Isolation, cDNA cloning, and overexpression of a 33-kD cell surface glycoprotein that binds to the globular 'heads' of C1q

This work describes the functional characterization, cDNA cloning, and expression of a novel cell surface protein. This protein designated gC1q-R, was first isolated from Raji cells and was found to bind to the globular 'heads' of C1q molecules, at physiological ionic strength, and also to inhibit complement-mediated lysis of sheep erythrocytes by human serum. The NH 2-terminal amino acid sequence of the first 24 residues of the C1q- binding protein was determined and this information allowed the synthesis of two degenerate polymerase chain reaction primers for use in the preparation of a probe in the screening of a B cell cDNA library. The cDNA isolated, using this probe, was found to encode a pre-pro protein of 282 residues. The NH 2 terminus of the protein isolated from Raji cells started at residue 74 of the predicted pre-pro sequence. The cDNA sequence shows that the purified protein has three potential N-glycosylation residues and is a highly charged, acidic molecule. Hence, its binding to C1q may be primarily but not exclusively due to ionic interactions. The 'mature' protein, corresponding to amino acid residues 74-282 of the predicted pre-pro sequence, was overexpressed in Escherichia coli and was purified to homogeneity. This recombinant protein was also able to inhibit the complement-mediated lysis of sheep erythrocytes by human serum and was shown to be a tetramer by gel filtration in nondissociating conditions. Northern blot and RT-PCR studies showed that the C1q-binding protein is expressed at high levels in Raji and Daudi cell lines, at moderate levels in U937, Molt-4, and HepG2 cell lines, and at a very low level in the HL60 cell line. However, it is not expressed in the K562 cell line. Comparison of gC1q-R NH 2-terminal sequence with that of the receptor for the collagen-like domain of C1q (cC1q-R) showed no similarity. Furthermore, antibodies to gC1q-R or an 18-amino acid residue- long NH 2-terminal synthetic gC1q-R peptide did not cross-react with antibodies to cC1q-R. Anti-gC1q-R immunoblotted a 33-kD Raji cell membrane protein, whereas anti cC1q-R recognized a molecule of ~60 kD. The NH 2- terminal sequence of gC1g-R appears to be displayed extracellularly since anti-gC1g-R peptide reacted with surface molecules on lymphocytes, polymorphonuclear leukocytes, and platelets, as assessed by flow cytometric and confocal laser scanning microscopic analyses. In addition, all or part of the gC1q binding domain may reside within the 24 amino acid stretch of the NH 2-terminal sequence of gC1q-R since the 18 amino acid residue long- synthetic peptide corresponding to this region inhibited serum C1q hemolytic activity. The data presented in this report suggest that there are at least two types of C1q-R which appear to be expressed on the same type of cells and these receptors individually or in concert may contribute to the diversity of C1q-mediated responses.published_or_final_versio

Cell-specific deletion of C1qa identifies microglia as the dominant source of C1q in mouse brain

BACKGROUND: The complement cascade not only provides protection from infection but can also mediate destructive inflammation. Complement is also involved in elimination of neuronal synapses which is essential for proper development, but can be detrimental during aging and disease. C1q, required for several of these complement-mediated activities, is present in the neuropil, microglia, and a subset of interneurons in the brain. METHODS: To identify the source(s) of C1q in the brain, the C1qa gene was selectively inactivated in the microglia or Thy-1(+) neurons in both wild type mice and a mouse model of Alzheimer’s disease (AD), and C1q synthesis assessed by immunohistochemistry, QPCR, and western blot analysis. RESULTS: While C1q expression in the brain was unaffected after inactivation of C1qa in Thy-1(+) neurons, the brains of C1qa (FL/FL) :Cx3cr1 (CreERT2) mice in which C1qa was ablated in microglia were devoid of C1q with the exception of limited C1q in subsets of interneurons. Surprisingly, this loss of C1q occurred even in the absence of tamoxifen by 1 month of age, demonstrating that Cre activity is tamoxifen-independent in microglia in Cx3cr1 (CreERT2/WganJ) mice. C1q expression in C1qa (FL/FL) : Cx3cr1 (CreERT2/WganJ) mice continued to decline and remained almost completely absent through aging and in AD model mice. No difference in C1q was detected in the liver or kidney from C1qa (FL/FL) : Cx3cr1 (CreERT2/WganJ) mice relative to controls, and C1qa (FL/FL) : Cx3cr1 (CreERT2/WganJ) mice had minimal, if any, reduction in plasma C1q. CONCLUSIONS: Thus, microglia, but not neurons or peripheral sources, are the dominant source of C1q in the brain. While demonstrating that the Cx3cr1 (CreERT2/WganJ) deleter cannot be used for adult-induced deletion of genes in microglia, the model described here enables further investigation of physiological roles of C1q in the brain and identification of therapeutic targets for the selective control of complement-mediated activities contributing to neurodegenerative disorders. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12974-017-0814-9) contains supplementary material, which is available to authorized users

The role of complement in ocular pathology

Functionally active complement system and complement regulatory proteins are present in the normal human and rodent eye. Complement activation and its regulation by ocular complement regulatory proteins contribute to the pathology of various ocular diseases including keratitis, uveitis and age-related macular degeneration. Furthermore, a strong relationship between age-related macular degeneration and polymorphism in the genes of certain complement components/complement regulatory proteins is now well established. Recombinant forms of the naturally occurring complement regulatory proteins have been exploited in the animal models for treatment of these ocular diseases. It is hoped that in the future recombinant complement regulatory proteins will be used as novel therapeutic agents in the clinic for the treatment of keratitis, uveitis, and age-related macular degeneration

A transcriptome-wide association study among 97,898 women to identify candidate susceptibility genes for epithelial ovarian cancer risk

Large-scale genome-wide association studies (GWAS) have identified approximately 35 loci associated with epithelial ovarian cancer (EOC) risk. The majority of GWAS-identified disease susceptibility variants are located in non-coding regions, and causal genes underlying these associations remain largely unknown. Here we performed a transcriptome-wide association study to search for novel genetic loci and plausible causal genes at known GWAS loci. We used RNA sequencing data (68 normal ovarian-tissue samples from 68 individuals and 6,124 cross-tissue samples from 369 individuals) and high-density genotyping data from European descendants of the Genotype-Tissue Expression (GTEx V6) project to build ovarian and cross-tissue models of genetically regulated expression using elastic net methods. We evaluated 17,121 genes for their cis-predicted gene expression in relation to EOC risk using summary statistics data from GWAS of 97,898 women, including 29,396 EOC cases. With a Bonferroni-corrected significance level of P<2.2×10-6, we identified 35 genes including FZD4 at 11q14.2 (Z=5.08, P=3.83×10-7, the cross-tissue model; 1 Mb away from any GWAS-identified EOC risk variant), a potential novel locus for EOC risk. All other 34 significantly-associated genes were located within 1 Mb of known GWAS-identified loci, including 23 genes at 6 loci not previously linked to EOC risk. Upon conditioning on nearby known EOC GWAS-identified variants, the associations for 31 genes disappeared and 3 genes remained (P<1.47 x 10-3). These data identify one novel locus (FZD4) and 34 genes at 13 known EOC risk loci associated with EOC risk, providing new insights into EOC carcinogenesis

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

Structural similarity between bovine conglutinin and bovine lung surfactant protein D and demonstration of liver as a site of synthesis of conglutinin

Conglutinin is a Ca2+-dependent, carbohydrate-binding, serum protein which contains an N-terminal collagen-like region and a C-terminal, C-type lectin domain. To date, conglutinin, which appears to play an important role in defence mechanisms, has been fully described, by protein sequence analysis, only in the bovine system. To allow comparison of lung surfactant protein D (SP-D) with conglutinin, within one species, a full-length cDNA clone for SP-D has been isolated from a bovine lung library. The derived amino acid sequence for bovine SP-D shows a higher (78%) level of identity to the sequence of conglutinin than to the sequence of human or rat SP-D (67 and 65% respectively). However, SP-D and conglutinin are known to have different carbohydrate-binding specificities, therefore some of the 16 residues conserved in the C-type lectin domains of all three species of SP-D, but which are not conserved in conglutinin, appear likely to be involved in determination of specificity. The use of a polymerase chain reaction (PCR)-derived DNA probe for bovine SP-D in Northern blotting studies yielded a signal from bovine liver mRNA as well as the expected signal from bovine lung mRNA. Since SP-D appears to be a lung-specific protein, it seems probable that the liver is the primary site of synthesis of conglutinin.link_to_subscribed_fulltex