human complement protein c8γ

Review

Human complement protein C8Q

Steven F. Schreck, Chasta Parker, Mnason E. Plumb, James M. Sodetz *Department of Chemistry and Biochemistry and the School of Medicine, University of South Carolina, Columbia, SC 29208, USA

Received 4 November 1999; received in revised form 14 January 2000; accepted 8 February 2000

Abstract

Human C8Q is a 22 kDa subunit of complement component C8, which is one of five components (C5b, C6, C7, C8, C9)that interact to form the cytolytic membrane attack complex (MAC) of complement. C8 contains three nonidentical subunits(K, L, Q) that are products of different genes. These subunits are arranged asymmetrically to form a disulfide-linked C8K-Qdimer that is noncovalently associated with C8L. C8K and C8L are homologous to C6, C7 and C9 and together these proteinscomprise what is referred to as the `MAC protein family'. By comparison, C8Q is distinct in that it belongs to the lipocalinfamily of small, secreted proteins which have the common ability to bind small hydrophobic ligands. While specific roles havebeen identified for C8K and C8L in the formation and function of the MAC, a function for C8Q and the identity of its ligandare unknown. This review summarizes the current status of C8Q structure and function and the progress made from efforts todetermine its role in the complement system. ß 2000 Elsevier Science B.V. All rights reserved.

Keywords: Human complement; C8; C8Q ; Cell lysis; Membrane attack complex

1. Introduction

Human C8Q is a subunit of the eighth componentof complement (C8), which is one of ¢ve components(C5b, C6, C7, C8, C9) that interact as a consequenceof complement activation to form a cytolytic macro-molecular complex referred to as the membrane at-tack complex (MAC) [1,2]. C8 is an oligomeric pro-tein composed of an K (Mr = 64 000), L (Mr = 64 000)and Q (Mr = 22 000) subunit [3]. These subunits aresynthesized independently from di¡erent genes andare secreted in the form of a disul¢de-linked C8K-Qdimer that is noncovalently associated with C8L [4].Studies using puri¢ed or recombinant forms of each

subunit have revealed distinct roles for C8K and C8Lin the formation and function of the MAC; however,a function for C8Q has yet to be identi¢ed [5,6].

Of the 35 proteins, enzymes, receptors and regula-tory components of the human complement system,C8Q is unique in that it is the only lipocalin. Amongthe lipocalins it is exceptional because it is one ofonly a few that form a covalent complex with anoth-er protein i.e. C8K. The lipocalins are a widely dis-tributed family of secreted proteins that are similar insize (approx. 20 kDa) and have the common abilityto bind small hydrophobic ligands, e.g. retinol,pheromones, odorants, etc.[7]. They exhibit consider-able sequence diversity (approx. 20^30% identity);however, each contains short sequence motifs thatare characteristic of all family members. The crystalstructures of several lipocalins suggest that despitesequence diversity, all lipocalins have a commonoverall folding pattern [7]. This pattern is referred

0167-4838 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 7 - 4 8 3 8 ( 0 0 ) 0 0 1 5 5 - 2

Abbreviations: MAC, membrane attack complex; BLG, L-lac-toglobulin; RBP, serum retinol binding protein

* Corresponding author. Fax: (803) 7779521;E-mail : [email protected]

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Biochimica et Biophysica Acta 1482 (2000) 199^208www.elsevier.com/locate/bba

to as the `lipocalin fold' and it consists of eight anti-parallel L-strands arranged to form a L-barrel struc-ture with a distinct ligand binding pocket. Sequencedi¡erences within the pocket presumably in£uencepocket size and binding speci¢city, hence the lipoca-lins exhibit considerable variation in ligand bindingproperties.

This review summarizes current knowledge abouthuman C8Q structure and function and describes re-sults from studies aimed at identifying a role for thisprotein in the complement system. To understandC8Q as a complement protein and as a lipocalin, itmust be described within the context of the MACand C8, which are the focus of the ¢rst part of thisreview.

2. Assembly of the MAC

Assembly of the MAC begins with proteolyticcleavage of component C5 by enzymes produced asa consequence of cell surface activation of the com-plement system (Fig. 1). Once formed, the productC5b interacts with C6 through a metastable bindingsite to form a soluble C5b-6 dimer in the vicinity ofthe activating cell. Subsequent binding of C7 produ-ces C5b-7, a trimeric complex that expresses a tran-sient, high-a¤nity lipid binding site [8]. This complexphysically associates with the target membrane whereit subsequently binds C8 to form tetrameric C5b-8on the surface. C5b-8 facilitates binding and poly-merization of multiple C9 molecules to form C5b-9,the pore-like structure referred to as the MAC [9].

Components of the MAC are commonly referredto as the `terminal components' of the complementsystem and the pathway of assembly as the `terminalpathway'. The individual components are hydro-philic proteins; however, when combined they form

an amphipathic complex that is capable of binding toand disrupting local membrane organization [10].The increase in membrane permeability leads to os-motic lysis of simple cells such as erythrocytes orinitiation of a variety of intracellular signaling eventsin the case of nucleated cells [11]. In bacteria, theMAC disrupts the outer membrane thereby increas-ing permeability and inducing lethal changes in theinner membrane [2].

Aside from cleavage of C5, all steps in the terminalpathway are nonenzymatic. The components circu-late independently but interact in a noncovalent yethighly speci¢c and sequential manner once C5b isproduced. As each intermediate is formed, bindingspeci¢city is altered and directed towards the nextcomponent in the pathway. Once combined, the af-¢nity between components is high despite the non-covalent nature of their interaction. Dissociation canonly be accomplished by solubilizing the membraneand/or denaturing the MAC. Although the compo-nents are well characterized, details regarding theirinteractions with each other and the membrane arestill poorly understood.

3. Proteins of the MAC

Human C5 (Mr = 190 000) is a structural homo-logue of complement components C3 and C4 andis composed of an K (Mr = 115 000) and L(Mr = 75 000) chain linked by a disul¢de bond. C5bis formed when the C5a peptide is cleaved fromthe K chain. Human C6 (Mr = 105 000) and C7(Mr = 92 000) are single-chain proteins that are sim-ilar in size and structural organization. Human C8(Mr = 151 000) has the most complex structure of theMAC components and is the only one assembledfrom multiple gene products. Human C9 (Mr =

Fig. 1. Assembly of the MAC. Activation of complement leads to formation of C5b-9, the cytolytic membrane attack complex ofcomplement, or MAC.

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72 000) is a single-chain protein that has the uniqueability to polymerize during MAC formation. Thecharacteristic pore-like appearance of the MAC isattributed to the association of as many as 12^18C9s that self-polymerize to form a circular structurein the membrane [9].

Human C6, C7, C8K, C8L and C9 are structurallyand genetically related proteins that together com-prise the MAC protein family [6,12]. They have asimilar structural organization and share regions ofhighly conserved sequence (Fig. 2). Their genes arealso conserved with respect to length of exons andthe location and phases of exon boundaries. A dis-tinctive feature of the MAC family is the presence oftandemly arranged modules approx. 40^80 residuesin length which are located at the N- and C-terminalends of each protein. These modules are widely dis-tributed among a number of functionally unrelatedproteins [13,14]. C6 and C7 contain C-terminal mod-ules not found in C8K, C8L and C9, hence the larger

size of these two components. Although not a mod-ule, the internal segment of each protein is desig-nated MACPF to emphasize the sequence similaritybetween this region of the MAC proteins and thepore-forming protein perforin. Human C8Q is distinctin that it is not modular nor homologous to anyprotein in the complement system.

3.1. The C8K and C8L subunits

Following dissociation and puri¢cation in the pres-ence of high ionic strength bu¡ers, the C8K-Q andC8L subunits retain a high a¤nity for each otheras evidenced by their ability to fully recombine andform active C8 [15,16]. Puri¢cation of stable forms ofC8K-Q and C8L has facilitated e¡orts to determinetheir role in C8 function. Furthermore, the abilityto selectively cleave the interchain disul¢de bond inC8K-Q and the availability of recombinant forms ofC8K and C8Q have enabled the role of these two

Fig. 2. Structural organization of the MAC protein family. Shown are maps based on sequences of the mature proteins and the mod-ule boundaries listed in the SWISS-PROT Protein Sequence Data Bank. Abbreviations correspond to thrombospondin type I (T1),low-density lipoprotein receptor class A (LA), epidermal growth factor (EG), complement control protein (CP) and Factor I (FM)modules. The membrane attack complex/perforin segment is designated MACPF. Numbers correspond to the ¢rst residue in eachmodule. Dots above each map indicate the approximate location of Cys residues, which are highly conserved. All are involved in in-trachain disul¢de bonds except Cys164 in C8K, which is linked to C8Q. The shaded region £anking Cys164 corresponds to a 17-residueinsertion that is unique to C8K. Hexagonal symbols designate Asn residues that are potential N-glycosylation sites.

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S.F. Schreck et al. / Biochimica et Biophysica Acta 1482 (2000) 199^208 201

subunits to be examined independently. Based onevidence described below, a model for C8 has beenproposed which recognizes the existence of severaldistinct functional sites on C8K and C8L (Fig. 3)[6]. With a few exceptions, the exact location ofeach site is unknown.

C8K contains several functionally important sitesincluding one that mediates the interaction betweenC8K-Q and C8L. The existence of this site was re-vealed when binding studies showed that puri¢edor recombinant C8K can associate with C8L in solu-tion and form a stable C8K-C8L complex [17,18].Recent experiments with chimeric and truncatedforms of recombinant C8K have established thatthis interaction is primarily dependent on a site(s)located within the MACPF segment of C8K [19].Together these results suggest that C8Q does not me-diate the binding of C8K-Q to C8L. A second site(s)on C8K is involved in interactions with C8Q. C8K andC8Q retain the ability to interact noncovalently aftercleavage of the interchain disul¢de bond in C8K-Q[20]. This suggests C8K and C8Q contain mutuallyrecognized binding sites. The signi¢cance of thesesites presumably relates to the fact that C8K andC8Q are synthesized independently and must associ-ate co- or posttranslationally before disul¢de bondformation can occur. A third site on C8K binds C9and directs incorporation of this component into theMAC. Evidence for such a site comes from the ob-

servation that C8K alone is capable of forming a 1:1complex with C9 in solution [21]. A fourth site lo-cated within residues 320^415 of C8K functions as abinding site for CD59, the membrane-associated reg-ulatory protein that inhibits formation of a function-al MAC on homologous cells [22].

C8K is also thought to contain one or more lipidbinding sites that become accessible as C8 is incor-porated into the MAC. Segments de¢ning these sitesappear to interact directly with the lipid bilayer oftarget cells. Evidence for this comes from photolab-eling studies using membrane-restricted probes toidentify components of the MAC that are insertedinto the bilayer [23]. In both C5b-8 and the MAC,the extent of labeling suggests that C8K is insertedinto the membrane bilayer. Whether multiple seg-ments are inserted and whether they assume a trans-membrane orientation within the MAC is unknown.

C8L also has several functional sites, one of whichmediates incorporation of C8 into the MAC by bind-ing to the C5b-7 complex [15]. A second site mediatesC8L interaction with the C8K chain as indicated byits high a¤nity for both C8K-Q and C8K [24]. A thirdsite(s) consists of one or more segments that interactwith the bilayer of the target membrane. Evidencefor the latter site also comes from photolabeling ex-periments which suggest that C8L in either C5b-8 orthe MAC is in contact with the bilayer but to a lesserextent than C8K.

Fig. 3. Functional sites in C8. (Left panel) Schematic representation of sites of interaction (hatched) in each subunit of C8. Sites arearbitrarily located within each subunit but are depicted as distinct and nonoverlapping because they function simultaneously withinthe MAC. Membrane interaction sites are designated `m'. (Right panel) Relative location of the C8 subunits within membrane-boundC5b-8 and the MAC. The spatial arrangement in relation to the other MAC proteins is based on cumulative data described in thetext and [6]. C9 is depicted as a polymer composed of an arbitrary number of monomeric units.

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4. Physical properties, synthesis and structure of C8QQ

The amino acid sequence of human C8Q was de-duced from analysis of human liver cDNA clonesand found to exhibit weak but signi¢cant similarityto members of the lipocalin family [4,25]. The matureprotein contains 182 residues and has a calculatedmass of 20 327 Da. The N-terminus is blocked witha pyroglutamyl residue. The protein contains no car-bohydrate and has one intrachain disul¢de bond(Cys76-Cys168). A third Cys (Cys40) is involved inlinkage to Cys164 in C8K [26]. The only other speciesof C8Q sequenced thus far is rabbit [27]. It also con-tains 182 residues and is 81% identical in sequence tohuman C8Q. The most notable di¡erence is the pres-ence of a potential N-glycosylation site at position153. Electrophoretic mobility di¡erences suggestthis site is glycosylated in rabbit C8Q [27].

The gene for human C8Q has been sequenced and aputative transcription initiation site has been identi-¢ed [28]. The gene is 1.8 kb in length and containsseven exons. A comparison of exon lengths andboundaries with those of other lipocalins reveals ahigh degree of conservation that implies an ancestralrelationship. The gene is located on chromosome9q34.3 near a cluster of other lipocalin genes [29],which suggests that C8Q arose by duplication of alipocalin family member. Hereditary de¢ciencies ofC8 have been described and are classi¢ed accordingto whether the level of C8K-Q or C8L is reduced inthe serum of a¡ected individuals. Such de¢cienciesare generally associated with recurrent neisserial in-fections [30]. With respect to C8K-Q de¢ciency, de-fects have thus far only been found in the C8Kgene [31,32].

The hepatocyte is the primary site of synthesis ofall three C8 subunits and is the principal source ofserum C8. Secondary sites of synthesis include mono-cytes, macrophages, ¢broblasts, astrocytes and endo-thelial cells. Biosynthetic studies using isolatedhepatocytes indicate that C8K-Q is assembled intra-cellularly and although some may be secreted inde-pendently, the majority combines with C8L and issecreted as C8 [33]. With respect to C8K and C8Q,each can be produced separately as a recombinantprotein in transfected cells [18]; however, there isno evidence for independent secretion from normalcells nor has either subunit been detected in free form

in the circulation. Regarding this, a recent analysis ofC8 expression in human tissue revealed the presenceof transcripts for C8Q but not C8K in fetal and adultkidney [34]. Although protein expression was notmeasured, these results could be interpreted as sup-port for the independent production of C8Q in vivo.Since most lipocalins are secreted independently,such a possibility is not unreasonable and is onethat deserves further investigation.

Aside from predictions based on amino acid se-quence, little is known about the tertiary structureof C8Q. Models based on coordinates for the retinolbinding lipocalin L-lactoglobulin (BLG) predict thatC8Q has a characteristic lipocalin-like structure [26].The location of Cys40 is predicted to be in loop L1which is a large loop near the opening of the bindingpocket [7]. The binding pocket is predicted to besimilar in size to that in BLG and lined with mostlyhydrophobic residues, some of which are conservedin both BLG and serum retinol binding protein(RBP). Because the sequence of C8Q is more similarto RBP than BLG (27% vs. 21.3% identity) and co-ordinates for the former lipocalin are now available[35], C8Q was recently modeled against RBP (L. Leb-ioda and J.M. Sodetz, unpublished data). Resultspredict a binding pocket comparable in size to theone in RBP; however, it is somewhat more hydro-philic and it contains several side chains that arecapable of hydrogen bonding.

The crystal structure of C8Q has not yet been re-ported; however, it is expected to be available soon.A mutant recombinant form of human C8Q in whichCys40 is replaced with Gly has been successfully pro-duced in insect cells [18]. The protein has been char-acterized, crystallized and di¡racted to 1.3 Aî resolu-tion (S.F. Schreck, C. Parker, L. Lebioda and J.M.Sodetz, unpublished data). Production of recombi-nant human C8Q in Escherichia coli has also beenreported [34]. Detailed features of the binding siteshould become apparent when the crystal structureis available and this information may provide insightinto the characteristics of the ligand.

5. Function of C8QQ

A ligand for C8Q has not been identi¢ed nor has itbeen shown unequivocally that C8Q has the capacity

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to bind small molecules. A speci¢c role for C8Q in theformation and function of the MAC has likewise notbeen identi¢ed. Several possibilities have been inves-tigated and the results from these studies are de-scribed below. Although the precise function of C8Qis still unknown, the range of possibilities has beennarrowed considerably.

C8Q apparently is not a major contributor to thebinding interaction between C8K-Q and C8L as evi-denced by the ability of C8K alone to bind C8L. Todetermine if C8Q is essential for the formation andfunction of the MAC itself, the C8K-C8L complexwas tested in hemolytic assays that used sheep eryth-rocytes as target cells [18]. These cells were preparedwith human C5b-7 on the surface so they would lysein the presence of functional C8 and C9. The C8K-C8L complex exhibited signi¢cant albeit reduced he-molytic activity towards these cells (approx. 15% ofC8). This indicates C8Q is not absolutely required forexpression of C8 hemolytic activity. Furthermore, itsuggests that C8Q is not required for C8 binding toC5b-7, which is consistent with the conclusion thatC8L mediates this interaction. It also suggests thatC8Q is not required for incorporation of C9 into theMAC, which is considered to be the role of C8Kwithin C5b-8. Interestingly, addition of recombinantC8Q to the C8K-C8L complex increases its hemolyticactivity to near-normal levels [18]. Thus, C8Q maynot be required for lysis but it does enhance MACactivity. It may do this by inducing a conformationalchange in C8K that increases its a¤nity for C5b-7and/or C9, or it may facilitate formation of a morelytically active MAC.

The observation that C8Q is not absolutely re-quired for C8 hemolytic activity is also consistentwith results from photolabeling experiments. C8Q isthe only constituent of C5b-8 and the MAC that isnot labeled by membrane-restricted probes whenthese complexes are formed on erythrocytes [23].This indicates that it is not closely associated withthe membrane bilayer and for this reason it is shownon the periphery of the MAC in Fig. 3. Whether ithas a major role in mediating the e¡ects of MAC onnucleated cells or in the expression of MAC bacter-iolytic activity remains to be established.

A role for C8Q in the regulation of MAC activityhas also been suggested. It is well established thathost cells are resistant to attack by their own comple-

ment [36]. Protection from `homologous' MAC isprovided by CD59, a membrane-associated comple-ment regulatory protein that binds to C8K and C9and inhibits formation of a fully functional MAC[37]. A second MAC regulatory protein (C8 bindingprotein) was described over 10 years ago and shownto bind to C8Q on nitrocellulose; however, this pro-tein was never fully characterized [38]. To obtainfunctional evidence for C8Q involvement in MACregulation, the C8K-C8L complex and C8 were com-pared for their ability to lyse human erythrocytes onwhich human C5b-7 was deposited [39]. Each boundto C5b-7 on these cells and mediated incorporationof the same amount of C9. However, both were rel-atively ine¡ective in lysing the cells. Thus, the pres-ence or absence of C8Q has no e¡ect on the restrictedactivity of C8 towards homologous cells. This sug-gests that C8Q does not interact directly with mem-brane-associated complement regulatory proteins orhave a role in regulating MAC activity.

This conclusion is signi¢cant with respect to thesuggestion that C8Q may be bivalent because of itsreported ability to bind C8K and the putative C8binding protein [40]. Indeed, some lipocalins are bi-valent and capable of binding one macromolecularligand near the open end of the L-barrel and a sec-ond one at the opposite or closed end. C8Q has beendescribed as one such lipocalin; however, only withinthe context of C8 binding protein. In light of theabove results with C8KWC8L, it is questionablewhether C8Q is actually bivalent.

A role for C8Q in the biosynthesis of C8 has alsobeen proposed. As the MAC is assembled on targetcells, C8K presumably is inserted in the membranebilayer. Therefore, it must contain one or more hy-drophobic sites that are capable of interacting withlipid. Sequences de¢ning these sites would only be-come exposed as C8 is incorporated into the MAC.Once it was identi¢ed as a lipocalin, C8Q was pro-posed to bind to such a site on C8K and therebyshield it from premature membrane interactions dur-ing intracellular processing [5]. Recent studies sug-gest that this is not the primary function of C8Q.Functional C8K can be produced independently asa recombinant protein, therefore C8Q is not requiredfor proper folding, processing or secretion [18]. Mod-els of C8Q support this conclusion in that the pre-dicted binding pocket is too deep for a single amino

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acid side chain and too small to accommodate multi-ple side chains associated with a loop. Thus it seemsunlikely that a speci¢c hydrophobic segment of C8Kis the natural `small' ligand for C8Q.

It is evident that C8K and C8Q contain mutuallyrecognizable binding sites. Both are synthesized in-dependently and therefore must associate intracellu-larly in order to form a disul¢de linkage. Further-more, mutant recombinant forms of each that areincapable of forming an interchain disul¢de bondretain the ability to interact noncovalently [18]. Re-garding this, it is of interest that when sequences ofall ¢ve MAC protein family members are aligned,C8K is unique in that it contains an insertion of 17residues at positions 159^175 (Fig. 4) [6]. This seg-ment includes Cys164 that is normally linked to C8Q.Insertions/deletions (indels) in otherwise homologousproteins are frequently associated with sites of pro-tein-protein interaction because they often involveresidues located in loops at the protein surface. Forthe C3/C4/C5 family, indels have been correlatedwith distinct binding functions performed by eachfamily member [41]. In the case of C8K, the aboveindel may constitute the binding site for C8Q and/orprovide an available, surface-exposed Cys residue.Recent studies suggest it does both. Insertion ofthis sequence in the corresponding region of C8Land co-expression of this chimera with C8Q yields adisul¢de-linked `C8L-Q' dimer [42]. This atypicaldimer is secreted from transfected cells at a levelcomparable to that observed for C8K-Q. Becausethis sequence is su¤cient to mediate intracellular in-teraction with C8Q and disul¢de bond formation, itlikely de¢nes the binding site for this subunit.

The search for a small ligand for C8Q has thusfar been limited to the retinoids. The similarity inpredicted structure between C8Q and BLG led one

group to examine the binding of radiolabeled reti-noids to puri¢ed C8K-Q [26]. C8K-Q was used becauseof the inability to isolate C8Q with its internal disul-¢de bond intact. Based on the observed binding ofretinol to C8K-Q, it was concluded that C8Q bindsretinol and therefore is a retinol transport protein.However, it should be noted that binding experi-ments were performed in 2 M NaCl. This reportedlywas necessary to disrupt electrostatic interactionswithin C8K-Q and thereby provide access to the pu-tative ligand binding site on C8Q. Analysis of thedata indicates the amount of ligand bound was ex-tremely small under these conditions (1 retinol:6000C8K-Q). Considering the high ionic strength used, thehydrophobic nature of retinol and the small amountof ligand bound, it is questionable whether these re-sults are signi¢cant.

A more recent study used recombinant C8Q andmethods commonly employed with well-character-ized retinol binding proteins to determine if retinolis a ligand [18]. These methods included absorbancespectroscopy, £uorescence emission and £uorescencequenching. Under conditions of physiological ionicstrength and where speci¢c binding to BLG wasreadily observed, retinol binding to puri¢ed C8K-Qor recombinant C8Q was not detected. Based on theseexperiments, it was concluded that retinol is not aligand for either C8K-Q or C8Q. As for the smallamount of binding observed by others at high ionicstrength, it is likely the result of nonspeci¢c parti-tioning into hydrophobic sites on C8K-Q.

6. Perspective

Perhaps the most intriguing aspect of C8Q relatesto the biological context in which it occurs, namely

Fig. 4. Indel sequence within C8K. An alignment of amino acid sequences corresponding to exon 5 of each member of the MAC pro-tein family is shown. Alignments were made based on the entire sequence of each protein. The ¢rst residue is a partial Gly(y) becausethe exon 4/5 boundary is a phase 2. Numbers identify the insertion in C8K. Cys164 is underlined.

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as part of C8 and the MAC. It remains unclear whyseven di¡erent gene products are needed to producea complex whose principal function is to disruptmembrane organization. It is especially interestingthat a lipocalin such as C8Q is part of this complex.While the studies described above have eliminatedsome possible functions for C8Q, the results are basedlargely on simple membranes and in vitro systems.Other possibilities to consider concern the MAC andits function on complex membranes.

For example, C8Q may be necessary for the forma-tion of a functional MAC on Gram-negative bacte-ria, which are considered to be its primary target. Itis conceivable that C8Q has a role in facilitating theassembly or stabilization of the MAC on bacterialmembranes by binding to a speci¢c lipid component.Such a role would explain the need for a lipocalinwithin the MAC.

Related to its possible function in bacterial killingis the prospect that C8Q may modulate the local in-£ammatory response in a manner similar to thatproposed for NGAL, the neutrophil gelatinase-asso-ciated lipocalin produced in human neutrophils.NGAL is present in neutrophils in an unassociatedform and as a disul¢de-linked heterodimer with the92 kDa form of gelatinase [43]. It has been suggestedthat NGAL released from activated neutrophilsmight function in vivo by binding small lipophilicin£ammatory mediators or lipopolysaccharides andthereby inhibit the in£ammatory response [43]. C8Qmay perform a similar function once MAC is depos-ited on the bacterial surface. By being located on theperiphery of the MAC it may bind and sequesterproin£ammatory mediators that either are releasedfrom neutrophils or are produced as byproductsfrom membrane degradation.

Nucleated cells are generally refractory to MAClysis; however, the MAC can elicit signi¢cant non-lytic stimulatory responses even when deposited onhost cells. Deposition of MAC on the membranesurface promotes the in£ux of calcium and triggersa variety of intracellular responses, some of whichpromote resistance to lysis by elimination of com-plexes through endocytosis or exocytosis [11,44].Some of these responses are proin£ammatory andinvolve release of mediators from neutrophils andmacrophages as well as platelets and endothelialcells. Considering the complexity of these mem-

branes, it may be that C8Q is directly involved inthe mechanism by which the MAC elicits these re-sponses, perhaps through interactions with mem-brane lipid. Alternatively, it may regulate these re-sponses by binding released mediators such as wassuggested above for the MAC on bacterial mem-branes.

All of these possibilities are speculative and to alarge extent based on observations made with intactC8. With the ability to produce recombinant formsof each human C8 subunit and the C8K-C8L com-plex, it should be possible to more precisely de¢nethe biological role of C8Q in the complement systemand to search more deliberately for its putative smallligand.

Acknowledgements

The authors' laboratory is supported by grant GM42898 from the National Institutes of Health, USA.

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