascade of serum proteins and receptors which forms a vital arm of innate immunity and enhances the adaptive immune response. This work establishes the chromosomal localization of four key genes of the murine complement system. Mapping was performed using a novel and rapid PCR restriction length polymorphism method which was developed to exploit the murine expressed sequence tag (EST) database. This technique circumvents the laborious cDNA or genomic cloning steps of other mapping methods by relying on EST data and the prediction of exon-intron boundaries. This method can be easily applied to the genes of other systems, ranging from the interests of the individual researcher to large-scale gene localization projects. Here the complement system, probably one of the most well-characterized areas of immunology, was used as a model system. It was shown that the C3a receptor C1r and C1s genes form an unexpected complement gene cluster towards the telomeric end of chromosome 6. The second mannose binding lectin-associated serine protease gene was mapped to the telomeric end of chromosome 4, which is distinct from other complement-activating serine proteases. These results provide new insights into the evolution of this group of proteins.

Animals↗
PubMed1982

Structural features of the first component of human complement, C1, as revealed by surface iodination.

Lactoperoxidase-catalysed surface iodination and sucrose-gradient ultracentrifugation were used to investigate the structure of human complement component C1. 1. Proenzymic subcomponents C1r and C1s associated to form a trimeric C1r2-C1s complex (7.6 S) in the presence of EDTA, and a tetrameric Clr2-C1s2 complex (9.1 S) in the presence of Ca2+. Iodination of the 9.1 S complex led to a predominant labelling of C1r (70%) over C1s (30%), essentially located in the b-chain moiety of C1r and in the a-chain moiety of C1s. 2. Reconstruction of proenzymic soluble C1 (15.2 S) from C1q, C1r and C1s was partially inhibited when C1s labelled in its monomeric form was used and almost abolished when iodinated C1r was used. Reconstruction of fully activated C1 was not possible, whereas hybrid C1q-C1r2-C1s2 complex was obtained. 3. Iodination of proenzymic or activated C1 bound to IgG-ovalbumin aggregates led to an equal distribution of the radioactivity between C1q and C1r2-C1s2. With regard to C1q, the label distribution between the three chains was similar whether C1 was in its proenzymic or activated form. Label distribution in the C1r2-C1s2 moiety of C1 was the same as that obtained for isolated C1r2-C1s2, and this was also true for the corresponding activated components. However, two different labelling patterns were found, corresponding to the proenzyme and the activated states.

Centrifugation, Density Gradient↗
PubMed1989

Control of immune complexes by the classical pathway.

The association between inherited deficiencies of the classical pathway complement components (C1q, C1r, C1s, C4, C2 and C3) and immune complex disease shows that complement is involved in protection against the development of immune complex disease (ICD). This protection is conferred by the ability of the complement system to keep antigen antibody complexes (IC) small and soluble. Two mechanisms exist, prevention of immune precipitation (PIP), which inhibits the formation of large insoluble lattices when IC are formed in the presence of complement (nascent IC), and solubilisation of preformed immune precipitates (SOL). PIP is probably the more important as it is unlikely that, in vivo, IC are ever formed in the absence of complement. PIP displays an absolute dependency upon the classical pathway while SOL is alternative pathway dependent. However, for optimal efficiency SOL requires an intact classical pathway. Thus the classical pathway plays a role in both PIP and SOL. The end result of both processes is the covalent binding of C3b to the IC lattice, which not only keeps IC soluble, but permits binding to CR1 for removal from the circulation. The sera of patients with ICD contain a factor (s) which inhibits PIP. The sera of RA patients inhibits PIP and purified IgM-RF has been shown to inhibit this function. However a second inhibitor of PIP has recently been purified, a glycoprotein (Mr 60 kd) (gp60) which is present in normal serum and in increased concentration in RA sera. Gp60 binds to the Fc piece of IgG, but not to IgA or IgM, and competes with C1q for a binding site on IgG Fc. Thus gp60 appears to act by preventing binding and activation of C1 by IgG containing IC.

Antigen-Antibody Complex↗
PubMed1991

Atypical structured glomerular deposits: an immunohistochemical study.

Atypical structured glomerular deposits were identified in sub-epithelial, sub-endothelial and mesangial areas in biopsy tissue from a female, aged 33, who presented with anaemia and was found to have proteinuria and microscopic haematuria. Histological examination showed that the deposits were periodic acid Schiff positive and silver negative whilst stains for amyloid were negative. Immunofluorescent staining for all immunoglobulins was negative, and only C3 showed moderate labelling. Conventional electron microscopy revealed that all deposits contained microtubular structures of variable length but with an average diameter of 25 nm and a periodicity of approximately 16 nm. The glomerular basement membrane was interrupted in many areas by deposits, and also contained 'myelin-like' structures. Free microtubular structures were also seen in the urinary space. Immunoelectron microscopy using protein-A-gold confirmed the immunofluorescent findings with immunoglobulins and fibrinogen, showed marked positive labelling of deposits with C1s and C3d and also intense labelling of coiled microtubular structures with C9. Other complement components C1q, C1r, C3c, C4 and C5 showed weak or negative results. Although these organised glomerular deposits contain complement components, their pathogenesis remains uncertain.

Adult↗
PubMed1999

Gene structure of the P100 serine-protease component of the human Ra-reactive factor.

The Ra-reactive factor (RaRF) is a complement dependent anti-microbial factor that reacts with numerous microorganisms such as viruses, bacteria, fungi and protozoa. It is a complex of a mannan-binding lectin (MBL) and the serine protease, P100 (MASPI). P100 activates the C4 component of the complement system and its domain organization is similar to C1r and C1s. In this study, determination was made of the structure of the human P100 gene which was found longer than 67 kbp and to be comprised of 16 exons. Its non-protease region consisted of 10 exons, as in the case of C1r and C1s, and the introns were found present in the boundary separating two CUB domains, an EGF-like domain and two CCP domains and each CUB and CCP domain contained extra internal introns. The serine protease region was comprised of 6 exons in contrast to C1r and C1s, either of which consists of a single exon. The exon-intron structure was found to reflect the evolution of these molecules and P100 to have derived earlier in the stage of evolution than C1r or C1s.

Amino Acid Sequence↗
PubMed1978

The role of C1s, C1r and properdin in the initiation of the C3b-dependent feedback mechanism of the complement system.

The influence of activated C1s, C1r and properdin in the fluid phase initiation of the C3b-dependent feedback mechanism of the human complement was studied. It was found that C1s caused conversion of C3 and factor B in a normal serum, but not in a serum genetically deficient in C4 or in a serum to which Na2EDTA had been added. When a normal serum was incubated with C1r before incubation with C1s, only C3 was converted, whereas factor B remained in the unaltered native state. Properdin did not influence the C1s mediated conversion of C3 and factor B. When activated properdin was added to a properdin-depleted serum, both C3 and factor B were converted. Activated properdin was also incubated with purified C3 and purified C3b. It was shown that C3 was converted to C3b, but C3b was not degraded despite prolonged incubation.

Chromatography, Affinity↗
PubMed1978

The first component of complement. A quantitative comparison of its biosynthesis in culture by human epithelial and mesenchymal cells.

Epithelial and mesenchymal cells synthesized and secreted all three subcomponents of the first component of complement (C1): C1q, C1r, and C1s. Quantitatively, however, columnar and transitional epithelial cells secreted 400--3,700 times more hemolytically active C1 than monocytes or fibroblasts. Only columnar epithelial cells synthesized C1 subcomponents with subunit structures similar to their serum counterparts. Transitional epithelial cells, fibroblasts, and monocytes produced C1q and C1s with subunits of apparent molecular weights larger than reported values. C1r from all cell lines was physiochemically similar to serum C1r.

Cells, Cultured↗
PubMed1989

Deficiency of the first component of human complement.

C1 deficiency results from an absence or lowering of the level of one or more of the proteins C1q, C1r and C1s, which are the subcomponents of the C1 complex of the classical pathway of the serum complement system. The major clinical pattern shown in such deficiency states is an inability to deal effectively with immune complexes, resulting in the typical symptoms associated with immune-complex-related diseases and a great susceptibility to recurrent bacterial infections. Both acquired and genetic deficiencies of the C1 subcomponents have been reported; the possible genetic deficiencies appear quite rare, with only 14 reports of C1q deficiency (involving 24 people) and six reports of C1r/C1s deficiency (involving 11 people) appearing in the literature to date.

Complement Activation↗
PubMed2001

The role of the individual domains in the structure and function of the catalytic region of a modular serine protease, C1r.

The first enzymatic event in the classical pathway of complement activation is autoactivation of the C1r subcomponent of the C1 complex. Activated C1r then cleaves and activates zymogen C1s. C1r is a multidomain serine protease consisting of N-terminal alpha region interacting with other subcomponents and C-terminal gammaB region mediating proteolytic activity. The gammaB region consists of two complement control protein modules (CCP1, CCP2) and a serine protease domain (SP). To clarify the role of the individual domains in the structural and functional properties of the gammaB region we produced the CCP1-CCP2-SP (gammaB), the CCP2-SP, and the SP fragments in recombinant form in Escherichia coli. We successfully renatured the inclusion body proteins. After renaturation all three fragments were obtained in activated form and showed esterolytic activity on synthetic substrates similar to each other. To study the self-activation process in detail zymogen mutant forms of the three fragments were constructed and expressed. Our major statement is that the ability of autoactivation and C1s cleavage is an inherent property of the SP domain. We observed that the CCP2 module significantly increases proteolytic activity of the SP domain on natural substrate, C1s. Therefore, we propose that CCP2 module provides accessory binding sites. Differential scanning calorimetric measurements demonstrated that CCP2 domain greatly stabilizes the structure of SP domain. Deletion of CCP1 domain from the CCP1-CCP2-SP fragment results in the loss of the dimeric structure. Our experiments also provided evidence that dimerization of C1r is not a prerequisite for autoactivation.

Catalytic Domain↗
PubMed1987

Decay accelerating factor (DAF) peptide sequences share homology with a consensus sequence found in the superfamily of structurally related complement proteins and other proteins including haptoglobin, factor XIII, beta 2-glycoprotein I, and the IL-2 receptor.

Amino acid sequence data derived from tryptic peptides of the decay accelerating factor indicate that this complement regulatory protein contains a sequence with homology to the superfamily of structurally related complement proteins, including the C4 binding protein, factor H, complement receptor type 1, complement receptor type 2, Ba, C1r, and to their non-complement relatives, including beta 2-glycoprotein I, factor XIIIb, the alpha 1 chain of haptoglobin, and the interleukin 2 receptor. Identifying DAF as a member of the superfamily of structurally related complement proteins provides evidence that DAF may contain a functionally important C4b and C3b binding domain.

Amino Acid Sequence↗