The gamma subunit of the eighth complement component (C8) in rainbow trout

Of the 35 proteins, enzymes, receptors and regulatory components of the complement system, C8gamma is unique in that it is the only lipocalin. C8gamma is a subunit of the C8 molecule, which is one of the five components (C5b, C6, C7, C8 and C9) that interact as a consequence of complement activation to form the membrane attack complex. Until now, C8gamma has been characterized only in mammalian species. In order to elucidate the phylogeny of this molecule, we have cloned the C8gamma subunit in rainbow trout (Oncorhynchus mykiss), a teleost fish representing a critical point in the evolutionary divergence of the complement system. The deduced amino acid sequence of trout C8gamma shows significant identity (37%) to the human C8gamma homolog and much lower to the other known lipocalins. The lipocalin domain is present and all the cysteine residues are conserved. The trout C8gamma gene is probably present as a single copy in the trout genome showing a differential expression pattern among tissues investigated.     

In vitro expression of the beta subunit of human complement component C8

Human C8 is one of five components of the cytolytic C5b-9 complex of complement. It is an oligomeric protein composed of three subunits (α, β, γ) encoded in separate genes. These are arranged as a disulfide-linked α-γ dimer and a noncovalently associated β chain. Biosynthesis studies and analyses of humans with hereditary C8 deficiencies suggest that C8α-γ synthesis and secretion can occur independently of C8β, but that serum levels of C8β are dependent on C8α-γ. One aim of the present study was to determine if functional human C8β could be synthesized in the absence of C8α-γ. Human C8β expression constructs were prepared and used to produce recombinant C8β (rC8β) in insect and COS-7 cells. Both cell types secreted rC8β that was similar in size to human C8β and exhibited similar ability to associate with human C8α-γ and form functional C8. A mutant form of C8β in which N-glycosylation sites were eliminated was also expressed and found to be functionally similar to rC8β and human C8β. These results indicate that C8α-γ is not required for intracellular processing and secretion of C8β. Furthermore, N-linked carbohydrate on C8β is not necessary for association with C8α-γ or for C8 activity.     

Bacteriolytic activity of rainbow trout (Oncorhynchus mykiss) complement

The total bacteriolytic activity comprising of the classical, alternative and possible lectine pathways as well as the bacteriolytic activity of the alternative pathway (AP) of rainbow trout (Oncorhynchus mykiss) complement was assessed in temperatures ranging from 0 to 35 °C against a recombinant strain Escherichia coli containing two reporter genes gfp and lucFF. At 35 °C there was no difference between the total (TC) activity and the activity of the AP, but at 10 °C the TC was notably higher than the AP. Total activity peaked at 30 °C and gradually grew smaller towards 0 °C. The activity of the AP was similarly temperature-dependent, but CB50 value was found to be beyond measurable range at temperatures below 10 °C. When compared to human serum complement, the peak human TC activity at 37 °C was four times higher than the TC of rainbow trout at 30 °C. Human TC activity was 10.1-fold lower at 25 °C when compared to the activity at 37 °C. At 37 °C the human AP bacteriolytic activity was 4.5-fold less effective than human TC, but at 25 °C there was no difference between human TC and AP. In contrast to previous reports where AP activity of fish was assayed as hemolytic activity our study showed that the bacteriolytic activity of AP was lower than that of TC and very low at temperatures below 10 °C suggesting that the earlier proposed particular importance of AP in fish should be reconsidered.     

Anaphylatoxin-like molecules generated during complement activation induce a dramatic enhancement of particle uptake in rainbow trout phagocytes

Here we have identified a serum fraction containing 8-kDa molecules with an unexpected capacity to greatly enhance particle uptake in trout head kidney leukocytes (HKLs). This 8-kDa particle-uptake enhancing fraction (PUEF-8) was purified from complement-activated serum by gel filtration chromatography. Mass spectrometric analysis and reactivity of anti-trout C3-1 and C4 antibodies, indicated the presence of C3a, C4a and C5a molecules in PUEF-8. Using a newly developed flow cytometric assay that measures the capacity of cells to ingest fluorescent beads, we showed that PUEF-8 induced a striking enhancement (344±50% higher than the PBS control value) in the number of HKLs ingesting three or more beads. In contrast, the effect of PUEF-8 on peripheral blood leukocytes (PBLs) was almost negligible. Interestingly, PUEF-8 acted as a strong chemoattractant for both HKLs and PBLs. These findings suggest a novel role for the anaphylatoxins generated during complement activation in teleost fish.     

Sequence and expression analysis of rainbow trout CXCR2, CXCR3a and CXCR3b aids interpretation of lineage-specific conversion,loss and expansion of these receptors during vertebrate evolution

The chemokine receptors CXCR1–3 bind to 11 chemokines (CXCL1–11) that are clustered on the same chromosome in mammals but are largely missing in ray-finned fish. A second CXCR1/2, and a CXCR3a and CXCR3b gene have been cloned in rainbow trout. Analysis of CXCR1–R3 genes in lobe-finned fish, ray-finned fish and tetrapod genomes revealed that the teleostomian ancestor likely possessed loci containing both CXCR1 and CXCR2, and CXCR3a and CXCR3b. Based on this synteny analysis the first trout CXCR1/2 gene was renamed CXCR1, and the new gene CXCR2. The CXCR1/R2 locus was shown to have further expanded in ray-finned fish. In relation to CXCR3, mammals appear to have lost CXCR3b and birds both CXCR3a and CXCR3b during evolution. Trout CXCR1–R3 have distinct tissue expression patterns and are differentially modulated by PAMPs, proinflammatory cytokines and infections. They are highly expressed in macrophages and neutrophils, with CXCR1 and CXCR2 also expressed in B-cells.     

cDNA cloning and phylogenetic analysis of the sixth complement component in rainbow trout

The sixth complement protein (C6) is an essential component of the membrane attack complex (MAC); the end product of the lytic pathway of complement activation. The MAC complex constitutes a supramolecular assembly containing the five precursor proteins C5b, C6, C7, C8, and C9. Once assembled on the target surface it forms transmembrane channels that cause membrane damage and cytolysis of complement-opsonized pathogens. Besides mediating direct pathogen elimination, exposure of cells to sublytic doses of MAC can trigger diverse cellular responses such as, cell activation, induction of apoptosis, cell cycle re-entry and proliferation in various biological settings. The terminal complement components (C6-C9) are structurally related proteins, differing in size and complexity. In order to study their evolution, we report here the cloning and molecular characterization of C6 component in rainbow trout. The deduced amino acid sequence of trout C6 exhibits 55 and 44% identity with zebra fish and human orthologs, respectively. The 'domain' architecture of trout C6 resembles that of mammalian counterparts, and the cysteine backbone is also conserved. Finally, trout C6 gene appears to exist as a single copy in the trout genome, and is expressed in a wide range of trout tissues.     

Localization of rem2 in the central nervous system of the adult rainbow trout (Oncorhynchus mykiss)

Rem2 is member of the RGK (Rem, Rad, and Gem/Kir) subfamily of the Ras superfamily of GTP binding proteins known to influence Ca²⁺ entry into the cell. In addition, Rem2, which is found at high levels in the vertebrate brain, is also implicated in cell proliferation and synapse formation. Though the specific, regional localization of Rem2 in the adult mammalian central nervous system has been well-described, such information is lacking in other vertebrates. Rem2 is involved in neuronal processes where the capacities between adults of different vertebrate classes vary. Thus, we sought to localize the rem2 gene in the central nervous system of an adult anamniotic vertebrate, the rainbow trout (Oncorhynchus mykiss). In situ hybridization using a digoxigenin (DIG)-labeled RNA probe was used to identify the regional distribution of rem2 expression throughout the trout central nervous system, while real-time polymerase chain reaction (rtPCR) further supported these findings. Based on in situ hybridization, the regional distribution of rem2 occurred within each major subdivision of the brain and included large populations of rem2 expressing cells in the dorsal telencephalon of the cerebrum, the internal cellular layer of the olfactory bulb, and the optic tectum of the midbrain. In contrast, no rem2 expressing cells were resolved within the cerebellum. These results were corroborated by rtPCR, where differential rem2 expression occurred between the major subdivisions assayed with the highest levels being found in the cerebrum, while it was nearly absent in the cerebellum. These data indicate that rem2 gene expression is broadly distributed and likely influences diverse functions in the adult fish central nervous system.     

补体经典激活途径C3转化酶的体外组装及活性观察

目的：体外组装包含人C4分子的补体经典激活途径C3转化酶，并对其转化酶活性及衰变特性进行观察。方法：利用豚鼠血清功能纯C1、C2及溶血中间体EAC4^hu体外组装经典途径C3转化酶，观察不同C1、C2用量及孵育温度对C3转化酶形成和自发性衰变的影响，以及人红细胞膜抽提蛋白对C3转化酶衰变化的影响。结果：高剂量和低剂量的C1均会影响C3转化酶的形成，增加C2用量可增加C3转化酶的形成数量，C3转化酶的自发性衰变随孵育温度的升高而加速，人红细胞膜抽提蛋白可抑制C3转化酶的自发性衰变过程，结论：C1、C2用量及孵育温度是影响C3转化酶形成和自发性衰变的主要因素，体外组装的补体经典激活途径C3转化酶可应用于相关补体调控蛋白的活性检测。     

Evolutionary analysis of two complement C4 genes: Ancient duplication and conservation during jawed vertebrate evolution

The complement C4 is a thioester-containing protein, and a histidine (H) residue catalyzes the cleavage of the thioester to allow covalent binding to carbohydrates on target cells. Some mammalian and teleost species possess an additional isotype where the catalytic H is replaced by an aspartic acid (D), which binds preferentially to proteins. We found the two C4 isotypes in many other jawed vertebrates, including sharks and birds/reptiles. Phylogenetic analysis suggested that C4 gene duplication occurred in the early days of the jawed vertebrate evolution. The D-type C4 of bony fish except for mammals formed a cluster, termed D-lineage. The D-lineage genes were located in a syntenic region outside MHC, and evolved conservatively. Mammals lost the D-lineage before speciation, but D-type C4 was regenerated by recent gene duplication in some mammalian species or groups. Dual C4 molecules with different substrate specificities would have contributed to development of the antibody-dependent classical pathway.     

A new single-nucleotide polymorphism in the seventh component of complement (C7) gene

A new single-nucleotide polymorphism has been found in the 3′ untranslated region of the complement component C7 gene. It is present with similar frequencies in the Japanese and Germans. This polymorphism would be a useful marker in the genetic study of C6 and C7 deficiencies. Received: March 10, 1999 / Accepted: April 9, 1999     

Complement receptors type 1 (CR1, CD35) and 2 (CR2, CD21) cooperate in the binding of hydrolyzed complement factor 3 (C3i) to human B lymphocytes

The C3b-binding receptor, CR1/CD35, supports CR2/CD21-mediated activation of complement by human B lymphocytes, possibly by associating with CR2 to promote or stabilize the binding of hydrolyzed C3 (C3i), the primary component of the AP convertase, C3i-Bb. To evaluate this hypothesis, we examined the uptake kinetics and binding equilibria for C3i dimer interaction with human blood cells in the absence and presence of CR1- and CR2-blocking mAb. C3i displayed dual uptake kinetics to B lymphocytes, comprising of rapid binding to CR1 and slower binding to CR2. The forward rate constants (k(1)) for CR1 and CR2, operating independently, differed ca. 9-fold (k(1)=193+/-9.4 and 22.2+/-6.0 x 10(3) M(-1)s(-1), respectively). Equilibrium binding of C3i to B lymphocytes was also complex, varying in strength by ca. 13-fold over the C3i concentration range examined. The maximum association constant (K(a, max)=109+/-27.2 x 10(7) l/mole) was ca. 9- and 6-fold greater, respectively, than those for CR1 or CR2 acting alone (K(a)=13.2+/-5.3 and 18.5+/-3.5 x 10(7) l/mole). The high avidity of the CR1-CR2 complex for C3i is consistent with its rates of C3i uptake and release being determined by CR1 and CR2, respectively.     

Stringent regulation of complement lectin pathway C3/C5 convertase by C4b-binding protein (C4BP)

The complement lectin pathway, an essential component of the innate immune system, is geared for rapid recognition of infections as each C4b deposited via this pathway is capable of forming a C3/C5 convertase. In the present study, role of C4b-binding protein (C4BP) in regulating the lectin pathway C3/C5 convertase assembled on zymosan and sheep erythrocytes coated with mannan (E_Man) was examined. While the C4BP concentration for inhibiting 50% (IC₅₀) formation of surface-bound C3 convertase on the two surfaces was similar to that obtained for the soluble C3 convertase (1.05 nM), 3- and 41-fold more was required to inhibit assembly of the C5 convertase on zymosan (2.81 nM) and E_Man (42.66 nM). No difference in binding interactions between C4BP and surface-bound C4b alone or in complex with C3b was observed. Increasing the C4b density on zymosan (14,000–431,000 C4b/Zym) increased the number of C4b bound per C4BP from 2.87 to 8.23 indicating that at high C4b density all seven α-chains of C4BP are engaged in C4b-binding. In contrast, the number of C4b bound per C4BP remained constant (3.79 ± 0.60) when the C4b density on E_Man was increased. The data also show that C4BP regulates assembly and decay of the lectin pathway C3/C5 convertase more stringently than the classical pathway C3/C5 convertase because of a 7- to 13-fold greater affinity for C4b deposited via the lectin pathway than the classical pathway. C4BP thus regulates efficiently the four times greater potential of the lectin pathway than the classical pathway in generating the C3/C5 convertase and hence production of pro-inflammatory products, which are required to fight infections but occasionally cause pathological inflammatory reactions.     

An NcoI polymorphism in the human complement component 7 (C7) gene

A novel polymorphic site has been found in the 3′ untranslated region (UTR) of the human complement component 7 (C7) gene. The polymorphic site at 14-bp downstream from the TAG stop codon was either C or A (Nco I-digested), with allele frequencies of 0.660 and 0.340. This NcoI polymorphism would be useful to perform a DNA marker haplotype study in patients with deficiencies of the complement genes, such as C6, C7, C9, which are located closely on chromosome 5p13. Received: March 25, 1999 / Accepted: April 3, 1999     

The covalent binding story of the complement proteins C3 and C4 (I) 1972-1981

Alex Law and Paul Levine recall their work to establish the covalent bond between C3 and target surfaces. It started with a naive experiment by analyzing the membrane polypeptides of sheep erythrocytes bound with ¹²⁵I-labelled C3. They found complexes with molecular weight higher than the individual C3 polypeptides. These complexes survived all conditions designed to disrupt non-covalent interactions. They then showed that the bond was an ester, with an active acyl group on C3 which reacted with a hydroxyl group on the acceptor molecule. With the discovery of an internal thioester by Jim Prahl, Jamila Janatova, Brian Tack and their colleagues, it became clear that the reaction was by an acyl transfer from the thioester of C3 to the target hydroxyl group. Later on they showed that C4 also bound covalently to target molecules. By establishing a fluid phase system to study the kinetics of the binding reactions of C3 and C4, Alex was able to continue the work in the MRC Immunochemistry Unit in Oxford from 1981, to eventually determine the chemical mechanism of the binding reaction. In order to give some sense of reality, this article is written as a narrative from Alex, who did the experiments.Both Alex and Paul are retired. Pauls lives on Martha’s Vineyard where he writes occasional articles on science for one of the Island's newspapers. Alex lives in Hong Kong and tries to make some sense of the local politics.     

Ionic tethering contributes to the conformational stability and function of complement C3b

C3b, the central component of the alternative pathway (AP) of the complement system, coexists as a mixture of conformations in solution. These conformational changes can affect interactions with other proteins and complement regulators. Here we combine a computational model for electrostatic interactions within C3b with molecular imaging to study the conformation of C3b. The computational analysis shows that the TED domain in C3b is tethered ionically to the macroglobulin (MG) ring. Monovalent counterion concentration affects the magnitude of electrostatic forces anchoring the TED domain to the rest of the C3b molecule in a thermodynamic model. This is confirmed by observing NaCl concentration dependent conformational changes using single molecule electron microscopy (EM). We show that the displacement of the TED domain is compatible with C3b binding to Factor B (FB), suggesting that the regulation of the C3bBb convertase could be affected by conditions that promote movement in the TED domain. Our molecular model also predicts mutations that could alter the positioning of the TED domain, including the common R102G polymorphism, a risk variant for developing age-related macular degeneration. The common C3b isoform, C3bS, and the risk isoform, C3bF, show distinct energetic barriers to displacement in the TED that are related to a network of electrostatic interactions at the interface of the TED and MG-ring domains of C3b. These computational predictions agree with experimental evidence that shows differences in conformation observed in C3b isoforms purified from homozygous donors. Altogether, we reveal an ionic, reversible attachment of the TED domain to the MG ring that may influence complement regulation in some mutations and polymorphisms of C3b.     

Expression of interferon alpha/beta receptor in the liver of chronic hepatitis C patients

Interferon (IFN) demonstrates antiviral activity by binding to receptors on the cell surface. Expression of the IFN receptor in hepatocytes may be directly associated with a hepatitis C virus (HCV) infection and the response to IFN therapy. A competitive PCR method was developed to measure IFN alpha/beta (α/β) receptor mRNA in liver samples obtained by needle biopsy. Thirty-one patients with chronic hepatitis C (21 without cirrhosis, 10 with cirrhosis) and six normal subjects were used. Eighteen of the 21 patients without cirrhosis received the IFN therapy. Competitive PCR was carried out using IFN α/β receptor gene-specific primers and a specific competitor. Expression of the receptor was detected in all liver samples. There was no association between the expression level and serum alanine aminotransferase level, serum (2′–5′) oligo (A) synthetase level, amount of serum HCV RNA, or HCV genotype. The expression level in patients with chronic hepatitis was significantly higher than that in normal livers (P < 0.05) and in cirrhotic livers (P < 0.01). Seven of the 18 patients treated with IFN demonstrated a sustained response to IFN (sustained responders), and the remaining 11 did not (nonsustained responders). The expression level of IFN α/β receptor mRNA in the sustained responders was significantly higher than that in the nonsustained responders (P < 0.01). Thus, the expression of IFN α/β receptor mRNA may be one of the host factors influencing the response to IFN therapy. J. Med. Virol. 56:217–223, 1998. © 1998 Wiley-Liss, Inc.     

High frequency of CD8αα homodimer-bearing T cells in human fetal intestine

Immunohistochemistry on frozen sections was used to identify CD8αα cells and CD8αβ cells in human intestine. As observed previously, CD8αβ cells predominate (>95%) in tonsil and post-natal intestine. However in human fetal intestine (16–24 weeks gestation), almost half the CD8⁺ cells in the lamina propria are CD8αα, and many CD8αα cells can be identified in the epithelium. In contrast, in the T cell zones of the Peyer's patches, CD8αβ cells are dominant. The CD8αα cells are virtually all αβ T cell receptor positive. By analogy with the murine system, these CD8αα cells in the fetal gut may be directly derived from the marrow, undergoing thymus-independent differentiation in the gut mucosa.     

β8 integrin and band 4.1B cooperatively regulate morphogenesis of the embryonic heart

Morphogenesis of the heart is regulated by various cues, including growth factors and extracellular matrix (ECM) proteins. The mechanisms by which cardiac cells properly integrate these cues to regulate growth, differentiation, and migration remain poorly understood. Here we have used genetic strategies in mice to identify αvβ8 integrin and its cytoskeletal adaptor protein, Band 4.1B, as essential regulators of cardiac morphogenesis. We demonstrate that approximately 60% of mouse embryos genetically null for β8 integrin and Band 4.1B display cardiovascular phenotypes and die by E11.5. This premature death is due, in part, to defective development of the cardiac outflow tract (OFT), with reduced expression of smooth muscle α‐actin (SMAα‐actin) in OFT cells derived from the cardiac neural crest. These data are the first to identify cell adhesion and signaling pathways regulated by αvβ8 integrin and Band 4.1B as essential for normal formation and function of the heart during embryogenesis. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.