Hans J. Müller-Eberhard

Anaphylatoxin inactivator of human plasma: its isolation and characterization as a carboxypeptidase

The failure of human serum to give rise to anaphylatoxin activity could be attributed to the presence of a potent inactivator of anaphylatoxin in human serum. The inactivator was isolated and characterized as an alpha-globulin with a molecular weight of approximately 310,000. It was found to abolish the activity of both anaphylatoxins, which are derived respectively from the third and the fifth component of complement, and of bradykinin. Inactivation of C3-derived anaphylatoxin and of bradykinin was accompanied by release of C-terminal arginine from these peptides. The anaphylatoxin inactivator was shown to hydrolyze the synthetic substrates hippuryl-L-arginine and hippuryl-L-lysine and to be inhibited by ethylenediaminetetraacetate (EDTA) or phenanthroline. These observations indicate that the anaphylatoxin inactivator constitutes a metal-dependent enzyme resembling in specificity pancreatic carboxypeptidase B.

Anaphylatoxins: C3a and C5a

Publisher Summary This chapter discusses the structural and functional characterization of anaphylatoxins, spasmogenic substances released during complement activation. These low molecular weight peptide fragments of C3 and C5 elicit a variety of cellular responses, which implies that they play a significant role in inflammation and acute allergic reactions. Their stimulation of multiple cell types at pico- and femtomolar concentrations suggests a hormone-like action via specific cell surface receptors. By using the information available defining the amino acid sequences of these molecules it is possible to synthesize oligopeptides with the properties of anaphylatoxins and these compounds serve as effective tools for analyzing further the mechanism(s) by which these potent substances function. They are functionally defined by their actions on the vasculature, smooth muscle, mast cells, and certain types of peripheral blood cells. The most important contribution of anaphylatoxin research to biology is the realization that complement reaction products act as hormone-like messengers that profoundly affect the function of certain cells. A variety of cell types is activated by the complement reaction products C3a and C5a, whether they are acting as spasmogens, chemotaxins, or cause release reactions.

Molecular biology and chemistry of the alternative pathway of complement.

Publisher Summary This chapter presents the proteins of the alternative pathway and their special properties as enzymes, enzyme substrates, and regulators. The participation with the classical pathway components, C5–C9, in membrane attack is considered. The surprising discovery of the activation of the alternative pathway by many systems other than antibody is emphasized along with the key role played by C3 and its component parts. The alternative pathway of complement activation is closely linked to natural resistance to infections. The chapter discusses biological manifestations of alternative pathway activation. This area of research has been facilitated by isolated cytolytic alternative pathway that includes the five proteins of the membrane attack pathway. Thus, antibody-independent killing by the cytolytic pathway of bacterial and animal cells, including virus-infected human cells, is illuminated. The biomedical relevance of the alternative pathway as part of the complement system is unquestionable. Once activated, it can mark particles for phagocytosis, induce killing of nucleated cells or of bacteria, and cause lysis of enveloped viruses or virus-infected cells. It generates biologically active protein fragments, such as Bb, C3a, C3b, and C5a that influence function and behavior of inflammatory cells and cells of the immune system.

Chemistry and Reaction Mechanisms of Complement

Publisher Summary This chapter discusses the chemistry and reaction mechanisms of complement. Complement constitutes the principal, immunologically relevant effector system that is present in blood serum. It consists of nine components or eleven distinct serum proteins. Membranes are the primary target of complement. They are irreversibly damaged, sustaining distinct ultrastructural lesions, by direct attack that requires participation of all nine complement components. Isolation of many of these components makes possible the analysis of the chemistry and dynamics of the complement reactions themselves and the understanding of the protein-protein interactions and enzyme activations involved. The effects of complement, primarily on cell surface membranes, eventuate in a spectrum of changes ranging from cell lysis to directed migration, histamine release, and susceptibility to phagocytosis, all of which are partially described in molecular terms. Complement research has become an active, rapidly moving, and exciting field of the biological sciences. New methods and tools are available to the biologist and clinician for the investigation of the physiogenic and pathogenic role of complement.

The reaction of monomeric and aggregated immunoglobulins with Cl

Abstract The relative capacity of different immunoglobulin classes and subclasses in different states of aggregation to bind the first component of complement (C l ) was studied. Both monomeric γG and 7S γM immunoglobulins were found to be able to bind C l . The binding capacity of γG myeloma proteins was dependent upon the γG subclass, the order of reactivity being γ G3 > γ Gl > γ G2 > γ G4. No differentiation into functional subclasses was possible on the examination of 12 different monomeric γM preparations. The relative C l binding capacity of γM polymers was: 7S γ M = 1, 19S γ M = 15, 27–35S γ M = 64, and > 35S γ M = 116. Comparable amounts of the 7S subunit of γM and γG bound C l to a similar extent, and aggregated γM and γG were also comparable in C l binding, suggesting that the differences in the relative C l binding capacity of these immunoglobulins is dependent upon the degree of polymerization that is present in the native state of these proteins. The Fc fragment of γCl was as efficient as the intact protein in fixing C l , when compared on a molar basis; human F(ab′) 2 and the cyanogen bromide treated Fc fragment were unable to bind C l .

The Potential Pathogenic Role of Complement in Dengue Hemorrhagic Shock Syndrome

Abstract In Bangkok, Thailand, 49 of 127 patients with dengue hemorrhagic fever experienced shock. The concentration of nine complement proteins measured in serial serum samples decreased during shock with the exception of C9. C3 and C5 were reduced to 20 to 40 per cent of normal in severe cases. Decrease of plasma fibrinogen, appearance of fibrinogen split products and thrombocytopenia indicated occurence of intravascular coagulation. Metabolic studies of C3 and C1q, performed on 24 patients, indicated a markedly enhanced fractional catabolic rate especially during shock. These results support the concept that activation of complement can constitute a major factor in the pathogenesis of dengue hemorrhagic shock. (N Engl J Med 289:996–1000, 1973)

The alternative pathway of complement activation.

Publisher Summary This chapter presents the alternative pathway of complement activation. Two pathways for the recruitment of complement and its biological activities exist in plasma and serum. The first is termed as the “classical pathway,” which is triggered by contact of C1 with immune complexes containing antibodies of the immunoglobulin G (IgG) or immunoglobulin M (IgM) class. Recognition of immune complexes by C1 is followed by the assembly of the activation unit (C2, C3, and C4) and the membrane attack unit (C5, C6, C7, C8, and C9). The second pathway, the alternative or properdin pathway, is activated by naturally occurring polysaccharides and lipopolysaccharides and by aggregates of immunoglobulin A (IgA). Initiation of the alternative pathway typically appears to require particulate activators. The initial events seem to occur on the surface of the activating particle. The chapter also presents a molecular concept of the entire properdin pathway. This concept envisages a single enzyme to fulfill the various functions of the pathway by being modulated sequentially by different factors.

Metabolism of third complement component (C3) in nephritis. Involvement of the classic and alternate (properdin) pathways for complement activation.

Abstract The mechanism of depression of the third complement component (C3) was studied in 20 patients with systemic lupus erythematosus and nephritis (13 with low C3), and 41 patients (21 with low C3) with chronic glomerulonephritis. The metabolism of C3 was studied by examining the plasma turnover of radioiodinated C3. The serum levels of the fourth component (C4) and properdin factor B (involved respectively in the classic and alternate pathways to C3 activation) were also examined. In patients with low serum C3, catabolic rates of C3 were elevated. Twelve of 13 patients with systemic lupus erythematosus and nephritis and low C3 had depressed levels of C4, and eight of these 12 had a decrease in serum factor B. Seven of 21 patients with chronic glomerulonephritis and low C3 had depressed serum levels of factor B, whereas serum C4 was normal in the entire group. The findings suggest that in systemic lupus erythematosus, the classic pathway is activated, with recruitment of the alternate pathway, whereas...

Complement metabolism in man: hypercatabolism of the fourth (C4) and third (C3) components in patients with renal allograft rejection and hereditary angioedema (HAE)

Abstract Highly purified and radioiodinated human C4 and (or) C3 were administered to patients with renal allografts in rejection, with hereditary angioedema (HAE), with chronic glomerulonephritis, and to control subjects. The latter group included normal individuals, anephric patients before transplantation, and stable renal allograft recipients. The catabolic rates of these complement proteins were determined by analysis of the disappearance of plasma protein-bound radioactivity (km), and by direct measurement of urinary excretion of radioactivity (ku). The correlation coefficient between these two methods was 0.96. The mean ±2 SD for catabolic rates in the control subjects was 0.9-2.7% plasma pool/hr for C4 and 0.9-2.0% plasma pool/hr for C3. Patients experiencing renal allograft rejection had unstable levels of C4 and C3, and exhibited moderate hypercatabolism of both proteins. One patient with chronic glomerulonephritis had hypercatabolism of C4 and C3 in the presence of stable normal serum levels. In patients with HAE who had extremely low levels of C4, catabolic rates for C4 were markedly elevated (3.7, 5.8, 7.0 and 8.8%/hr). Analysis of plasma curves in HAE revealed a three component disappearance curve instead of the two component curve in control subjects receiving the same preparation. Even though C3 levels were normal, moderate hypercatabolism of C3 was also present in HAE (2.6, 2.8, 2.8, and 3.2% of pool/hr). The marked hypercatabolism of C4 in HAE constitutes the first direct evidence for the in vivo destruction by uninhibited C1 esterase of its natural substrate C4. The moderate hypercatabolism of C3 is consistent with the in vivo formation of C3-convertase.

A comparison of methods for the molecular quantitation of the fourth component of human complement

Abstract Quantitation of C′4 by the effective molecule titration procedure yielded values which were approximately 300 times lower than values based on nitrogen determination or on immunochemical analysis. To explore the reason for the disparate results, a quantitative analysis of the reaction mechanism of C′4 in immune hemolysis was performed employing highly purified, radioactively labeled C′4. It was found that the majority of the C′4 molecules escape detection by effective molecule titrations due to (a) inefficient binding of C′4 to the cell surface, and (b) hemolytic inefficiency of specifically cell bound C′4. Two factors were experimentally derived which permit conversion of the number of effective C′4 molecules to the absolute number of C′4 molecules. The number of effective molecules per μg of C′4 was similar for isolated C′4 and for C′4 in fresh human serum.