Disease-associated N-terminal complement factor H mutations perturb cofactor and decay-accelerating activities

Many mutations associated with atypical hemolytic uremic syndrome (aHUS) lie within complement control protein modules 19-20 at the C terminus of the complement regulator factor H (FH). This region mediates preferential action of FH on self, as opposed to foreign, membranes and surfaces. Hence, speculation on disease mechanisms has focused on deficiencies in regulation of complement activation on glomerular capillary beds. Here, we investigate the consequences of aHUS-linked mutations (R53H and R78G) within the FH N-terminal complement control protein module that also carries the I62V variation linked to dense-deposit disease and age-related macular degeneration. This module contributes to a four-module C3b-binding site (FH1-4) needed for complement regulation and sufficient for fluid-phase regulatory activity. Recombinant FH1-4(V62) and FH1-4(I62) bind immobilized C3b with similar affinities (K(D) = 10-14 μM), whereas FH1-4(I62) is slightly more effective than FH1-4(V62) as cofactor for factor I-mediated cleavage of C3b. The mutant (R53H)FH1-4(V62) binds to C3b with comparable affinity (K(D) ～12 μM) yet has decreased cofactor activities both in fluid phase and on surface-bound C3b, and exhibits only weak decay-accelerating activity for C3 convertase (C3bBb). The other mutant, (R78G)FH1-4(V62), binds poorly to immobilized C3b (K(D) >35 μM) and is severely functionally compromised, having decreased cofactor and decay-accelerating activities. Our data support causal links between these mutations and disease; they demonstrate that mutations affecting the N-terminal activities of FH, not just those in the C terminus, can predispose to aHUS. These observations reinforce the notion that deficiency in any one of several FH functional properties can contribute to the pathogenesis of this disease.     

Recombinant generation of two fragments of the rat complement inhibitory factor H [FH(SCR1-7) and FH(SCR1-4)] and their structural and functional characterization in comparison to FH isolated from rat serum

Factor H (FH) is the predominant soluble inhibitor of the complement system. With a concentration of 200-800 microg/ml in human and rat plasma it acts as a cofactor for the soluble factor I (FI)-mediated cleavage of the component C3b to iC3b. Furthermore it competes with factor B for binding to C3b and C3(H2O) and promotes the dissociation of the C3bBb complex. FH is a monomer of about 155 kDa which comprises 20 short consensus repeats (SCR), each of which is composed of approximately 60 amino acid (aa) residues. Two functional fragments of FH comprising the SCR1-4 or SCR1-7 were generated using either the Baculovirus system or stably transfected human embryonal kidney cells, respectively. These fragments, as well as FH purified from rat serum, were first analyzed for their relative molecular weights (Mr) using non-reducing or reducing SDS-PAGE. The Mr of the FH variants differed by about 20% depending on the experimental conditions employed. Only the Mr of proteins separated under reducing conditions were in accordance with the MW calculated from the aa sequence. Analyses of the glycosylation patterns using PAS-staining showed a lack of staining of the recombinant variants (SCR1-4 and SCR1-7) in contrast to FH(SCR1-20) from serum. Using a complement hemolysis assay (CH50-assay) all three variants exhibited a molar complement inhibitory activity of FH(1-20)/FH(1-7)/FH(1-4) of about 3/1/1. These data support the postulated model of FH bearing three binding sites for its ligand C3b, from which one is located in the SCR1-4, whereas the other two are located in the SCR8-20.     

Stabilization of the amplification convertase of complement by monoclonal antibodies directed against human factor B

Three IgM mouse monoclonal antibodies (MoAb), 5B5-A, 2D2-B, and 5B12-A, were prepared by fusion of spleen cells from mice immunized with human B with the SP 2/0 myeloma cell line. They were assessed for their effect on cell-bound and fluid-phase amplification convertases of complement (C) with purified proteins in vitro. 5B5-A and 2D2-B were similar in their effects on cell-bound preformed C3bBb in that they bound to cell-bound C3bBb, stabilized the C3bBb convertase, and rendered the C3bBb convertase relatively resistant to the plasma protein H. These two MoAb were also able to enhance C3 consumption in vitro in reaction mixtures containing C3b, C3, B, and D. At the same time, they presumably stabilized the C3 convertase and caused relative sparing of B hemolytic activity in the reaction mixtures. In contrast, 5B12-A, which also bound to Bb and C3bBb, did not induce any stabilization, but rather caused accelerated decay of cell-bound C3bBb. These results indicate that MoAb against B can have C3NeF-like activity. On the other hand, not all MoAb against B have stabilizing activity on the C3bBb convertase.     

A corresponding tyrosine residue in the C2/factor B type A domain is a hot spot in the decay acceleration of the complement C3 convertases

The cleavage of C3 by the C3 convertases (C3bBb and C4b2a) determines whether complement activation proceeds. Dissociation (decay acceleration) of these central enzymes by the regulators decay-accelerating factor (DAF), complement receptor 1 (CR1), factor H, and C4-binding protein (C4BP) controls their function. In a previous investigation, we obtained evidence implicating the alpha4/5 region of the type A domain of Bb (especially Tyr338) in decay acceleration of C3bBb and proposed this site as a potential interaction point with DAF and long homologous repeat A of CR1. Because portions of only two DAF complement control protein domains (CCPs), CCP2 and CCP3, are necessary to mediate its decay of the CP C3 convertase (as opposed to portions of at least three CCPs in all other cases, e.g. CCPs 1-3 of CR1), DAF/C4b2a provides the simplest structural model for this reaction. Therefore, we examined the importance of the C2 alpha4/5 site on decay acceleration of C4b2a. Functional C4b2a complexes made with the C2 Y327A mutant, the C2 homolog to factor B Y338A, were highly resistant to DAF, C4BP, and long homologous repeat A of CR1, whereas C2 substitutions in two nearby residues (N324A and L328A) resulted in partial resistance. Our new findings indicate that the alpha4/5 region of C2a is critical to decay acceleration mediated by DAF, C4BP, and CR1 and suggest that decay acceleration of C4b2a and C3bBb requires interaction of the convertase alpha4/5 region with a CCP2/CCP3 site of DAF or structurally homologous sites of CR1 and C4BP.     

Mutations of the type A domain of complement factor B that promote high-affinity C3b-binding

Factor B is a zymogen that carries the catalytic site of the complement alternative pathway convertases. During C3 convertase assembly, factor B associates with C3b and is cleaved at a single site by factor D. The Ba fragment is released, leaving the active complex, C3bBb. During the course of this process, the protease domain becomes activated. The type A domain of factor B, also part of Bb, is similar in structure to the type A domain of the complement receptor and integrin, CR3. Previously, mutations in the factor B type A domain were described that impair C3b-binding. This report describes "gain of function" mutations obtained by substituting factor B type A domain amino acids with homologous ones derived from the type A domain of CR3. Replacement of the betaA-alpha1 Mg2+ binding loop residue D254 with smaller amino acids, especially glycine, increased hemolytic activity and C3bBb stability. The removal of the oligosaccharide at position 260, near the Mg2+ binding cleft, when combined with the D254G substitution, resulted in increased affinity for C3b and iC3b, a C3b derivative. These findings offer strong evidence for the direct involvement of the type A domain in C3b binding, and are suggestive that steric effects of the D254 sidechain and the N260-linked oligosaccharide may contribute to the regulation of ligand binding.     

Localization of macrophage agglutination factor activity to the gelatin-binding domain of fibronectin

We previously proposed that macrophage agglutination factor (MAggF, a T cell-derived guinea pig lymphokine) is a fibronectin (FN). We now show MAggF binding to gelatin and to peritoneal macrophages is mediated by domains similar to corresponding domains of plasma FN. MAggF activity in lymphokine concentrates prepared by two different methods differed nearly 10-fold in m.w. on gel filtration chromatography. Despite this difference, MAggF dose-activity curves of both preparations were parallel, and MAggF in both preparations bound reversibly to gelatin and to monoclonal anti-guinea pig FN immunoadsorbents. MAggF activity in one preparation was inhibited by the addition of soluble monoclonal antibody specific for the gelatin-binding domain of human FN; inhibitory activity of this antibody was blocked by purified guinea pig plasma FN or partially purified MAggF from the other preparation. Measured MAggF activity of both preparations was reduced in a dose-dependent manner by pretreatment of indicator macrophages with monoclonal anti-human monocyte FN receptor antibody or F(ab')2 fragments or with guinea pig plasma FN. Neither anti-FN receptor antibody nor plasma FN interacted directly with MAggF. Indirect immunofluorescence studies confirmed the presence of uncomplexed plasma membrane receptors for FN on indicator macrophages in MAggF-responsive populations that were able to bind added FN. Our identification of MAggF as lymphokine FN provides a basis for future biochemical analysis of delayed hypersensitivity inflammatory reactions.     

Structural analysis of the C-terminal region (modules 18-20) of complement regulator factor H (FH)

Factor H (FH) is a soluble regulator of the human complement system affording protection to host tissues. It selectively inhibits amplification of C3b, the activation-specific fragment of the abundant complement component C3, in fluid phase and on self-surfaces and accelerates the decay of the alternative pathway C3 convertase, C3bBb. We have determined the crystal structure of the three carboxyl-terminal complement control protein (CCP) modules of FH (FH18-20) that bind to C3b, and which additionally recognize polyanionic markers specific to self-surfaces. These CCPs harbour nearly 30 disease-linked missense mutations. We have also deployed small-angle X-ray scattering (SAXS) to investigate FH18-20 flexibility in solution using FH18-20 and FH19-20 constructs. In the crystal lattice FH18-20 adopts a "J"-shape: A ~122-degree tilt between the structurally highly similar modules 18 and 19 precedes an extended, linear arrangement of modules 19 and 20 as observed in previously determined structures of these two modules alone. However, under solution conditions FH18-20 adopts multiple conformations mediated by flexibility between CCPs 18 and 19. We also pinpoint the locations of disease-associated missense mutations on the module 18 surface and discuss our data in the context of the C3b:FH interaction.     

Decay-accelerating factor (DAF), complement receptor 1 (CR1), and factor H dissociate the complement AP C3 convertase (C3bBb) via sites on the type A domain of Bb.

The AP C3 convertase, C3bBb(Mg(2+)), is subject to irreversible dissociation (decay acceleration) by three proteins: DAF, CR1, and factor H. We have begun to map the factor B (fB) sites critical to these interactions. We generated a panel of fB mutations, focusing on the type A domain because it carries divalent cation and C3b-binding elements. C3bBb complexes were assembled with the mutants and subjected to decay acceleration. Two critical fB sites were identified with a structural model. 1) Several mutations centered at adjacent alpha helices 4 and 5 (Gln-335, Tyr-338, Ser-339, Asp-382) caused substantial resistance to DAF and CR1-mediated decay acceleration but not factor H. 2) Several mutations centered at the alpha 1 helix and adjoining loops (especially D254G) caused resistance to decay acceleration mediated by all three regulators and also increased C3b-binding affinity and C3bBb stability. In the simplest interpretation of these results, DAF and CR1 directly interact with C3bBb at alpha 4/5; factor H likely interacts at some other location, possibly on the C3b subunit. Mutations at the C3b.Bb interface interfere with the normal dissociation of C3b from Bb, whether it is spontaneous or promoted by DAF, CR1, or factor H.     

The Scl1 protein of M6-type group A Streptococcus binds the human complement regulatory protein, factor H, and inhibits the alternative pathway of complement

Non-specific activation of the complement system is regulated by the plasma glycoprotein factor H (FH). Bacteria can avoid complement-mediated opsonization and phagocytosis through acquiring FH to the cell surface. Here, we characterize an interaction between the streptococcal collagen-like protein Scl1.6 of M6-type group A Streptococcus (GAS) and FH. Using affinity chromatography with immobilized recombinant Scl1.6 protein, we co-eluted human plasma proteins with molecular weight of 155 kDa, 43 kDa and 38 kDa. Mass spectrometry identified the 155 kDa band as FH and two other bands as isoforms of the FH-related protein-1. The identities of all three bands were confirmed by Western immunoblotting with specific antibodies. Structure-function relation studies determined that the globular domain of the Scl1.6 variant specifically binds FH while fused to collagenous tails of various lengths. This binding is not restricted to Scl1.6 as the phylogenetically linked Scl1.55 variant also binds FH. Functional analyses demonstrated the cofactor activity of the rScl1.6-bound FH for factor I-mediated cleavage of C3b. Finally, purified FH bound to the Scl1.6 protein present in the cell wall material obtained from M6-type GAS. In conclusion, we have identified a functional interaction between Scl1 and plasma FH, which may contribute to GAS evasion of complement-mediated opsonization and phagocytosis.     

Haemophilus influenzae interacts with the human complement inhibitor factor H

Pathogenic microbes acquire human complement inhibitors to circumvent the innate immune system. In this study, we identify two novel host-pathogen interactions, factor H (FH) and factor H-like protein 1 (FHL-1), the inhibitors of the alternative pathway that binds to Hib. A collection of clinical Haemophilus influenzae isolates was tested and the majority of encapsulated and unencapsulated bound FH. The isolate Hib 541 with a particularly high FH-binding was selected for detailed analysis. An increased survival in normal human serum was observed with Hib 541 as compared with the low FH-binding Hib 568. Interestingly, two binding domains were identified within FH; one binding site common to both FH and FHL-1 was located in the N-terminal short consensus repeat domains 6-7, whereas the other, specific for FH, was located in the C-terminal short consensus repeat domains 18-20. Importantly, both FH and FHL-1, when bound to the surface of Hib 541, retained cofactor activity as determined by analysis of C3b degradation. Two H. influenzae outer membrane proteins of approximately 32 and 40 kDa were detected with radiolabeled FH in Far Western blot. Taken together, in addition to interactions with the classical, lectin, and terminal pathways, H. influenzae interferes with the alternative complement activation pathway by binding FH and FHL-1, and thereby reducing the complement-mediated bactericidal activity resulting in an increased survival. In contrast to incubation with active complement, H. influenzae had a reduced survival in FH-depleted human serum, thus demonstrating that FH mediates a protective role at the bacterial surface.     

Phospholipases as possible virulence factor in Vibrio parahaemolyticus

Phospholipase C activity was elevated in pathogenic Vibrio parahaemolyticus isolated from patients. Phospholipase A activity was more pronounced in the nonpathogenic V. parahaemolyticus strains isolated from water. Extracts of the strains containing phospholipase C and A activity but no thermostable direct haemolysin (TDH) were capable of producing lesions in guinea pig skin indicating the presence of a toxic factor other than TDH. It is suggested that the toxic factor may be phospholipase C since the purified enzyme from Clostridium perfringens produced a similar reaction in guinea pig skin.     

Production of nephritic factor of the alternative complement pathway by Epstein Barr virus-transformed B cell lines derived from a patient with membranoproliferative glomerulonephritis

Nephritic factor of the alternative complement pathway (C3NeF) is an IgG autoantibody which binds to and stabilizes the C3 convertase (C3bBb) enzyme, and which has been detected mainly in sera from patients with membranoproliferative glomerulonephritis (MPGN) and partial lipodystrophy. To study the production of C3NeF, mononuclear cells isolated from the peripheral blood of patients with MPGN and C3NeF activity in their sera were infected with Epstein Barr virus (EBV) to establish active B lymphocyte cell lines. By using a modified C3NeF screening assay, we detected C3NeF activity in the supernatant of a B cell line derived from a patient with MPGN Type II, but in none of the supernatants of B cell lines derived from normal individuals. C3NeF-positive supernatants were investigated for their ability to conserve classical or alternative pathway C3 convertase activity by using EAC3bBb and EAC4b2a stabilization assays. C3NeF-positive supernatants stabilized the C3bBb convertase activity, but not the C4b2a convertase activity. Studies of the supernatants, using anti-human IgG affinity columns, showed that the C3NeF activity was in the IgG fraction; furthermore, C3NeF antibody agglutinated sheep erythrocytes coated with C3bBb, but not with C3b alone. On gel electrophoresis, both heavy and light chains of the C3NeF were comparable in size to that of normal human IgG molecules. We conclude that C3NeF, produced in vitro by EBV-transformed B cell lines derived from a patient with MPGN Type II, is functionally identical to the conventional C3NeF in serum. In vitro preparation of homogeneous NeF(s) should greatly facilitate the studies of the role of these autoantibodies in complement dysmetabolism.     

Decay-accelerating factor must bind both components of the complement alternative pathway C3 convertase to mediate efficient decay

Decay-accelerating factor (DAF; CD55) inhibits the complement (C) cascade by dissociating the multimolecular C3 convertase enzymes central to amplification. We have previously demonstrated using surface plasmon resonance (Biacore International) that DAF mediates decay of the alternative pathway C3 convertase, C3bBb, but not of the inactive proenzyme, C3bB, and have shown that the major site of interaction is with the larger cleavage subunit factor B (Bb) subunit. In this study, we dissect these interactions and demonstrate that the second short consensus repeat (SCR) domain of DAF (SCR2) interacts only with Bb, whereas SCR4 interacts with C3b. Despite earlier studies that found SCR3 to be critical to DAF activity, we find that SCR3 does not directly interact with either subunit. Furthermore, we demonstrate that properdin, a positive regulator of the alternative pathway, does not directly interact with DAF. Extending from studies of binding to decay-accelerating activity, we show that truncated forms of DAF consisting of SCRs 2 and 3 bind the convertase stably via SCR2-Bb interactions but have little functional activity. In contrast, an SCR34 construct mediates decay acceleration, presumably due to SCR4-C3b interactions demonstrated above, because SCR3 alone has no binding or functional effect. We propose that DAF interacts with C3bBb through major sites in SCR2 and SCR4. Binding to Bb via SCR2 increases avidity of binding, concentrating DAF on the active convertase, whereas more transient interactions through SCR4 with C3b directly mediate decay acceleration. These data provide new insights into the mechanisms involved in C3 convertase decay by DAF.     

Structural analysis of engineered Bb fragment of complement factor B: insights into the activation mechanism of the alternative pathway C3-convertase

The C-terminal fragment, Bb, of factor B combines with C3b to form the pivotal C3-convertase, C3bBb, of alternative complement pathway. Bb consists of a von Willebrand factor type A (vWFA) domain that is structurally similar to the I domains of integrins and a serine protease (SP) domain that is in inactive conformation. The structure of the C3bBb complex would be important in deciphering the activation mechanism of the SP domain. However, C3bBb is labile and not amenable to X-ray diffraction studies. We engineered a disulfide bond in the vWFA domain of Bb homologous to that shown to lock I domains in active conformation. The crystal structures of Bb(C428-C435) and its inhibitor complexes reveal that the adoption of the "active" conformation by the vWFA domain is not sufficient to activate the C3-convertase catalytic apparatus and also provide insights into the possible mode of C3-convertase activation.     

Characterization of a macrophage chemotactic lymphokine produced by purified protein derivative stimulation in vitro and in vivo

Using an immunoadsorbent column conjugated with anti-macrophage chemotactic factor-c (anti-MCF-c), MCF-c which has been separated and highly purified from a delayed-type hypersensitivity reaction (DHR) site, shares common antigenicity with the major macrophage chemotactic lymphokine released from purified protein derivative (PPD)-stimulated lymphocytes and also macrophage chemotactic lymphokine from phytohemagglutinin (PHA)-stimulated lymphocytes. Using a combination of the immunoadsorbent column and Sephadexgel chromatography these two lymphokines are purified to homogeneity from PPD- or PHA-stimulated guinea pig lymphocyte culture supernatants. These observations, taken in conjunction with the similarity in biological activities, physicochemical properties, and antigenicities, suggest that these two mediators are very similar, or possibly identical. MCF-c with chemotactic activity for macrophages seemed to exist as complexes with serum protein at the skin site of PPD-induced DHR in guinea pigs. The active substance, separated from the complexes under acid conditions, is indistinguishable from the major macrophage chemotactic lymphokine released by PPD stimulation with respect to antigenicity, heat stability, and non-diffusibility. They both have a molecular weight of about 12, 500. The chemotactic lymphokine formed comparable complexes with serum protein under neutral conditions; however, this complex dissociated in acid without loss of activity.     

Structure of the N-terminal region of complement factor H and conformational implications of disease-linked sequence variations

Factor H is a regulatory glycoprotein of the complement system. We expressed the three N-terminal complement control protein modules of human factor H (FH1-3) and confirmed FH1-3 to be the minimal unit with cofactor activity for C3b proteolysis by factor I. We reconstructed FH1-3 from NMR-derived structures of FH1-2 and FH2-3 revealing an approximately 105-A-long rod-like arrangement of the modules. In structural comparisons with other C3b-engaging proteins, factor H module 3 most closely resembles factor B module 3, consistent with factor H competing with factor B for binding C3b. Factor H modules 1, 2, and 3 each has a similar backbone structure to first, second, and third modules, respectively, of functional sites in decay accelerating factor and complement receptor type 1; the equivalent intermodular tilt and twist angles are also broadly similar. Resemblance between molecular surfaces is closest for first modules but absent in the case of second modules. Substitution of buried Val-62 with Ile (a factor H single nucleotide polymorphism potentially protective for age-related macular degeneration and dense deposit disease) causes rearrangements within the module 1 core and increases thermal stability but does not disturb the interface with module 2. Replacement of partially exposed (in module 1) Arg-53 by His (an atypical hemolytic uremic syndrome-linked mutation) did not impair structural integrity at 37 degrees C, but this FH1-2 mutant was less stable at higher temperatures; furthermore, chemical shift differences indicated potential for small structural changes at the module 1-2 interface.     

Trypsin-activated complex of human factor B with cobra venom factor (CVF), cleaving C3 and C5 and generating a lytic factor for unsensitized guinea pig erythrocytes. I. Generation of the activated complex.

A complex formed between cobra venom factor (CVF) and isolated human factor B (B) was found to be converted by trypsin to a stable enzyme, CVF-B which cleaved the third component (C3) and the fifth component (C5) of human complement. The formation of CVF-B by trypsin required divalent cations, whereas the formation of the lytic factor from human serum occurred even in the presence of EDTA. CVF-B purified by gel filtration could initiate the hemolysis of unsensitized guinea pig erythrocytes when incubated with human complement components C5 to C9 in 0.01 M EDTA buffer. C3 was not required for the lysis of guinea pig erythrocytes initiated by CVF-B because of the beta1C precipitation line formed between human serum and anti-beta1C antibody did not inhibit the hemolysis by CVF-B in agarose gel. Treatment of beta1C and beta1F globulins in whole human serum with CVF-B in the presence of 0.01 M EDTA converted them to components with higher mobilities on immunoelectrophoresis.     

Tumor necrosis factor as an interleukin 1-dependent differentiation inducing factor (D-factor) for mouse myeloid leukemic cells

Experiments were conducted to purify the differentiation-inducing factor (D-factor), which induces differentiation of mouse myeloid leukemic cell line, Ml, into macrophage-like cells, in a conditioned medium of guinea pig peritoneal macrophages stimulated with lipopolysaccharide. On gel filtration under high performance liquid column chromatography (HPLC), D-factor eluted at the position of 45-15 KD. By the subsequent separation on DEAE HPLC the D-factor activity disappeared. However, in the presence of recombinant human IL 1 alpha the D-factor activity appeared at a position where tumor necrosis factor (TNF) eluted. Even after fractionation on hydroxyapatite HPLC the IL 1-dependent D-factor was co-chromatographed with TNF. Recombinant human TNF as well as the partially purified guinea pig TNF induced differentiation of Ml cells in conjunction with either the partially purified guinea pig IL 1 or recombinant human IL 1 alpha, although these factors by themselves did not induce differentiation. These findings suggest that a part of D-factor activity in the conditioned medium resulted from the cooperative effects between TNF and IL 1.     

Characterization of the oncornavirus particles in the plasma of guinea pigs with L2C leukemia.

The inoculation of L2C guinea pig leukemia cells into strain 2 guinea pigs results in the death of the animals within 12 to 15 days. Death is preceded by the simultaneous appearance in the plasma of (i) elevated leukocyte levels, (ii) extracellular virus particles, and (iii) a particle-associated RNA-directed DNA polymerase. This enzyme activity has a cation preference identical to that of the type B bromodeoxyuridine-induced guinea pig virus, i.e., an Mg2+ optimum at 20 mM and no activity using Mn2+. Competitive molecular hybridization studies also revealed that the plasma of leukemic guinea pigs contained approximately 2 X 10(9) genome equivalents per ml of an RNA that is homologous to the RNA of the bromodeoxyuridine-induced guinea pig virus. Morphological observations indicate that most, but not all, of the extracellular particles observed in leukemia plasma are derived from the intracisternal particles seen in the L2C tumor cells. The possibilities that either two viral populations are present or that the in vivo morphogenesis of the type B bromodexoyuridine-inducible guinea pig virus is markedly different from its in vitro morphogenesis are discussed.     

Evidence for a role of the amino-terminal region in the biological activity of the classical anaphylatoxin, porcine C5a des-Arg-74

The presence of methionyl residues at positions 1 and 17 in porcine classical anaphylatoxin (e.g. C5a des-Arg-74) permits chemical cleavage of the factor with cyanogen bromide to generate two defined fragments. Peptides corresponding to the amino-terminal fragment, CN-I, and the carboxyl-terminal peptide, CN-II, were purified from the CNBr digest of C5a des-Arg-74 by reverse-phase high performance liquid chromatography. The isolated derivatives were assessed for their abilities to cause contraction of isolated guinea pig ileal smooth muscle, guinea pig lung parenchymal strips, and degranulation of guinea pig polymorphonuclear neutrophils. In each assay, CN-I was devoid of biological activity at concentrations greater than 10(-6) M. In contrast, the carboxyl-terminal 56-residue fragment, CN-II, possessed weak (10(-6) versus 10(-9) M for classical anaphylatoxin) agonist activity in each of the assay systems. Our data suggest that structural information contained in the amino-terminal 17 residues of the C5a des-Arg-74 molecule contributes to the biological potency of the intact factor, but is not an essential component of the active site. Whether the structural information in residues 1-17 relates to receptor binding directly or serves to stabilize the conformation of the 18-73-fragment containing the active center of the molecule is yet to be determined.