CHAPTER FIVE
5.2 Cl-inhibitor
Cl-inhibitor is a plasma protein of the serine proteinase inhibitor superfamily (serpins) and is synthesized in the liver (Bock et al., 1986). Themolecul e ishighly glycosyl ated with N- and 0- link ed carbohydrate s cons titute a total of49% ofits molecular weight (Bock et al., 1986). Thus of the 104 kDa molecul e only 52 kDa constitutes the core protein (Figure 5.1)(Bock et al., 1986). Deglycosylation with N-glycanas e and 0- glycanase, or both, does not have any major effect on its functional activity (Reboul et al., 1987). It also has two disulphide bonds, one connecting residues 101-406 and the otherconnects residu es 108-183 of the molecule(Bocket al., 1986).
Figure 5.1 Schematic diagram of mature, Cl-inhibitor. Nand C indicatethe N-and C-tennini of the 478-residue polypeptidechain.The disulphide bridge pattern is shown with bars;
residue numbers refer to disulfide-bounded cysteine residues.Diamonds marks known sites ofoligosaccharides (Asn-3, -47,-59,-216, -23 1, and -330,Ser-42, Thr-26,-49, -61, -66, -70 and -74). The peptide bond cleaved by Cls during complex formation is indicatedwith anarrow (Bocket al., 1986).
In the complement cascade Cl-inhibitor requires complex formation of Cl-inhibitor with C1s for activation (Brower and Harpel, 1981). Like other serpins, the active site of Cl-inhibitor is locat edin an exposed loop nearthe C-tenninusof the molecule (Knauper et al., 1991). C1s binds Arg 444 in its PI site and Thr445 in the P"! site(Figure 5.1), during .complex formation, releasing a small C-tenninal fragment and forming a covalent inactive C1s: Cl-inhibitor complex (Bock et al., 1986; Salvesen et al., 1985).
PMN elastasemay cleave Cl-inhibitor at residues 37 (I-L), 40 (V-S) and 440 (I-S) ofthe N- and C-tenninal region and result in the destruction of inhibitory activity (Bock et al., 1986) (Figure 5.2). MMP-8 and MMP-9 cleave residues 439 (A-I) and 441 (S-V) closer to the reactive site (Figure 5.2)(Knauper et al., 1991; Pemberton et al., 1989).
Inactivation of Cl-inhibitor by MMPs can beprevented by the addition ofEDTA and 1, 10-phenanthroline(Knauperet al., 1991) or peptide inhibitors (Grey et al., 1992).
Figure5.2
1 13 33 S S 438 V R· T L L V F E 476P R A478
(NH2) N-P-N .. . E-S-L-Q ... F-V-E-P-¥L-E-l -.... -S-A-I-S- -A- - - - - - - ... - - -
(COOH) A A
tit
C E
Proteolytic modifi cation of human Cl-inhibitor by va r ious proteolytic enzymes as indicated byarrows (adaptedfrom Knaupe retal .,1991).
A, HumanPMN elastase E,Human PMNcollage nase
C, HumanPMNgelatinas e (mainproduct) D,Pseudomonasaeruginosa
E, HumanPMNgelatinase (minor product)
*
Active siteCl -inhibitor removes active Clr and Cls bybinding and forming a complex with each molecule (Figure 5.3)(Zicc ardi and Coope r, 1979). Dissociation of Clr and Cls from Clq results in the initiation of classical complement pathway resulting in poreformation in the surface of the bound microorganisms or microorganism bound Clq may bind to one of the Clqreceptors (ClqRs) and phagocytosis and respiratoryburst may ensue.
5.3 Clq, Clqreceptors and cellular responses
Cl q a 462 kDa molecule has six A, B, and C polypeptide chains, each chain is composed ofapproximately 225 residues which form a triple helical coil (Nicholson- Weller and Klickstein, 1999; Ruiz et al., 1999)(Figure5.3). The amino terminal half of each chain has a collagen-like region (CLR), while the carboxy terminal half of the molecule has a globular lectin region (Nicholson-Wellerand Klickstein, 1999) making it a member of the "collectin" family (Figure 5.3). The two domains are separated by a kink region where the globularstructurestarts to diverge (Ruiz et al., 1999).
Itis residues 14-26 of the A chain of the globular lectin region of Cl q thatbinds to the surfaceof bacteriaand initiatesthe classicalpathwayof complement opsonization (Jiang
et al., 1994) (Figure 5.3). In initiation ofrespiratory burst, however, it is the C chainthat is required (Ruiz et al., 1999) (Figure 5.3). Previously activation of complement was thought to be initiat ed only by C1q bound to the Fe region of antibodies. However, recently C1q has been shown to bind directly to molecules involved in many inflammatory diseases and molecules such as DNA, Alzheimer's proteins (Eggleton et al., 1998),viral compone nts, lipid A, othe rcompo nents of bacteri a and parasites (Bobak et al., 1987), ~-amyl oid protein (Jiang et al., 1994) and C-reactive protein (CRP)(Eggleton et al., 1998). The binding site on such molecules is distinct from the mannose-binding lecithin (MBL) pathway which binds mannose residues, and is assoc iatedwith two serine proteinases (MASP-1and MASP-2). Thismimics the activity seen in Cl q-C1r2C1S2complex (Eggleton et al., 1998).HeretheMBLcomplex bindsto a pathog en surface and MASP-1 and MASP-2 are activated to cleave C4 and C2 (Eggleton et al., 1998) forming C3 convertase from C2b bound to C4b, as in the Cl q- C1r and C1s classicalpath way(Figure 1.3).
During Cl q-mediated opsoni zation, the collagen-like region of C1q (C1qCLR), with a molecular weight of 180 kDa, is necessary to trigger C1q-mediated phagocytosis and superoxide production by PMNs (Eggleton et al., 1998; Ruiz et al., 1995; 1999) and even MMP-cleaved C1q fragments may trigger respir ator y burst (Ruiz et al., 1999).
Whether binding initiates phagocytosis or superoxid e production, however, depends on which C1q receptor is bound. Binding of C1qCLR to the C1qRp receptor (126 kDa) results in initiation of phagocytosis while binding ofC1qCLR to theC1qRO'2-receptors (60 kDa) generates superoxideproduction (Ruiz et al.,1999;Kishor eand Reid, 2000).
Like C1q, other collectins such as MBL and pulmonary surfactant protein A (SP-A) have been shown to enhanc e Fe receptor- or compleme nt receptor-mediated phagocytosis. Howe ver, neither SP-A nor MBP stimulate superoxide production in PMNs (Ruiz et al., 1995; Goodman and Tenner, 1992). They were,therefore, considered less important in the curr ent studyand steps were taken to ensure onlythe Clq-mediated system was studied.
Receptive surface
COOH, Globular lectin regio
<0
NH2,Collagen like domain
(B)
KINK region
+
2~
(A)
+
~
rs sr4~
(C)
Figure 5.3 Early assembly of the complement system.A) After binding of Cl to the surface of the particle or antibody molecule, Cl-inhibitor (In) dissociates Clr (r) and Cls (s) to initiate the classical complement pathway (Adapted from Ziccardi and Cooper, 1979). B) The Clq molecule. C) A single triple helical structure of Clq (adapted from Ruiz et al., 1995).
Itis known that during stimulation of PMNs by C1q-opsonized particles, phagocytosis and respiratory burst is initiated (Eggleton et al., 1998; Tenner and Cooper, 1982;
Eggleton et al., 1998; Goodman and Tenner, 1992). Primary and secondary granule release does not occur (Goodman and Tenner, 1992) but MMP-9 granules may be released. Whether TIMP-1 is released after such stimulation is, however, unknown.
Not much is known about the signalling mechanism of Cl q-mediated phagocytosis and respiratory burst triggered by ClqRp- and Cl qk.Oyreceptors. Unfortunately in previous experiments performed by Tenner and Cooper (1982), to study such effects, Cl q-coated latex beads that were too large to be internalized by PMNs were used. Therefore, C1q- mediated phagocytosis was not observed and results were left incomplete. Such experiments were,therefore,repeated during the current study using smaller beads.