Alexandra Davies

Membrane defence against complement lysis: the structure and biological properties of CD59

The complement system is an important branch of the innate immune response, constituting a first line of defence against invading microorganisms which activate complement via both antibody-dependent and-independent mechanisms. Activation of complement leads to (a) a direct attack upon the activating cell surface by assembly of the pore-forming membrane attack complex (MAC), and (b) the generation of inflammatory mediators which target and recruit other branches of the immune system. However, uncontrolled complement activation can lead to widespread tissue damage in the host, since certain of the activation products, notably the fragment C3b and the C5b-7 complex, can bind nonspecifically to any nearby cell membranes. Therefore it is important that complement activation is tightly regulated. Our own cells express a number of membrane-bound control proteins which limit complement activation at the cell surface and prevent accidental complement-mediated damage. These include decay-accelerating factor, complement receptor 1 and membrane cofactor protein, all of which are active at the level of C3/C5 convertase formation. Until recently, cell surface control of MAC assembly had been attributed to a single 65-kD membrane protein called homologous restriction factor (alternatively named C8-binding protein and MAC-inhibiting protein). However a second MAC-inhibiting protein has since been discovered and it is now clear that this protein plays a major role in the control of membrane attack. This review charts the rapid progress made in elucidating the protein and gene structure, and the mechanism of action of this most recently discovered complement inhibitor, CD59.

Streptococcal Inhibitor of Complement Inhibits Two Additional Components of the Mucosal Innate Immune System: Secretory Leukocyte Proteinase Inhibitor and Lysozyme

ABSTRACT Streptococcal inhibitor of complement (SIC) is a 31-kDa extracellular protein of a few, very virulent, strains of Streptococcus pyogenes (particularly M1 strains). It is secreted in large quantities (about 5 mg/liter) and inhibits complement lysis by blocking the membrane insertion site on C5b67. We describe investigations into the interaction of SIC with three further major components of the innate immune system found in airway surface liquid, namely, secretory leukocyte proteinase inhibitor (SLPI), lysozyme, and lactoferrin. Enzyme-linked immunosorbent assays showed that SIC binds to SLPI and to both human and hen egg lysozyme (HEL) but not to lactoferrin. Studies using 125I-labeled proteins showed that SIC binds approximately two molecules of SLPI and four molecules of lysozyme. SLPI binding shows little temperature dependence and a small positive enthalpy, suggesting that the binding is largely hydrophobic. By contrast, lysozyme binding shows strong temperature dependence and a substantial negative enthalpy, suggesting that the binding is largely ionic. Lysozyme is precipitated from solution by SIC. Further studies examined the ability of SIC to block the biological activities of SLPI and lysozyme. An M1 strain of group A streptococci was killed by SLPI, and the antibacterial activity of this protein was inhibited by SIC. SIC did not inhibit the antiproteinase activity of SLPI, implying that there is specific inhibition of the antibacterial domain. The antibacterial and enzymatic activities of lysozyme were also inhibited by SIC. SIC is the first biological inhibitor of the antibacterial action of SLPI to be described and may prove to be an important tool for investigating this activity in vivo. Inhibition of the antibacterial actions of SLPI and lysozyme would be advantageous to S. pyogenes in establishing colonization on mucosal surfaces, and we propose that this is the principal function of SIC.

Streptococcal inhibitor of complement (SIC) inhibits the membrane attack complex by preventing uptake of C567 onto cell membranes.

Streptococcal inhibitor of complement (SIC) was first described in 1996 as a putative inhibitor of the membrane attack complex of complement (MAC). SIC is a 31 000 MW protein secreted in large quantities by the virulent Streptococcus pyogenes strains M1 and M57, and is encoded by a gene which is extremely variable. In order to study further the interactions of SIC with the MAC, we have made a recombinant form of SIC (rSIC) in Escherichia coli and purified native M1 SIC which was used to raise a polyclonal antibody. SIC prevented reactive lysis of guinea pig erythrocytes by the MAC at a stage prior to C5b67 complexes binding to cell membranes, presumably by blocking the transiently expressed membrane insertion site on C7. The ability of SIC and clusterin (another putative fluid phase complement inhibitor) to inhibit complement lysis was compared, and found to be equally efficient. In parallel, by enzyme‐linked immunosorbent assay both SIC and rSIC bound strongly to C5b67 and C5b678 complexes and to a lesser extent C5b‐9, but only weakly to individual complement components. The implications of these data for virulence of SIC‐positive streptococci are discussed, in light of the fact that Gram‐positive organisms are already protected against complement lysis by the presence of their peptidoglycan cell walls. We speculate that MAC inhibition may not be the sole function of SIC.

Subversion of the innate immune response by micro-organisms.

A microbe becomes a pathogen by successfully evading the host’s immune responses, and the microbial strategies for so doing are legion. They include methods to avoid recognition by the immune system—for example, by antigenic variation as shown by influenza and HIV and by parasites or plasmodia, or by acquiring a host coat as is done by worms and retroviruses. Another major mechanism is to avoid the effector mechanisms of the immune response. This can be done by subverting cytotoxic T cells by the production of decoy HLA molecules; or by subverting Fc function by producing Fc receptor homologues; or by subverting complement by producing homologues of complement control proteins (CCPs). Some viruses also have developed methods of subverting apoptosis in the cells that they infect. This paper concentrates on the innate immune response. The definition of this term is a little fuzzy. Fearon and Locksley regard all mechanisms using germline coded molecules as being innate which therefore includes natural antibodies with germline V regions.1 Perhaps a more conventional definition is that innate mechanisms are those that are not specifically altered by prior exposure to the same pathogen. In the first part of this paper some examples of subversion of the complement system will be described. The second half describes some much newer work from our laboratory that takes us into aspects of the innate immune response on mucosal surfaces that have not so far been described. Figure 1 shows a greatly simplified view of complement activation by micro-organisms. Regulation occurs principally at two points—the first is action of the C3 converting enzymes and the second, action of the membrane attack complex (MAC). Figure 1 Principal activities of the complement system. Regulation of the C3 converting enzymes is produced by a number of proteins (fig 2), all of which are based on …

Complement and immunity to viruses

Complement is one of the first lines of host defence to be faced and countered by viruses as they struggle to establish an infection. As an important arm of the humoral immune response, the complement system is immediately ready to target and eliminate virus particles and to interact with the surface of virus‐infected cells to mark them for destruction by other branches of the immune response. Nevertheless, some viruses are still very successful human pathogens. This article will discuss the role of complement in antiviral immunity, the mechanisms by which complement may be activated by viruses or virus‐infected cells, and explore some of the strategies which viruses have evolved to subvert the immune response, including mechanisms by which complement activation may be prevented or aborted.

Antigen S1, encoded by the MIC1 gene, is characterized as an epitope of human CD59, enabling measurement of mutagen-induced intragenic deletions in the AL cell system

S1 cell membrane antigen is encoded by the MIC1 gene on human chromosome 11. This antigen has been widely used as a marker for studies in gene mapping or in analysis of mutagen-induced gene deletions/mutations, which utilized the human-hamster hybrid cell-line, AL-J1, carrying human chromosome 11. Evidence is presented here which identifies S1 as an epitope of CD59, a cell membrane complement inhibiting protein. E7.1 monoclonal antibody, specific for the S1 determinant, was found to react strongly with membrane CD59 in Western blotting, and to bind to purified, urinary form of CD59 in ELISAs. Cell membrane expression of S1 on various cell lines always correlated with that of CD59 when examined by immunofluorescent staining. In addition, E7.1 antibody inhibited the complement regulatory function of CD59. Identification of S1 protein as CD59 has increased the scope of the AL cell system by enabling analysis of intragenic mutations, and multiplex PCR analysis of mutated cells is described, showing variable loss of CD59 exons.

Molecular Basis for the Interaction Between AP4 β4 and its Accessory Protein, Tepsin

The adaptor protein 4 (AP4) complex (ϵ/β4/μ4/σ4 subunits) forms a non‐clathrin coat on vesicles departing the trans‐Golgi network. AP4 biology remains poorly understood, in stark contrast to the wealth of molecular data available for the related clathrin adaptors AP1 and AP2. AP4 is important for human health because mutations in any AP4 subunit cause severe neurological problems, including intellectual disability and progressive spastic para‐ or tetraplegias. We have used a range of structural, biochemical and biophysical approaches to determine the molecular basis for how the AP4 β4 C‐terminal appendage domain interacts with tepsin, the only known AP4 accessory protein. We show that tepsin harbors a hydrophobic sequence, LFxG[M/L]x[L/V], in its unstructured C‐terminus, which binds directly and specifically to the C‐terminal β4 appendage domain. Using nuclear magnetic resonance chemical shift mapping, we define the binding site on the β4 appendage by identifying residues on the surface whose signals are perturbed upon titration with tepsin. Point mutations in either the tepsin LFxG[M/L]x[L/V] sequence or in its cognate binding site on β4 abolish in vitro binding. In cells, the same point mutations greatly reduce the amount of tepsin that interacts with AP4. However, they do not abolish the binding between tepsin and AP4 completely, suggesting the existence of additional interaction sites between AP4 and tepsin. These data provide one of the first detailed mechanistic glimpses at AP4 coat assembly and should provide an entry point for probing the role of AP4‐coated vesicles in cell biology, and especially in neuronal function.

AP-4 vesicles unmasked by organellar proteomics to reveal their cargo and machinery

Adaptor protein 4 (AP-4) is an ancient membrane trafficking complex, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder of unknown aetiology. We apply multiple unbiased proteomic methods, including ‘Dynamic Organellar Maps’, to find proteins whose subcellular localisation depends on AP-4. We identify three highly conserved transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, and two AP-4 accessory proteins, RUSC1 and RUSC2. We demonstrate that AP-4 deficiency causes missorting of ATG9A in diverse cell types, including neuroblastoma and AP-4 patient-derived cells, as well as dysregulation of autophagy. Furthermore, we show that RUSC2 facilitates the microtubule plus-end-directed transport of AP-4-derived, ATG9A-positive vesicles from the TGN to the cell periphery. Since ATG9A has essential functions in neuronal homeostasis, our data not only uncover the ubiquitous function of the AP-4 pathway, but also begin to explain the molecular pathomechanism of AP-4 deficiency.Adaptor protein 4 (AP-4) is an ancient membrane trafficking complex, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder of unknown aetiology. We apply unbiased proteomic methods, including Dynamic Organellar Maps, to find proteins whose subcellular localisation depends on AP-4. We identify three transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, and two AP-4 accessory proteins, RUSC1 and RUSC2. We demonstrate that AP-4 deficiency causes missorting of ATG9A in diverse cell types, including patient-derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A-positive vesicles from the TGN to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the ATG9A reservoir required for autophagosome biogenesis. Our study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy.

Clathrin heavy chain 22 contributes to the control of neuropeptide degradation and secretion during neuronal development.

The repertoire of cell types in the human nervous system arises through a highly orchestrated process, the complexity of which is still being discovered. Here, we present evidence that CHC22 has a non-redundant role in an early stage of neural precursor differentiation, providing a potential explanation of why CHC22 deficient patients are unable to feel touch or pain. We show the CHC22 effect on neural differentiation is independent of the more common clathrin heavy chain CHC17, and that CHC22-dependent differentiation is mediated through an autocrine/paracrine mechanism. Using quantitative proteomics, we define the composition of clathrin-coated vesicles in SH-SY5Y cells, and determine proteome changes induced by CHC22 depletion. In the absence of CHC22 a subset of dense core granule (DCG) neuropeptides accumulated, were processed into biologically active ‘mature’ forms, and secreted in sufficient quantity to trigger neural differentiation. When CHC22 is present, however, these DCG neuropeptides are directed to the lysosome and degraded, thus preventing differentiation. This suggests that the brief reduction seen in CHC22 expression in sensory neural precursors may license a step in neuron precursor neurodevelopment; and that this step is mediated through control of a novel neuropeptide processing pathway.

AP-4 vesicles contribute to spatial control of autophagy via RUSC-dependent peripheral delivery of ATG9A

Adaptor protein 4 (AP-4) is an ancient membrane trafficking complex, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder of unknown aetiology. We apply unbiased proteomic methods, including ‘Dynamic Organellar Maps’, to find proteins whose subcellular localisation depends on AP-4. We identify three transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, and two AP-4 accessory proteins, RUSC1 and RUSC2. We demonstrate that AP-4 deficiency causes missorting of ATG9A in diverse cell types, including patient-derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A-positive vesicles from the trans-Golgi network to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the “ATG9A reservoir” required for autophagosome biogenesis. Our study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy.Adaptor protein complex 4 (AP-4) deficiency causes a severe neurological disorder via an unknown mechanism. Here, the authors reveal cargo and machinery of the AP-4 transport pathway, and propose that AP-4 mediates spatial regulation of autophagy through peripheral delivery of ATG9A.