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PubMed2000

Proteolytic activities of two types of mannose-binding lectin-associated serine protease.

Mannose (or mannan)-binding lectin (MBL) is an oligomeric serum lectin that plays a role in innate immunity by activating the complement system. In human, two types of MBL-associated serine protease (MASP-1 and MASP-2) and a truncated protein of MASP-2 (small MBL-associated protein; sMAP or MAp19) are complexed with MBL. To clarify the proteolytic activities of MASP-1 and MASP-2 against C4, C2, and C3, we isolated these two types of MASP in activated forms from human serum by sequential affinity chromatography. On an anti-MASP-1 column, MASP-2 passed through the column in the presence of EDTA and high salt concentration, whereas MASP-1 was retained. Isolated MASP-1 and MASP-2 exhibited proteolytic activities against C3 and C4, respectively. C2 was activated by both MASPs. C1 inhibitor (C1 INH), an inhibitor for C1r and C1s, formed equimolar complexes with MASP-1 and MASP-2 and inhibited their proteolytic activities.

Binding, Competitive↗
PubMed1987

A sensitive specific hemolytic assay for proenzyme C1.

The traditional hemolytic assay of the functional activity of C1, the first component of the classical complement pathway, was modified to permit differentiation between proenzyme (unactivated) C1 and the activated state of the enzyme (C1). A two-step assay was developed to quantitate proenzyme C1. The C1 sample to be assayed was first preincubated with C1 inhibitor, a process that specifically inhibits the enzymatic activity of C1 without affecting the subsequent activation of proenzyme C1 by EAC4, a model immune complex. Since the rate of reaction between C1 inhibitor, a serum regulatory protein, and C1 is concentration-dependent, this step is performed at high C1 and C1 inhibitor concentrations. Subsequent dilutions of the sample prevents C1 inhibitor-mediated inactivation of the C1 that is activated during the C1 hemolytic assay. Thus, in the presence of C1 inhibitor, the level of C1 hemolytic activity specifically reflects the activity of proenzyme C1, while in the absence of C1 inhibitor, the hemolytic activity reflects the total activity of C1. Both the absolute and the relative amounts of the proenzyme (unactivated) and activated C1 can thereby be quantitated in most samples. Furthermore, a partially purified C1 inhibitor reagent, easily prepared from serum, was shown to function identically to the purified C1 inhibitor, obviating the need for a multistep isolation procedure for this protein. Using this simple yet sensitive assay to investigate the efficiency of reconstitution of C1 activity from the purified components C1q, C1r, and C1s, we also find evidence for temperature- and concentration-dependent reaction steps in the formation of functional C1.

Animals↗
PubMed1976

Evidence that C1s participates in the alternative complement pathway.

Purified C1s, subcomponent of C1, induced electrophoretic conversion of factor B and consumption of hemolytic C3 and C5 in sera genetically deficient in C2 or C4. Blocking of the hemolytic activity of C1s in C2-deficient serum by F(ab')2 anti-C1s resulted in inhibition of the alternative pathway, as indicated by the failure of zymosan or cobra venom factor to induce comsumption of C3 and C6. Zymosan also failed to activate the alternative pathway when C1s was absorbed from C1r-deficient serum using a solid immunoabsorbent. These data, showing that C1s participates in the alternative pathway under certain experimental conditions, suggest the interesing possibility that C1s is important in the activation sequence of both the classical and the alternative pathway more generally.

Animals↗
PubMed1989

Identification of a human non-interferon lymphokine activating monocyte complement biosynthesis.

A monocyte-stimulating activity produced by mitogen-induced mononuclear cells has been defined by its ability to enhance the synthesis in vitro of complement C1 subcomponents, C2 and C3. A lymphokine responsible for this activity was purified from culture supernatants of peripheral blood mononuclear cells activated by staphylococcal enterotoxin A. From 0.5 litre of supernatant the purification procedure [(NH4)2SO4 precipitation, phenyl-Sepharose chromatography and preparative electrofocusing] yielded about 100 pmol of purified lymphokine. Its pI is 7.9 and its Mr, estimated by SDS/polyacrylamide-gel electrophoresis, is 14,600, 27,000 and 56,000, the high-Mr species representing oligomeric forms of the Mr-14,600 molecule. Its amino acid analysis reveals a high percentage of hydrophobic amino acids (34%); the absence of histidine residues suggests that it is a novel monocyte-activating lymphokine. It enhances C1r and C1s biosynthesis at a pretranslational level. From its structure and activity this lymphokine appears different from gamma-interferon.

Amino Acids↗
PubMed1980

Mechanism of antibody-independent activation of the first component of complement (Cl) on retrovirus membranes.

Murine leukemia viruses activate human C1 in the absence of specific antibody. Such activation requires the binding of C1 to the viral surface through two subcomponents, C1q and C1s. This conclusion is based on the following results. (1) Isolated human C1q and C1s bind the same membrane protein on virions. (2) Binding one subcomponent is independent of the other. (3) Only dimeric C1s binds, whereas monomeric C1s, prepared by dissociation with ethylenediaminetetraacetate (EDTA), has no affinity for the virus. (4) The activated C1s dimer, C1s, does not attach to the virus. (5) Saturation of C1s binding sites on the viral surface does not prevent binding of macromolecular C1, but such bound C1 is not activated. (6) No exchange occurs between C1s bound to the viral membrane and C1s contained in C1, which in turn is attached via C1q to the same virus. Therefore activation occurs only when both C1q and C1s in the same C1 complex in contact with the viral activator. Human C1r has no affinity for the virus nor does guinea pig C1s. The latter result explains why guinea pig serum does not function in antibody-independent virolysis.

Animals↗
PubMed2006

Complement levels and activity in the normal and LPS-injured lung.

Complement, a complex protein system, plays an essential role in host defense through bacterial lysis, stimulation of phagocytosis, recruitment of immune cells to infected tissue, and promotion of the inflammatory response. Although complement is most well-characterized in serum, complement activity is also present in the lung. Here we further characterize the complement system in the normal and inflamed lung. By Western blot, C5, C6, and factor I were detected in bronchoalveolar lavage (BAL) at lower levels than in serum, whereas C2 was detected at similar levels in BAL and serum. C4 binding protein (C4BP) was not detectable in BAL. Exposure to lipopolysaccharide (LPS) elevated levels of C1q, factor B, C2, C4, C5, C6, and C3 in human BAL and C3, C5, and factor B in mouse and rat BAL. Message for C1q-B, C1r, C1s, C2, C4, C3, C5, C6, factor B, and factor H, but not C9 or C4BP, was readily detectable by RT-PCR in normal mouse lung. Exposure to LPS enhanced factor B expression, decreased C5 expression, and did not affect C1q-B expression in mouse and rat lung. BAL from rats exposed to LPS had a greater ability to deposit C3b onto bacteria through complement activation than did BAL from control rats. In summary, these data demonstrate that complement levels, expression, and function are altered in acute lung injury and suggest that complement within the lung is regulated to promote opsonization of pathogens and limit potentially harmful inflammation.

Animals↗
PubMed2001

Structure and function of complement activating enzyme complexes: C1 and MBL-MASPs.

The complement system is a major effector arm of the immune defense contributing to the destruction of invading pathogens. There are three possible routes of complement cascade activation: the classical, the alternative and the lectin pathways. The activation of the classical and lectin pathways is initiated by supramolecular complexes, which resemble each other. Each complex has a recognition subunit (C1q in the classical and mannose-binding lectin (MBL) in the lectin pathway), which associates with serine protease zymogens (C1q with C1r and C1s, and MBL with MBL-associated serine proteases: MASP-1, MASP-2) to form the C1 and MBL-MASPs complexes, respectively. As the recognition subunits bind to activator structures, subsequent activation of the serine protease zymogens occurs. The precise structure of the complexes and the exact mechanism of their activation have not been solved, yet. In this review we summarize the recent advances about the structure and function of the individual subcomponents of both complexes achieved by genetic engineering, molecular modeling, physico-chemical and functional studies. Special emphasis will be laid on the serine proteases: the role of the individual domains in the assembly of the C1s-C1r-C1r-C1s tetramer and in the control of the protease activity will be discussed. We will then focus on recent functional models of the supramolecular complexes. The question of how a non-enzymatic signal (the binding of C1q or MBL to activators) can be converted into enzymatic events (activation of serine protease zymogens) will be addressed. The similarities and differences between C1 and MBL-MASPs will also be discussed.

Complement Activation↗
PubMed1998

Selective complement C1s deficiency caused by homozygous four-base deletion in the C1s gene.

The complement system plays an important role in defense mechanisms by promoting the adherence of microorganisms to phagocytic cells and lysis of foreign organisms. Deficiencies of the first complement components, C1r/C1s, often cause systemic lupus erythematosus-like syndromes and severe pyogenic infections. Up to now no genetic analysis of the C1r/C1s deficiencies has been carried out. In the present work, we report the first genetic analysis of selective C1s deficiency, the patient having a normal amount of C1r. C1s RNA with a normal size was detected in patient's subcutaneous fibroblasts (YKF) by RNA blot analysis and RT-PCR. The amount of C1s RNA was approximately one-tenth of the RNA from the human chondrosarcoma cell line, HCS2/8. In contrast, the levels of C1r and beta-actin RNA of YKF were similar to that of HCS2/8. Sequence analysis of C1s cDNA revealed a deletion at nucleotides 1087-1090 (TTTG), creating a stop codon (TGA) at position 94 downstream of the mutation site. Direct sequencing of the gene between the primers designed on intron 9 and exon 10 indicated the presence of the deletion on exon 10 of the gene. Quantitative Southern blot hybridization suggested the mutation was homozygous. The 4-bp deletion on exon 10 was also found in the patient's heterozygous mother who had normal hemolytic activity.

Base Sequence↗
PubMed1987

Molecular cloning of cDNA for human complement component C1s. The complete amino acid sequence.

The complete amino acid sequence (673 residues plus 15 residues of leader sequence) of human complement component C1s has been determined by nucleotide sequencing of cDNA clones from a human liver library probed with synthetic oligonucleotides. Much of the sequence is supported by independent amino acid sequence information. The cDNA sequence contains an anomalous "intron-like" sequence, including a stop codon, that can be discounted because of the amino acid sequence evidence. The N-terminal chain (422 residues) of C1s, like that of C1r with which it is broadly homologous, contains five domains: domains I and III are homologous to one another and to similar regions in C1r, domain II is homologous to the epidermal growth factor sequence found in C1r and several other proteins, and domains IV and V are homologous to one another and to the 60-residue repeating sequence found in C1r, C2, factor B, C4-binding protein and some apparently unrelated proteins. The sequence of the C-terminal chain (251 residues) agrees with that already established to be the "serine protease" domain of C1s.

Amino Acid Sequence↗
PubMed2002

Structural biology of C1.

The classical complement pathway is a major element of innate immunity against infection, and is also involved in immune tolerance, graft rejection and various pathologies. This pathway is triggered by C1, a multimolecular protease formed from the association of a recognition protein, C1q, and a catalytic subunit, the calcium-dependent tetramer C1s-C1r-C1r-C1s, which comprises two copies of each of the modular proteases C1r and C1s. All activators of the pathway are recognized by the C1q moiety of C1, a process that generates a conformational signal that triggers self-activation of C1r, which in turn activates C1s, the enzyme that mediates specific cleavage of C4 and C2, the C1 substrates. Early work based on biochemical and electron microscopy studies has allowed characterization of the domain structure of the C1 subcomponents and led to a low-resolution model of the complex in which the elongated C1s-C1r-C1r-C1s tetramer folds into a compact, figure-of-8-shaped conformation upon interaction with C1q. The strategy used over the past decade was based on a dissection of the C1 proteins into modular segments to characterize their function and solve their three-dimensional structure by X-ray crystallography or NMR spectroscopy. This approach allows deep insights into the structure-function relationships of C1, particularly with respect to the assembly of the C1 complex and the mechanisms underlying its activation and proteolytic activity.

Animals↗
PubMed1998

Inhibition of activation of the classical pathway of complement by human neutrophil defensins.

Defensins are small, cationic antimicrobial peptides that are present in the azurophilic granules of neutrophils. Earlier studies have shown that defensins may influence complement activation by specific interaction with activated C1, C1q, and C1-inhibitor. In the present study, we show that the defensin human neutrophil peptide-1 (HNP-1) is able to inhibit activation of the classical complement pathway by inhibition of C1q hemolytic activity. The binding site for HNP-1 on C1q is most likely located on the collagen-like stalks, as a clear, dose-dependent binding of HNP-1 to either intact C1q or to the collagen-like stalks of C1q was demonstrated using enzyme-linked immunosorbent assay (ELISA). Besides binding of HNP-1 to C1q, also a limited binding to C1 and to a mixture of C1r and C1s was observed, whereas no binding to C1-inhibitor was found. Because binding of HNP-1 to C1-inhibitor has been suggested in earlier studies, we also assessed the binding of HNP-1 to mixtures of C1-inhibitor with either C1r/ C1s or C1. No binding was found. Using a competition ELISA, it was found that HNP-1, but not protamine, inhibited binding of biotin-labeled HNP-1 to C1q in a dose-dependent fashion. In the fluid phase, preincubation of HNP-1 with C1q resulted in complex formation of HNP-1 and C1q and generation of stable complexes. In conclusion, HNP-1 is able to bind to C1q in the fluid phase and inhibits the classical complement pathway. This mechanism may be involved in the control of an inflammatory response in vivo.

Binding Sites↗
PubMed1985

Antibody-independent interaction between the first component of human complement, C1, and the outer membrane of Escherichia coli D31 m4.

The heptoseless mutant of Escherichia coli, E. coli D31 m4, binds C1q and C1 at 0 degrees C and at low ionic strength (I0.07). Under these conditions, the maximum C1q binding averages 3.0 X 10(5) molecules per bacterium, with a Ka of 1.4 X 10(8) M-1. Binding involves the collagen-like region of C1q, as shown by the capacity of C1q pepsin-digest fragments to bind to E. coli D31 m4, and to compete with native C1q. Proenzyme and activated forms of C1 subcomponents C1r and C1s and their Ca2+-dependent association (C1r-C1s)2 do not bind to E. coli D31 m4. In contrast, the C1 complex binds very effectively, with an average fixation of 3.5 X 10(5) molecules per bacterium, and a Ka of 0.25 X 10(8) M-1, both comparable with the values obtained for C1q binding. C1 bound to E. coli D31 m4 undergoes rapid activation at 0 degrees C. The activation process is not affected by C1-inhibitor, and only slightly inhibited by p-nitrophenyl p'-guanidinobenzoate. No turnover of the (C1r-C1s)2 subunit is observed. Once activated, C1 is only partially dissociated by C1-inhibitor. Our observations are in favour of a strong association between C1 and the outer membrane of E. coli D31 m4, involving mainly the collagen-like moiety of C1.

Cell Membrane↗
PubMed1983

The binding properties of human complement component C1q. Interaction with mucopolysaccharides.

Quantitative measurements have been made of the interaction of human complement subcomponent C1q with mucopolysaccharides. The binding of C1q to heparin was quantitatively examined by utilizing an assay that employs a 125I-labeled low molecular weight heparin glycosaminoglycan (LMW-Hep) (Mr = 8500). Two classes of binding sites were detected. The first class of sites bound 2.02 mol of LMW-Hep/mol of C1q with a Kd of 76.6 nM. The second class of sites complexes with 12 mol of LMW-Hep with a Kd of 1.01 microM. The higher affinity-binding site for LMW-Hep could be assigned to the collagenous region of C1q (C1q-c); 2.2 mol of 125I-LMW-Hep were bound/mol of purified isolated C1q-c with a Kd = 381 nM. In contrast, the isolated C1q globular region did not bind to 125I-LMW-Hep. The binding of LMW-Hep to C1q and the C1q-c region was confirmed by fluorescence polarization experiments; C1q and C1q-c bound 2.3 and 2.02 mol of fluorescamine-labeled LMW-Hep/mol of protein, respectively. A variety of mucopolysaccharides were able to inhibit interaction of C1q with 125I-LMW-Hep, the most effective being heparan sulfate and dermatan sulfate. LMW-Hep (2.5 nM) inhibited the ability of C1q (0.5 nM) to recombine with C1r (1.4 nM) and C1s (1.6 nM) to form hemolytically active C1. At 250 mM, LMW-Hep inhibited the hemolytic activity of reconstituted C1. The ability of mucopolysaccharides to interact with purified C1q suggests a role for such molecules in the regulation of the first component of complement.

Centrifugation, Density Gradient↗
PubMed1990

Protective and inactivating effects of neutrophil myeloperoxidase on C1q activity.

This study investigated the interaction between neutrophil myeloperoxidase (MPO) and the C1q component of the complement system. Using a dot-spot assay, MPO was found to bind to C1q in a dose-dependent manner. The specificity of this reaction was proved by the inhibitory effect of F(ab')2 antibodies to C1q and by the inability of MPO to bind to C1r, C1s and IgG. The interaction between MPO and C1q did not influence the enzymatic activity of the peroxidase but resulted in a more stable C1q as assessed by hemolytic assay for C1q. The protective effect of MPO on C1q did not require the presence of H2O2 in the reaction mixture nor was it inhibited by sodium azide, whereas it was abolished by heating the peroxidase. Lactoferrin and lysozyme, unlike MPO, were ineffective in protecting C1q from functional decay. Addition of H2O2 and chloride to MPO and C1q led to a complete inactivation of C1q, which could not be induced by H2O2 alone. The hypochlorite, which is known to be generated during the reaction of MPO with H2O2 and chloride, exhibited a similar inactivating effect on C1q, which was prevented by an external source of methionine.

Chlorides↗
PubMed1987

Mechanism of killing of Giardia lamblia trophozoites by complement.

Only antibodies of the IgM class support the lytic effect of complement on Giardia lamblia (GL). We sensitized GL trophozoites (SGL) at 4 degrees C with serum containing anti-GL antibodies or IgM purified from this serum, and either normal human serum (NHS), complement 2-deficient human serum (C2d-HS), or C4-deficient guinea pig serum was used as source of complement. SGL were killed by NHS (86%) and by the deficient sera (50 and 40%, respectively), suggesting activation of the alternative pathway. However, the reaction was inhibited by Mg-EGTA. These observations led to studies of the role of C1. The lytic effect of NHS and C2d-HS on SGL was abolished by immunochemically depleting C1 from these sera, and reconstituted by adding purified C1q plus C1r and C1s. Factor B-depleted C2d-HS also lost its capacity to mediate killing, but reconstitution with factor B led to a dose-dependent increase in the killing of SGL. We next investigated the participation of the membrane attack complex in this system. SGL carrying C5b to C7 were lysed when incubated with C8 alone (56%); the addition of C9 further increased killing (98%), while C9 in the absence of C8 had no effect. We concluded that although activation of the classical pathway produces lysis of SGL, lysis may also proceed through a unique pathway of complement activation that requires C1 and factor B, but is independent of C4 and C2. Lysis of SGL can be accomplished by C5b to C8 in the absence of C9.

Animals↗
PubMed1997

Expression and characterization of a 159 amino acid, N-terminal fragment of human complement component C1s.

A 159 residue, N-terminal fragment of the human C1s complement component, C1s alpha(159), was expressed in the baculovirus, insect cell system. The protein was abundantly produced 3 days after infection, reaching levels as high as 40 microg/ml in cell culture media. It had a molecular weight of 18,100 (+/-4.9) Da by laser desorption mass spectrometry, close to the theoretical value of 18,111 Da, confirmed by sequencing. Sedimentation equilibrium and gel filtration column chromatography showed that C1s alpha(159) was a monomer in the presence of EDTA, and a dimer in the presence of Ca2+. The C1s alpha(159)2 dimer had a sedimentation coefficient of 3.1 S. When the C1s alpha(159)2 was mixed with Clq, there was little or no interaction. Likewise, unactivated C1r2 dimer had a sedimentation coefficient of 6.8 S, and when mixed with C1q little or no interaction was observed. When C1s alpha(159)2 was mixed with the 6.8 S C1r2 in Ca2+, a 7.5 S complex was formed, presumably the C1s alpha(159) x C1r x C1r x C1s alpha(159) tetramer. When C1q, which migrated at 10.1 S was mixed with C1s alpha(159)2 and C1r2 in the presence of Ca2+, a C1-like complex, but containing C1s alpha(159) instead of C1s, was formed which migrated at 14.0 S. This C1-like molecule remained unactivated unless challenged with an ovalbumin-antiovalbumin immune complex. In the presence of immune complex, the C1r became activated. This suggested that the presence of the 159 amino acid C1s alpha domain, which held the C1r to the C1q, was sufficient to permit activation by an immune complex, even though the catalytic domains of C1s were not present.

Amino Acid Sequence↗
PubMed2002

The mannan-binding lectin-associated serine proteases (MASPs) and MAp19: four components of the lectin pathway activation complex encoded by two genes.

Mannan-binding lectin (MBL) and ficolins (L-ficolin and H-ficolin) initiate the lectin pathway of complement activation upon binding to microbial carbohydrates. The activation is mediated by associated serine proteases, termed MASPs, since they were discovered as MBL-associated serine proteases. The MASP family comprises three serine proteases, MASP-1, MASP-2 and MASP-3 and a non-enzymatic protein, MAp19. The MASPs show identical domain structure, shared also with C1r and C1s. MASP-1 and MASP-3 are alternative splice products of a single gene, MASP1/3, and have identical A chains, whereas they have individual B chains, encompassing the serine protease domain. MASP2 and MAp19 are alternative splice products of the MASP-2 gene, with MAp19 consisting of the first two domains of MASP-2 plus additional four amino acid residues. MASP-2 is the protease responsible for activating C4 and C2 to generate the C3 convertase, C4bC2b. The biological function of the remaining three proteins has not yet been resolved.

Animals↗