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.     

Recombinant C345C and factor I modules of complement components C5 and C7 inhibit C7 incorporation into the complement membrane attack complex

Complement component C5 binds to components C6 and C7 in reversible reactions that are distinct from the essentially nonreversible associations that form during assembly of the complement membrane attack complex (MAC). We previously reported that the approximately 150-aa residue C345C domain (also known as NTR) of C5 mediates these reversible reactions, and that the corresponding recombinant module (rC5-C345C) binds directly to the tandem pair of approximately 75-residue factor I modules from C7 (C7-FIMs). We suggested from these and other observations that binding of the C345C module of C5 to the FIMs of C7, but not C6, is also essential for MAC assembly itself. The present report describes a novel method for assembling a complex that appears to closely resemble the MAC on the sensor chip of a surface plasmon resonance instrument using the complement-reactive lysis mechanism. This method provides the ability to monitor individually the incorporation of C7, C8, and C9 into the complex. Using this method, we found that C7 binds to surface-bound C5b,6 with a K(d) of approximately 3 pM, and that micromolar concentrations of either rC5-C345C or rC7-FIMs inhibit this early step in MAC formation. We also found that similar concentrations of either module inhibited complement-mediated erythrocyte lysis by both the reactive lysis and classical pathway mechanisms. These results demonstrate that the interaction between the C345C domain of C5 and the FIMs of C7, which mediates reversible binding of C5 to C7 in solution, also plays an essential role in MAC formation and complement lytic activity.     

Crystal structure of C5b-6 suggests structural basis for priming assembly of the membrane attack complex

The complement membrane attack complex (MAC) forms transmembrane pores in pathogen membranes. The first step in MAC assembly is cleavage of C5 to generate metastable C5b, which forms a stable complex with C6, termed C5b-6. C5b-6 initiates pore formation via the sequential recruitment of homologous proteins: C7, C8, and 12–18 copies of C9, each of which comprises a central MAC-perforin domain flanked by auxiliary domains. We recently proposed a model of pore assembly, in which the auxiliary domains play key roles, both in stabilizing the closed conformation of the protomers and in driving the sequential opening of the MAC-perforin β-sheet of each new recruit to the growing pore. Here, we describe an atomic model of C5b-6 at 4.2 Å resolution. We show that C5b provides four interfaces for the auxiliary domains of C6. The largest interface is created by the insertion of an interdomain linker from C6 into a hydrophobic groove created by a major reorganization of the α-helical domain of C5b. In combination with the rigid body docking of N-terminal elements of both proteins, C5b becomes locked into a stable conformation. Both C6 auxiliary domains flanking the linker pack tightly against C5b. The net effect is to induce the clockwise rigid body rotation of four auxiliary domains, as well as the opening/twisting of the central β-sheet of C6, in the directions predicted by our model to activate or prime C6 for the subsequent steps in MAC assembly. The complex also suggests novel small molecule strategies for modulating pathological MAC assembly.     

Structure of human C8 protein provides mechanistic insight into membrane pore formation by complement

C8 is one of five complement proteins that assemble on bacterial membranes to form the lethal pore-like “membrane attack complex” (MAC) of complement. The MAC consists of one C5b, C6, C7, and C8 and 12–18 molecules of C9. C8 is composed of three genetically distinct subunits, C8α, C8β, and C8γ. The C6, C7, C8α, C8β, and C9 proteins are homologous and together comprise the MAC family of proteins. All contain N- and C-terminal modules and a central 40-kDa membrane attack complex perforin (MACPF) domain that has a key role in forming the MAC pore. Here, we report the 2.5 Å resolution crystal structure of human C8 purified from blood. This is the first structure of a MAC family member and of a human MACPF-containing protein. The structure shows the modules in C8α and C8β are located on the periphery of C8 and not likely to interact with the target membrane. The C8γ subunit, a member of the lipocalin family of proteins that bind and transport small lipophilic molecules, shows no occupancy of its putative ligand-binding site. C8α and C8β are related by a rotation of ∼22° with only a small translational component along the rotation axis. Evolutionary arguments suggest the geometry of binding between these two subunits is similar to the arrangement of C9 molecules within the MAC pore. This leads to a model of the MAC that explains how C8-C9 and C9-C9 interactions could facilitate refolding and insertion of putative MACPF transmembrane β-hairpins to form a circular pore.     

Expression and characterization of the C345C/NTR domains of complement components C3 and C5

Complement components C3, C4, and C5 are members of the thioester-containing alpha-macroglobulin protein superfamily. Within this superfamily, a unique feature of the complement proteins is a 150-residue-long C-terminal extension of their alpha-subunits that harbors three internal disulfide bonds. Previous reports have suggested that this is an independent structural module, homologous to modules found in other proteins, including netrins and tissue inhibitors of metalloproteinases. Because of its distribution, this putative module has been named both C345C and NTR. To assess the structures of these segments of the complement proteins, their relationships with other domains, and activities as independent structures, we expressed C345C from C3 and C5 in a bacterial strain that permits cytoplasmic disulfide bond formation. Affinity purification directly from cell lysates yielded recombinant C3- and C5-C345C with properties consistent with multiple intramolecular disulfide bonds and high beta-sheet contents. rC5-, but not rC3-C345C inhibited complement hemolytic activity, and surface plasmon resonance studies revealed that rC5-C345C binds to complement components C6 and C7 with dissociation constants of 10 and 3 nM, respectively. Our results provide strong evidence that this binding corresponds to the previously described reversible binding of C5 to C6 and C7, and taken together with earlier work, indicate that the C5-C345C module interacts directly with the factor I modules in C6 and C7. The high binding affinities suggest that complexes composed of C5 bound to C6 or C7 exist in plasma before activation and may facilitate assembly of the complement membrane attack complex.     

Functional insights from the structure of the multifunctional C345C domain of C5 of complement

The complement protein C5 initiates assembly of the membrane attack complex. This remarkable process results in lysis of target cells and is fundamental to mammalian defense against infection. The 150-amino acid residue domain at the C terminus of C5 (C5-C345C) is pivotal to C5 function. It interacts with enzymes that convert C5 to C5b, the first step in the assembly of the membrane attack complex; it also binds to the membrane attack complex components C6 and C7 with high affinity. Here a recombinant version of this C5-C345C domain is shown to adopt the oligosaccharide/oligonucleotide binding fold, with two helices packed against a five-stranded beta-barrel. The structure is compared with those from the netrin-like module family that have a similar fold. Residues critical to the interaction with C5-convertase cluster on a mobile, hydrophobic inter-strand loop that protrudes from the open face of the beta-barrel. The opposite, helix-dominated face of C5-C345C carries a pair of exposed hydrophobic side chains adjacent to a striking negatively charged patch, consistent with affinity for positively charged factor I modules in C6 and C7. Modeling of homologous domains from complement proteins C3 and C4, which do not participate in membrane attack complex assembly, suggests that this provisionally identified C6/C7-interacting face is indeed specific to C5.     

Distinct localization of the complement C5b‐9 complex on Gram‐positive bacteria

The plasma proteins of the complement system fulfil important immune defence functions, including opsonization of bacteria for phagocytosis, generation of chemo‐attractants and direct bacterial killing via the Membrane Attack Complex (MAC or C5b‐9). The MAC is comprised of C5b, C6, C7, C8, and multiple copies of C9 that generate lytic pores in cellular membranes. Gram‐positive bacteria are protected from MAC‐dependent lysis by their thick peptidoglycan layer. Paradoxically, several Gram‐positive pathogens secrete small proteins that inhibit C5b‐9 formation. In this study, we found that complement activation on Gram‐positive bacteria in serum results in specific surface deposition of C5b‐9 complexes. Immunoblotting revealed that C9 occurs in both monomeric and polymeric (SDS‐stable) forms, indicating the presence of ring‐structured C5b‐9. Surprisingly, confocal microscopy demonstrated that C5b‐9 deposition occurs at specialized regions on the bacterial cell. On Streptococcus pyogenes, C5b‐9 deposits near the division septum whereas on Bacillus subtilis the complex is located at the poles. This is in contrast to C3b deposition, which occurs randomly on the bacterial surface. Altogether, these results show a previously unrecognized interaction between the C5b‐9 complex and Gram‐positive bacteria, whichmight ultimately lead to a new model of MAC assembly and functioning.     

Complement lysis: evidence for an amphiphilic nature of the terminal membrane C5b-9 complex of human complement

The terminal, membrane-derived C5b-9 complex of human complement (C) is an apparently hollow, cylindrical macromolecule vertically oriented on the target membrane. In the present study, an antiserum to the complex has been used to probe its immunobiochemical properties. "Neoantigenic" determinants characteristic of the complex have been detected, which are absent on native C5-C9 molecules. Evidence that the C5b-9 complex is an amphiphilic molecule that possesses apolar, detergent-binding surfaces has been obtained by using charge-shift crossed immunoelectrophoresis, and by direct demonstration of Triton X-100 binding to the complex in quantitative immunoelectrophoresis. By the same criteria, serum C5, C6, and C9 are hydrophilic molecules. The results indicate that assembly of C5-C9 into the terminal membrane C5b-9 complex is accompanied by conformational changes in the individual C components that lead to the exposure of apolar molecular regions in the complex. It is proposed that this constitutes the basis for the lipid-binding properties of the macromolecule, which enable it to become inserted into biologic and artificial lipid membranes with apparent generation of a transmembrane pore.     

Structure of complement C6 suggests a mechanism for initiation and unidirectional, sequential assembly of membrane attack complex (MAC)

The complement membrane attack complex (MAC) is formed by the sequential assembly of C5b with four homologous proteins as follows: one copy each of C6, C7, and C8 and 12-14 copies of C9. Together these form a lytic pore in bacterial membranes. C6 through C9 comprise a MAC-perforin domain flanked by 4-9 "auxiliary" domains. Here, we report the crystal structure of C6, the first and longest of the pore proteins to be recruited by C5b. Comparisons with the structures of the C8αβγ heterodimer and perforin show that the central domain of C6 adopts a "closed" (perforin-like) state that is distinct from the "open" conformations in C8. We further show that C6, C8α, and C8β contain three homologous subdomains ("upper," "lower," and "regulatory") related by rotations about two hinge points. In C6, the regulatory segment includes four auxiliary domains that stabilize the closed conformation, inhibiting release of membrane-inserting elements. In C8β, rotation of the regulatory segment is linked to an opening of the central β-sheet of its clockwise partner, C8α. Based on these observations, we propose a model for initiation and unidirectional propagation of the MAC in which the auxiliary domains play key roles: in the assembly of the C5b-8 initiation complex; in driving and regulating the opening of the β-sheet of the MAC-performin domain of each new recruit as it adds to the growing pore; and in stabilizing the final pore. Our model of the assembled pore resembles those of the cholesterol-dependent cytolysins but is distinct from that recently proposed for perforin.     

Complement proteins C5b-9 induce transbilayer migration of membrane phospholipids.

Transbilayer migration of membrane phospholipid arising from membrane insertion of the terminal human complement proteins has been investigated. Asymmetric vesicles containing pyrene-labeled phosphatidylcholine (pyrenePC) concentrated in the inner monolayer were prepared by outer monolayer exchange between pyrenePC-containing large unilamellar vesicles and excess (unlabeled) small unilamellar vesicles, using bovine liver phosphatidylcholine-specific exchange protein. After depletion of pyrenePC from the outer monolayer, the asymmetric large unilamellar vesicles were isolated by gel filtration and exposed to the purified C5b-9 proteins at 37 degrees C. Transbilayer exchange of phospholipid between inner and outer monolayers during C5b-9 assembly was monitored by changes in pyrene excimer and monomer fluorescence. Membrane deposition of the C5b67 complex (by incubation with C5b6 + C7) caused no change in pyrenePC fluorescence. Addition of C8 to the C5b67 vesicles resulted in a dose-dependent decrease in the excimer/monomer ratio. This change was observed both in the presence and absence of complement C9. No change in fluorescence was observed for control vesicles exposed to C8 (in the absence of membrane C5b67), or upon C5b-9 addition to vesicles containing pyrenePC symmetrically distributed between inner and outer monolayers. These data suggest that a transbilayer exchange of phospholipid between inner and outer monolayers is initiated upon C8 binding to C5b67. The fluorescence data were analyzed according to a "random walk" model for excimer formation developed for the case where pyrenePC is asymmetrically distributed between lipid bilayers. Based on this analysis, we estimate that a net transbilayer migration of approximately 1% of total membrane phospholipid is initiated upon C8 binding to C5b67. The potential significance of this transbilayer exchange of membrane phospholipid to the biological activity of the terminal complement proteins is considered.     

Structure, function and control of complement c5 and its proteolytic fragments

As part of the innate immune system, the complement system recognises a wide range of non-self structures present on pathogens or altered self cells. Its activation elicits proteolytic cascades which eventually results in the cleavage of the C5 protein into two fragments, C5a and C5b. The small anaphylatoxin C5a induces a variety of biological responses upon binding to the 7TM receptors C5aR and the C5L2, while the large C5b fragment nucleates formation of the membrane attack complex capable of killing susceptible pathogens by the formation of a pore structure in association with complement components C6, C7, C8, and C9. A number of regulatory molecules help to control C5 mediated immune responses towards host cells, but in several major inflammatory conditions including sepsis and arthritis, C5a is believed to contribute significantly to disease etiology. Inhibition of membrane attack complex assembly is already approved for treatment of paroxysmal nocturnal haemoglobinuria and atypical hemolytic uremic syndrome. A number of recent crystal structures have provided a comprehensive insight into the architecture and properties of intact C5 and its fragments, and how pathogens interfere with their function. Here we review the functional and structural aspects of C5 and its fragments, the pathological conditions associated with them, and strategies employed by pathogens to interfere with the biological function of C5. Structural insight and elucidation of evasion strategies employed by pathogens present a unique opportunity for promoting the development of novel selective C5 inhibitors with therapeutic applications.     

Complement Regulatory Molecules on Human Myelin and Glial Cells: Differential Expression Affects the Deposition of Activated Complement Proteins

Abstract: The expression of decay-accelerating factor CD55, membrane cofactor protein CD46, and CD59 was studied on Schwann cells cultured from human sural nerve and myelin membranes prepared from human cauda equina and spinal cord. These proteins are regulatory membrane molecules of the complement system. CD55 and CD46 are inhibitors of C3 and C5 convertases and CD59 inhibits C8 and C9 incorporation into C5b-9 complex and C9-C9 polymerization. The presence of these proteins was assessed by using antibodies to each of the proteins by fluorescent microscopy, fluorescence-activated cell sorter analysis, and also sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis. Schwann cells in culture expressed CD55, CD46, and CD59. It is interesting that only CD59 was detected on myelin from both central and peripheral nerve tissue. The ability of these proteins to limit C3 peptide deposition and C9 polymerization in myelin was studied by western blot analysis. C3b deposition was readily detected on antibody-sensitized myelin incubated with normal human serum used as a source of complement but not with EDTA-treated or heat-inactivated serum. C3b deposition was not affected by anti-CD55 antibody. On the other hand, poly-C9 formation in myelin, which was maximum when 50% normal human serum was used, was increased four- to fivefold when myelin was preincubated with anti-CD59. Our data suggest that complement activation on myelin is down-regulated at the step of the assembly of terminal complement complexes, including C5b-9, due to the presence of CD59.     

Hydrolysis of myelin basic protein in human myelin by terminal complement complexes

The participation of terminal complement complexes (TCC) in demyelination has been shown in rodent cerebellar cultures. Since TCC modulates activities of various membrane-associated enzymes and increases the level of cellular Ca2+ we investigated whether TCC could activate Ca2+-dependent neutral proteases in myelin that would lead to hydrolysis of myelin basic protein (BP). Addition of antibody and C7-deficient serum plus C7 to sealed myelin vesicles of two to six bilayers caused significant BP hydrolysis compared to the hydrolysis caused by antibody and C7-deficient serum. Significant hydrolysis occurred at the stage of C5b6,7 assembly, which increased in magnitude at the C5b6-8 stage. C5b6-9 formation did not enhance the effect of C5b6-8. BP hydrolysis by C5b6,7 did not require Ca2+ whereas the effect of C5b6-8/C5b6-9 was, in part, Ca2+-dependent. We postulated that TCC formation in myelin membranes causes activation of myelin-associated neutral proteases with subsequent hydrolysis of BP as a consequence of complement peptide insertion and channel formation. Such processes may alter the structure of myelin and augment the action of other inflammatory cells and their products in demyelinating diseases that could ultimately lead to the loss of myelin.     

Membrane permeability to macromolecules mediated by the membrane attack complex

A simple and well-defined system of purified phospholipids and human complement proteins was used to study membrane permeability to macromolecules mediated by the membrane attack complex (MAC) of complement. Large unilamellar vesicles (LUVs) of phosphatidylcholine (PC) or phosphatidylserine (PS) containing trapped macromolecules [bovine pancreatic trypsin inhibitor (BPTI), thrombin, glucose-6-phosphate dehydrogenase (G6PD), and larger molecules] were used to monitor permeability. Membrane permeability to macromolecules was measured by thrombin inhibition by an external inhibitor or by separation of released molecules by gel filtration. Membrane-bound intermediates (C5b-8 or C5b-93) were stable for hours, and macromolecular permeability occurred without fragmentation, fusion, or aggregation of the vesicles. Quantitative membrane binding by C5b-7 as well as essentially quantitative release of thrombin was obtained for PS vesicles. MAC binding to PS-LUVs approximated the theoretical Poisson distribution curve for full release of vesicle contents by one complex per vesicle. Reactions with PC-LUVs occurred with some fluid-phase MAC assembly. Therefore, results from experiments with these vesicles were interpreted in a relative manner. However, the values obtained closely corroborated those obtained with PS-LUVs. At low C9/C5b-8 ratios, the size of the lesion was proportional to the C9 content of the MAC. Half-maximum release of BPTI, thrombin, and G6PD, by a single MAC per vesicle, required approximately 3,5, and 7 C9/C5b-8 (mol/mol), respectively. Larger molecules (greater than or equal to 118-A diameter) were not released from the vesicles. Release of G6PD (95.4-A diameter) required 45% of saturating C9. Therefore, it appeared that the last half of the bound C9 molecules did not increase pore size and the pore which released G6PD approached the diameter of the closed circular lesion measured (by others) in electron micrographs (approximately 100 A). The results were consistent with the formation of a stable membrane pore by a single complex per vesicle in which C9 molecules line only one side of the pore at low C9/C5b-8 ratios and maximum pore size is attained by incomplete, noncircular polymers of C9.     

Single-channel analysis of the conductance fluctuations induced in lipid bilayer membranes by complement proteins C5b-9

Summary Single-channel analysis of electrical fluctuations induced in planar bilayer membranes by the purified human complement proteins C5b6, C7, C8, and C9 have been analyzed. Reconstitution experiments with lipid bilayer membranes showed that the C5b-9 proteins formed pores only if all proteins were present at one side of the membrane. The complement pores had an average single-channel conductance of 3.1 nS at 0.15m KCl. The histogram of the complement pores suggested a substantial variation of the size of the single channel. The linear relationship between single-channel conductance at fixed ionic strength and the aqueous mobility of the ions in the bulk aqueous phase indicated that the ions move inside the complement pore in a manner similar to the way they move in the aqueous phase. The minimum diameter of the pores as judged from the conductance data is approximately 3 nm. The complement channels showed no apparent voltage control or regulation up to transmembrane potentials of 100 mV. At neutral pH the pore is three to four times more permeable for alkali ions than for chloride, which may be explained by the existence of fixed negatively charged groups in or near the pore. The significance of these observations to current molecular models of the membrane lesion formed by these cytolytic serum proteins is considered.     

Effect of complement proteins C5b-9 on blood platelets. Evidence for reversible depolarization of membrane potential

The carbocyanine dye 3,3'-dipropylthiodicarbocyanine iodide has been used to investigate changes in membrane potential (Em) which occur upon binding of complement proteins C5b-9 to the plasma membrane of blood platelets. Gel-filtered platelets exposed to C5b6 and C7 in serum-free medium show no change in Em from that of controls, as indicated by either 3,3,'-dipropylthiodicarbocyanine iodide fluorescence or by the distribution of [14C]tetraphenylphosphonium bromide. Addition of complement proteins C8 and C9 to the C5b67 platelets results in partial depolarization of Em, which spontaneously repolarizes to basal levels within 15-20 min at 37 degrees C. Under these conditions, C5b-9-treated platelets show no increase in lysis over complement-free controls. Isotonic replacement of external sodium by either potassium or choline alters both the rate and extent of membrane depolarization and inhibits the platelets' capacity to repolarize after C5b-9 assembly. Repolarization of Em to basal levels is also completely blocked by addition of ouabain, confirming that this recovery is mediated by the plasma membrane Na+/K+ pump. These results demonstrate that membrane binding of the C5b-9 proteins can induce a transient change in Em when bound to the plasma membrane at a sublytic concentration, providing a mechanism for target cell activation by these potentially cytolytic proteins.     

The relationship between channel size and the number of C9 molecules in the C5b-9 complex

We have recently shown by dose-response analyses with resealed erythrocyte ghosts that the channel formed by complement is a monomer of C5b-9 of the composition C5b61C71C81C9n, in which n = 1 for channels permitting passage of sucrose (0.9 nm molecular diameter) and n = 2 for channels allowing transit of inulin (3 nm molecular diameter) (1). We have now continued these experiments and expanded them by including ribonuclease A (molecular diameter, 3.8 nm) as a marker to assess whether additional C9 molecules enlarge the functional C5b-9 channel. Our results show that formation of C5b-9 channels displays one-hit characteristics with respect to C5b6 when tested by transmembrane passage of inulin or ribonuclease A. By contrast, analysis of dose-response curves of C9 indicate that n = 2-3 for channels allowing transit of inulin and n = 4 for channels allowing transit of ribonuclease A. We have also performed sieving experiments with ghosts carrying C5b-7 and containing two small markers, inositol and sucrose. Dose-response curves for C8 were performed in the presence of excess C9 to ensure conversion of all C5b-8 to C5b-9 channels. The results indicate that small channels (approximately 0.8 nm effective diameter) are not formed at high C9 multiplicity, thus confirming the results obtained with the larger markers, i.e., increase of C9 input leads to formation of larger channels.     

The C5 domain of the collagen VI alpha3(VI) chain is critical for extracellular microfibril formation and is present in the extracellular matrix of cultured cells

Collagen VI, a microfibrillar protein found in virtually all connective tissues, is composed of three distinct subunits, alpha1(VI), alpha2(VI), and alpha3(VI), which associate intracellularly to form triple helical heterotrimeric monomers then dimers and tetramers. The secreted tetramers associate end-to-end to form beaded microfibrils. Although the basic steps in assembly and the structure of the tetramers and microfibrils are well defined, details of the interacting protein domains involved in assembly are still poorly understood. To explore the role of the C-terminal globular regions in assembly, alpha3(VI) cDNA expression constructs with C-terminal truncations were stably transfected into SaOS-2 cells. Control alpha3(VI) N6-C5 chains with an intact C-terminal globular region (subdomains C1-C5), and truncated alpha3(VI) N6-C1, N6-C2, N6-C3, and N6-C4 chains, all associated with endogenous alpha1(VI) and alpha2(VI) to form collagen VI monomers, dimers and tetramers, which were secreted. These data demonstrate that subdomains C2-C5 are not required for monomer, dimer or tetramer assembly, and suggest that the important chain selection interactions involve the C1 subdomains. In contrast to tetramers containing control alpha3(VI) N6-C5 chains, tetramers containing truncated alpha3(VI) chains were unable to associate efficiently end-to-end in the medium and did not form a significant extracellular matrix, demonstrating that the alpha3(VI) C5 domain plays a crucial role in collagen VI microfibril assembly. The alpha3(VI) C5 domain is present in the extracellular matrix of SaOS-2 N6-C5 expressing cells and fibroblasts demonstrating that processing of the C-terminal region of the alpha3(VI) chain is not essential for microfibril formation.     

In-vitro activation of complement system by lactic acidosis in newborn and adults

INTRODUCTION: Complement activation occurs secondary to a variety of external stimuli. Lactic acidosis has been previously shown to activate the complement factors C3a and C5a. In the present investigation we examined the differential effect of lactic acidosis on anaphylatoxin levels in cord and adult blood. Furthermore we aimed to determine if the entire complement cascade could be activated by lactic acidosis. METHODS: Cord and adult blood samples (n = 20 each) were collected and incubated for one hour in either untreated condition or with the addition of lactate in two concentrations (5.5 mmol/l vs. 22 mmol/l). Following incubation, levels of C3a, C5a and sC5b-9, and blood gas parameters were determined. RESULTS: Anaphylatoxin (C3a and C5a) and sC5b-9 levels increased with the addition of lactate in a dose-dependent manner in cord and adult blood (C3a: 1 h, 5.5 mmo/l, 22 mmol/l: 418/498/622 microg/l in cord blood; 1010/1056/1381 microg/l in adult blood, p<0,05; similar results were found for C5a and sC5b-9). CONCLUSION: Lactic acidosis leads to an activation of the entire complement system in neonates and in adults. This activation is dose-dependent and more pronounced in adults as compared to neonates.     

The N-terminal N5 subdomain of the alpha 3(VI) chain is important for collagen VI microfibril formation

Collagen VI assembly is unique within the collagen superfamily in that the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains associate intracellularly to form triple helical monomers, and then dimers and tetramers, which are secreted from the cell. Secreted tetramers associate end-to-end to form the distinctive extracellular microfibrils that are found in virtually all connective tissues. Although the precise protein interactions involved in this process are unknown, the N-terminal globular regions, which are composed of multiple copies of von Willebrand factor type A-like domains, are likely to play a critical role in microfibril formation, because they are exposed at both ends of the tetramers. To explore the role of these subdomains in collagen VI intracellular and extracellular assembly, alpha 3(VI) cDNA expression constructs with sequential N-terminal deletions were stably transfected into SaOS-2 cells, producing cell lines that express alpha 3(VI) chains with N-terminal globular domains containing modules N9-N1, N6-N1, N5-N1, N4-N1, N3-N1, or N1, as well as the complete triple helix and C-terminal globular domain (C1-C5). All of these transfected alpha 3(VI) chains were able to associate with endogenous alpha 1(VI) and alpha 2(VI) to form collagen VI monomers, dimers, and tetramers, which were secreted. Importantly, cells that expressed alpha 3(VI) chains containing the N5 subdomain, alpha 3(VI) N9-C5, N6-C5, and N5-C5, formed microfibrils and deposited a collagen VI matrix. In contrast, cells that expressed the shorter alpha 3(VI) chains, N4-C5, N3-C5, and N1-C5, were severely compromised in their ability to form end-to-end tetramer assemblies and failed to deposit a collagen VI matrix. These data demonstrate that the alpha 3(VI) N5 module is critical for microfibril formation, thus identifying a functional role for a specific type A subdomain in collagen VI assembly.