Origin of mannose-binding lectin-associated serine protease (MASP)-1 and MASP-3 involved in the lectin complement pathway traced back to the invertebrate, amphioxus.

Mannose-binding lectin-associated serine proteases (MASPs) are involved in complement activation through the lectin pathway. To elucidate the phylogenetic origin of MASP and a primordial complement system, we cloned two MASP cDNAs from amphioxus (Branchiostoma belcheri) of the cephalochordates, considered to be the closest relative of vertebrates. The two sequences, orthologues of mammalian MASP-1 and MASP-3, were produced by alternative processing of RNA from a single gene consisting of a common H chain-encoding region and two L chain-encoding regions, a structure which is similar to that of the human MASP1/3 gene. We also isolated two MASP genes from the ascidian Halocynthia roretzi (urochordates) and found that each of them consists simply of an H chain-encoding region and a single L chain-encoding region. The difference in structure between the ascidian MASP genes and the amphioxus/mammalian MASP genes suggests that a prototype gene was converted to the MASP1/3-type gene possessing two L chain-encoding regions at an early stage of evolution before the divergence of amphioxus. This conclusion is supported by the presence of MASP-1 and MASP-3 homologues in almost all vertebrates, as demonstrated by the cloning of novel cDNA sequences representing lamprey (cyclostomes) MASP-1 and Xenopus MASP-3. The ancient origin of MASP-1 and MASP-3 suggests that they have crucial functions common to all species which emerged after cephalochordates.

Structure, function and molecular genetics of human and murine C1r.

C1r, the enzyme responsible for intrinsic activation of the C1 complex of complement, is a modular serine protease featuring an overall structural organization homologous to those of C1s and the mannan-binding lectin-associated serine proteases (MASPs). This review will initially summarize current information on the structure and function of C1r, with particular emphasis on the three-dimensional structure of its catalytic domain, which provides new insights into the activation mechanism of C1. The second part of this review will focus on recent discoveries dealing with a truncated, C1r-related protein, and the occurrence in the mouse of two isoforms, C1rA and C1rB, exhibiting tissue-specific expression patterns.

The role of the individual domains in the structure and function of the catalytic region of a modular serine protease, C1r.

The first enzymatic event in the classical pathway of complement activation is autoactivation of the C1r subcomponent of the C1 complex. Activated C1r then cleaves and activates zymogen C1s. C1r is a multidomain serine protease consisting of N-terminal alpha region interacting with other subcomponents and C-terminal gammaB region mediating proteolytic activity. The gammaB region consists of two complement control protein modules (CCP1, CCP2) and a serine protease domain (SP). To clarify the role of the individual domains in the structural and functional properties of the gammaB region we produced the CCP1-CCP2-SP (gammaB), the CCP2-SP, and the SP fragments in recombinant form in Escherichia coli. We successfully renatured the inclusion body proteins. After renaturation all three fragments were obtained in activated form and showed esterolytic activity on synthetic substrates similar to each other. To study the self-activation process in detail zymogen mutant forms of the three fragments were constructed and expressed. Our major statement is that the ability of autoactivation and C1s cleavage is an inherent property of the SP domain. We observed that the CCP2 module significantly increases proteolytic activity of the SP domain on natural substrate, C1s. Therefore, we propose that CCP2 module provides accessory binding sites. Differential scanning calorimetric measurements demonstrated that CCP2 domain greatly stabilizes the structure of SP domain. Deletion of CCP1 domain from the CCP1-CCP2-SP fragment results in the loss of the dimeric structure. Our experiments also provided evidence that dimerization of C1r is not a prerequisite for autoactivation.

Inhibitory effects of newly synthesized active center-directed trypsin-like serine protease inhibitors on the complement system.

OBJECTIVE: To obtain a synthetic anti-complement inhibitor which has stronger activity than FUT-175 (nafamostat mesilate), as a synthetic ester derivative containing amidino and guanidino groups. METHODS: We synthesized several modified compounds of FUT-175. The anti-complement activities were measured using synthetic substrates and complement-mediated hemolysis in vitro. The anti-complement activity in vivo was evaluated via Forssman systemic shock in guinea pigs. RESULTS: FUT-175 inhibited C1r and C1s with IC50s of 1.7x10(-6) and 3.2x10(-7) M, respectively. Inhibitory activities were decreased by substitution of the amidino group with a hydrogen atom (compound 2), but not the guanidino group with a hydrogen atom (compound 3). Compound 6, in which the benzene ring of compound 3 was substituted with a furan ring, inhibited C1r and the complement-mediated hemolysis in high-diluted serum with higher potency than FUT-175. The inhibitory activity of compound 6 in hemolysis was weakened in low diluted serum. Compound 7 had a guanidino group inserted into compound 6; however, Compound 7 strongly inhibited hemolysis even in low-diluted serum, and suppressed Forssman systemic shock more potently than both FUT-175 and compound 6. CONCLUSIONS: These data suggest that the 2-furylcarboxylic acid derivatives have a strong potential for inhibiting the activities of the complement, and the guanidino group was required to retain high inhibitory activities in vivo, and compound 7 is a hopeful anti-complement agent.

Structure and functions of the interaction domains of C1r and C1s: keystones of the architecture of the C1 complex.

C1r and C1s, the proteases responsible for activation and proteolytic activity of the C1 complex of complement, share similar overall structural organizations featuring five nonenzymic protein modules (two CUB modules surrounding a single EGF module, and a pair of CCP modules) followed by a serine protease domain. Besides highly specific proteolytic activities, both proteases exhibit interaction properties associated with their N-terminal regions. These properties include the ability to bind Ca2+ ions with high affinity, to associate with each other within a Ca2+-dependent C1s-C1r-C1r-C1s tetramer, and to interact with C1q upon C1 assembly. Precise functional mapping of these regions has been achieved recently, allowing identification of the domains responsible for these interactions, and providing a comprehensive picture of their structure and function. The objective of this article is to provide a detailed and up-to-date overview of the information available on these domains, which are keystones of the assembly of C1, and appear to play an essential role at the interface between the recognition function of C1 and its proteolytic activity.

Structural and functional studies on C1r and C1s: new insights into the mechanisms involved in C1 activity and assembly.

C1r and C1s, the enzymes responsible for the activation and proteolytic activity of the C1 complex of complement, are modular serine proteases featuring similar overall structural organizations, yet expressing very distinct functional properties within C1. This review will initially summarize available information on the structure and function of the protein modules and serine protease domains of C1r and C1s. It will then focus on the regions of both proteases involved in: (i) assembly of C1s-C1r-C1r-C1s, the Ca(2+)-dependent tetrameric catalytic subunit of C1; (ii) expression of C1 catalytic activities. Particular emphasis will be aid on recent structural and functional studies that provide new insights into the complex mechanisms involved in the assembly, activation, and proteolytic activity of C1.

Structure and function of the serine-protease subcomponents of C1: protein engineering studies.

Our protein engineering studies on human C1r and C1s revealed important characteristics of the individual domains of these multidomain serine-proteases, and supplied evidence about the cooperation of the domains to create binding sites, and to control the activation process. We expressed the recombinant subcomponents in the baculovirus-insect cell system and checked the biological activity. Deletions and point mutants of C1r were constructed and C1r-C1s chimeras were also produced. Our deletion mutants demonstrated that the N-terminal CUB domain and the EGF-like domain of C1r together are responsible for the calcium dependent C1r-C1s interaction. It seems very likely that these two modules form the calcium-binding site of the C1r alpha-fragment and participate in the tetramer formation. The deletion mutants also demonstrated that the N-terminal region of the C1r molecule contains essential elements involved in the control of activation of the serine-protease module. The substrate specificity of the serine-protease is also determined by the five N-terminal noncatalytic domain of C1r/C1s chimera, which contains the catalytic domain of C1s preceded by the N-terminal region of C1r, could replace the C1r in the hemolytically active C1 complex. The C1s/C1r chimera, in which the alpha-fragment of the C1r was replaced for that of the C1s exibits both C1r- and C1s-like characteristics. We stabilized the zymogen form of human C1r by mutating the Arg(463)-Ile(464) bond. Using our stable zymogen C1r we showed that one active C1r in the C1 complex is sufficient for the full activity of the entire complex. Further experiment with this mutant could provide us with important information about the structure of the C1 complex.

Functional effects of domain deletions in a multidomain serine protease, C1r.

The C1r subcomponent of the first component of complement is a complex, multidomain glycoprotein containing five regulatory or binding modules in addition to the serine protease domain. To reveal the functional role of the N-terminal regulatory domains, two deletion mutants of C1r were constructed. One mutant comprises the N-terminal half of domain I joined to the second half of the highly homologous domain III, resulting in one chimeric domain in the N-terminal region, instead of domains I-III. In the second mutant most of the N-terminal portion of domain I was deleted. Both deletion mutants were expressed in the baculovirus-insect cell expression system with yields typical of wild type C1r. Both mutants maintained the ability of the wild type C1r to dimerize. The folding and secretion of the recombinant proteins was not affected by these deletions, and C1-inhibitor binding was not impaired. The stability of the zymogen was significantly decreased however, indicating that the N-terminal region of the C1r molecule contains essential elements involved in the control of activation of the serine protease module. Tetramer formation with C1s in the presence of Ca2+ was abolished by both deletions. We suggest that the first domain of C1r is essential for tetramer formation, since the deletion of domain I from C1r impairs this interaction.

Receptor expression and functional status of cultured human eosinophils derived from umbilical cord blood mononuclear cells.

Selective use of recombinant human cytokines has enabled the culture of large numbers of eosinophils from human cord blood mononuclear cells, raising the possibility of their use as a model of eosinophil function. Cultured eosinophils (CE) were compared with normal-density peripheral blood eosinophils (PBE) in terms of their membrane receptor expression and function. Fc gamma R and CR1 expression of CE and PBE was similar. In contrast, the specific mean fluorescence for LFA-1 alpha, p150,95 alpha, ICAM-1, and HLA-DR was significantly elevated for CE compared with PBE. CE responded in PAF-induced chemotaxis in a similar fashion to PBE. CE gave higher numbers of both resting and platelet activating factor (PAF)-stimulated immunoglobulin G (IgG)- and C3b-dependent rosettes than PBE. CE and PBE had comparable capacity to kill IgG- and C-opsonized schistosomula in terms of both baseline values and PAF-induced enhancement of cytotoxicity. Baseline adherence by CE and PBE to plasma-coated glass was essentially the same, but stimulated adhesion (PAF) of CE was lower. Compared with PBE, CE generated less than half the amounts of extracellular and cell-associated PAF induced by calcium ionophore A23187 stimulation. Unlike PBE, CE did not generate PAF after exposure to IgG-coated Sepharose particles. CE stimulated with IgG-coated beads generated small quantities of LTC4, while A23187 stimulation resulted in approximately half the LTC4 levels observed with PBE. The total cell content of eosinophil peroxidase (EPO) was similar for CE and PBE. These data suggest that although CE and PBE have many phenotypic and functional properties in common there are quantitative differences that may be a consequence of their immaturity and/or the influence of the cytokines used in their culture.

Expression of hemolytically active human complement component C1r proenzyme in insect cells using a baculovirus vector.

The gene of human C1r has been expressed in a baculovirus-insect-cell system via the pAc373 transplacement vector. The full-length cDNA copy was inserted into the pAc373 vector downstream from the strong polyhedrin promoter of the baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda cells were cotransfected with the resultant plasmid, pAcC1r, and the wild-type AcNPV DNA. Recombinant viruses, which drove the expression of C1r protein, were selected by plaque morphology and ELISA. Insect cells infected with the recombinant virus produced and secreted human C1r protein, at a level of 1-2 mg/l of medium. The expressed C1r was isolated from the medium by chromatofocusing. On reducing gels only a single Coomassie-staining band was observed, and this band migrated at 80-83 kD characteristic of the unactivated C1r proenzyme. Its identification as C1r was immunologically confirmed on Western blots. C1 reconstituted from purified C1r expressed in insect cells together with human C1q and C1s proved biologically active in a hemolytic assay. Thus, the baculovirus-insect-cell system is capable of expressing and secreting a sophisticated, multifunctional human complement subcomponent in its biologically activatable form.