Biosynthesis of pro-C3, a precursor of the third component of complement.

Abstract

The complement system is composed of 11 plasma proteins that, together with proteins of the properdin system, constitute an important humoral effector of immunological reactions (1). Several native serum complement (C) proteins have been shown to consist of two or more covalently bound polypeptide subunits. These include Clq (2), C3 (3), C4 (4), C5 (3), and C8 (1). Recently, a precursor form of the fourth component of guinea pig C was identified in studies of the cell-free synthesis of C4 and in homogenates of liver (5). The precursor of C4 (pro-C4) is a single polypeptide chain of mol wt 200,000, which is converted into native serum C4, a three chain protein of similar molecular weight (4, 6).The possibility was considered that other multichain C proteins were also synthesized as single chain precursors in analogy to pro-C4 and to other known precursor proteins such as proinsulin (7). In order to test this hypothesis, we analyzed the size and subunit composition of the third component of C (C3) synthesized in tissue culture and under cell-free conditions. Materials and MethodsPhenylmethylsulfonylfluoride (PMSF), 1 reduced glutathione, HEPES, GTP, creatine phosphokinase (CPK), phosphorylase A, and bovine serum albumin (BSA) were purchased from Sigma Chemical Co., St. Louis, Mo. ATP (dipotassium salt) and phosphocreatine (dipotassium salt) were from Calbiochem, La Jolla, Calif. Ribonuclease-free sucrose and ovalbumin (OVA) were from Schwartz/Mann Div., Becton, Dickinson and Co., Orangeburg, N. Y. Tritiated leucine, 45 Ci/ mmol, and a 14C-labeled I~amino acid mixture consisting of equal amounts of 14C-leucine (325 mCi/ retool), 14C-valine (251 mCi/mmol), 14C-isoleucine (321 mCi/mmol), and l~C-lysine (282 mCi/mmol) were obtained from New England Nuclear, Boston,, Medium 199 Special (lacking valine, leucine, isoleucine, lysine, and glutamine), and glutamine were purchased from Microbiological Associates, Bethesda, Md.; fl-galactosidase and carbonic anhydraso from Worthington Biochemical Corp., Freehold, N. J.; NCS solubilizer from Amersham/Searle Corp., Chicago, Ill.

A C3(H20) recycling pathway is a component of the intracellular complement system

“…Uptake was rapid, saturable, and sensitive to competition with unlabeled C3(H 2 O), indicating a specific mechanism of loading. Figure 1A) were generated (14). This distinction between PBCs and their respective cell lines was not limited to B cells, but was true for several other PBCs tested, including CD4 + T cells versus Jurkat, a human T lymphocyte cell line (not shown).…”

Complement biosynthesis by the human hepatoma-derived cell line HepG2.

“…In the guinea pig (15,16) and the mouse (17,18) there is evidence that several multichain complement proteins are synthesized as single-chain precursor proteins. For the corresponding human complement proteins, the availability of a hepatoma-derived cell line HepG2 (19) permitted a similar analysis of the precursor, human pro-C3.…”

Pretranslational regulation of the synthesis of the third component of complement in human mononuclear phagocytes by the lipid A portion of lipopolysaccharide.

“…In the human, its plasma concentration is the highest of any complement component (150-300 mg/ 100 ml) and is regulated as an acute phase reactant. C3 is synthesized as a single-chain precursor protein (proC3) of -200 kD, which subsequently undergoes signal peptide cleavage and proteolytic cleavage by a plasminlike enzyme to its native two-chain form (5,6). Although the hepatocyte is the primary source of plasma C3, studies of C3 synthesis by monocytes from C3-deficient individuals (7) and synovial tissue from patients with rheumatoid arthritis (8) suggested that regulation of C3 synthesis is independent in monocytes and hepatocytes and that mononuclear phagocyte production of C3 plays a significant role in controlling tissue concentrations of this protein.…”