Domain structure and associated functions of subcomponents C1r and C1s of the first component of human complement.

Abstract

The serine protease subcomponents of the ac- (12)(13)(14). The (Clr, Cls)2 subunit is a very elongated structure of dimensions 51-59 nm, as first shown by electron microscopy (15) and confirmed by neutron-scattering studies in solution (16). However, the domain structure of (Clr, Cls)2 remains to be elucidated. The present study allows a detailed interpretation of the domain structure of Cir, Cls, and their associations Clr2, Cls2, and (Clr, Cls)2. MATERIALS AND METHODSProenzymic and activated forms of Clr and Cls were purified from human serum by using insoluble immune aggregates as described (5, 12). Calcium-dependent interactions of Clr and Cls before or after limited proteolysis were measured by sucrose gradient ultracentrifugation as described (17).Limited Proteolytic Cleavages. Autolytic cleavage of Cir (12, 18) was obtained by incubation of the protein (0.4-0.5 mg/ml) for 7 hr at 370C in 145 mM NaCl/5 mM triethanolamine HCl, pH 7.4. Limited cleavage of Cis was carried out using plasmin: Cis (1-1.5 mg/ml) in 145 mM NaCI/5 mM triethanolamine HCl, pH 7.4, was incubated with human plasmin (KABI, 25 units/mg) for up to 1 hr at 370C [enzyme/ substrate ratio (wt/wt), 1:50]. When indicated in the text, Clr and Cls were labeled with [1,3-3H]diisopropyl phosphorofluoridate (Amersham) either before or after proteolytic cleavage, as described (17). Fragments from limited proteolysis of Clr and Cis were separated by high-pressure gel permeation on a TSK-G3000 SW column (LKB) equilibrated in the same buffer as used for cleavage.Electron Microscopy. Proteins were dialyzed against 50 mM ammonium bicarbonate and glycerol was added to a final concentration of 75% (vol/vol). Protein samples (40 ,ug/ml) were sprayed onto freshly cleaved mica slides at room temperature. Samples were desiccated in an Edwards evaporator for 0.5-1 hr under a vacuum of at least 1o-, torr(1 torr = 133 Pa). Rotary shadowing with tantalum/tungsten was done with a 7°angle; the resulting replicas were coated with carbon, floated onto distilled water, and picked up on a 400-mesh copper grid. They were examined in 100 CX and 100 S Jeol electron microscopes at 80 kV. Dimensions of the molecules were corrected by subtracting 20 A to account for metal thickness (Clq observed both after rotary shadowing and negative staining was taken as a reference). RESULTSElectron Microscopy of Native Proteins. Pictures of Cir ( Fig. 1 A and B) 4477The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Deciphering complement mechanisms: The contributions of structural biology

“…2). That the C1r catalytic domain associates as a homodimer in solution has been demonstrated using various techniques (Villiers et al, 1985;Weiss et al, 1986;Lacroix et al, 2001). Likewise, the particular head-totail configuration seen in the structure is consistent with previous chemical cross-linking experiments and with the observation that deletion of the CCP 1 module prevents dimer formation (Lacroix et al, 1997(Lacroix et al, , 2001).…”

Neutron scattering study of the (γ‐B) catalytic domains of complement proteases Cl̄r and Cl̄s

“…The catalytic regions have initially been considered as domains on the basis of their resistance to proteolysis and their globular appearance in electron microscopy. Thus, treatment of Cls with plasmin yields a (y-B) fragment that is visualized as a monomeric globular structure [6], whereas limited proteolysis of the native Cir-Cir dimer [6-81 ends in a truncated (r-B)2 molecule visualized as twin domains. However, recent studies by differential scanning calorimetry [9] indicate that the (y-B) fragment of Cls comprises three independently folded domains, two in the y region (motifs IV and V) and one in the catalytic B chain.…”

Baculovirus-mediated Expression of Truncated Modular Fragments from the Catalytic Region of Human Complement Serine Protease C1s

“…Based on chemical cross-linking and three-dimensional homology modeling, it was shown recently that this module closely interacts with the serine protease domain on the side opposite to both the active site and the Arg 422 -Ile 423 bond cleaved upon activation (12). From a functional point of view, the fact that the proteolytic fragment ␥-B retains a functional active site (9) and studies based on the use of monoclonal antibodies indicating that the ␥ segment may be involved in C4 binding (13,14) together sug-gest that ␥-B may represent the catalytic region of C1 s. However, no precise information is available yet on the respective role of the serine protease domain and CCP modules in the various aspects of C1 s catalytic activity. The present study provides identification of the domains of C1s involved in its activation by C1 r, proteolytic activity toward C4 and C2, and reactivity toward C1 inhibitor, based on baculovirus-mediated expression of truncated recombinant fragments from the ␥-B region.…”

“…Limited proteolysis of C1 s with plasmin yields a C-terminal fragment ␥-B, comprising two contiguous complement control protein (CCP) modules, a 15-residue intermediary segment homologous to the activation peptide in chymotrypsinogen, and the serine protease domain (9). The CCP modules (originally known as short consensus repeats) are structural motifs of about 60 residues homologous to those mostly found in various complement regulatory proteins known to interact with components C3b and/or C4b (10).…”