Lectin Complement Research Articles

Role of early lectin pathway activation in the complement-mediated killing of Trypanosoma cruzi

The complement system is the first line of defence against pathogen infection and can be activated by the classic, alternative and lectin pathways. Trypanosoma cruzi, the causative agent of Chagas disease, has to evade complement system killing and invade the host cells to progress in infection. T. cruzi infectious stages resist complement-mediated killing by expressing surface receptors, which dissociate or prevent C3 convertase formation. Here, we present the first evidence that T. cruzi activates the complement lectin pathway. We detected rapid binding of mannan-binding lectin, H-ficolin, and L-ficolin to the surface of T. cruzi, and found that serum depleted of these molecules failed to kill parasites. Furthermore, lectin pathway activation by T. cruzi required the MBL-associated serine protease 2 (MASP2) activity resulting in C2 factor cleavage. In addition, we demonstrate that the infectious stage of T. cruzi inhibits the lectin pathway activation and complement killing expressing the complement C2 receptor inhibitor trispanning (CRIT) protein. Transgenic parasites overexpressing CRIT were highly resistant to complement-mediated killing. CRIT-derived peptides inhibited both C2 binding to the surface of T. cruzi and parasite killing. Biochemical studies revealed that the CRIT extracellular domain 1 inhibits MASP2 cleavage of C2 factor and thereby impairs C3 convertase formation. Our findings establish that the complement lectin pathway recognizes T. cruzi and provide molecular insights into how the infectious stage inhibits this activation to resist complement system killing.

Complement Protease MASP-1 Activates Human Endothelial Cells: PAR4 Activation Is a Link between Complement and Endothelial Function

Activation of the complement system can induce and enhance inflammatory reaction. Mannose-binding lectin-associated serine protease-1 (MASP-1) is an abundant protease of the complement lectin pathway; however, its physiological function is unclear. In this study, we demonstrate for the first time that MASP-1 is able to activate Ca(2+) signaling, NF-kappaB, and p38 MAPK pathways in cultured HUVECs. Activation was initiated by MASP-1 only; the related protease, MASP-2, had no such effect. The phenomenon was dependent on the proteolytic activity of MASP-1, suggesting modulation of endothelial cell function through a protease-activated receptor (PAR). Using synthetic peptide substrates representing the protease-sensitive regions of PARs, we were able to demonstrate that PAR4 is a target of MASP-1. The presence of functionally active PAR4 in HUVECs was demonstrated using PAR4 agonist peptide and mRNA quantification. Finally, we showed that the amount of membrane-bound intact PAR4 decreases after MASP-1 treatment. All of these results provide a novel link between the regulation of endothelial cell function and complement system activation, and they suggest that MASP-1-induced PAR4 activation could contribute to the development of the inflammatory reaction.

Complement protease MASP-1 activates human endothelial cells: PAR4 activation is a link between complement and endothelial function

Activation of the complement system can induce and enhance inflammatory reaction. Mannose-binding lectin-associated serine protease-1 (MASP-1) is an abundant protease of the complement lectin pathway; however, its physiological function is unclear. In this study, we demonstrate for the first time that MASP-1 is able to activate Ca 2+ signaling, NF-κB, and p38 MAPK pathways in cultured HUVECs. Activation was initiated by MASP-1 only; the related protease, MASP-2, had no such effect. The phenomenon was dependent on the proteolytic activity of MASP-1, suggesting modulation of endothelial cell function through a protease-activated receptor (PAR). Using synthetic peptide substrates representing the protease-sensitive regions of PARs, we were able to demonstrate that PAR4 is a target of MASP-1. The presence of functionally active PAR4 in HUVECs was demonstrated using PAR4 agonist peptide and mRNA quantification. Finally, we showed that the amount of membrane-bound intact PAR4 decreases after MASP-1 treatment. All of these results provide a novel link between the regulation of endothelial cell function and complement system activation, and they suggest that MASP-1-induced PAR4 activation could contribute to the development of the inflammatory reaction.

Stringent regulation of complement lectin pathway C3/C5 convertase by C4b-binding protein (C4BP)

The complement lectin pathway, an essential component of the innate immune system, is geared for rapid recognition of infections as each C4b deposited via this pathway is capable of forming a C3/C5 convertase. In the present study, role of C4b-binding protein (C4BP) in regulating the lectin pathway C3/C5 convertase assembled on zymosan and sheep erythrocytes coated with mannan (EMan) was examined. While the C4BP concentration for inhibiting 50% (IC50) formation of surface-bound C3 convertase on the two surfaces was similar to that obtained for the soluble C3 convertase (1.05nM), ∼3- and 41-fold more was required to inhibit assembly of the C5 convertase on zymosan (2.81nM) and EMan (42.66nM). No difference in binding interactions between C4BP and surface-bound C4b alone or in complex with C3b was observed. Increasing the C4b density on zymosan (14,000–431,000 C4b/Zym) increased the number of C4b bound per C4BP from 2.87 to 8.23 indicating that at high C4b density all seven α-chains of C4BP are engaged in C4b-binding. In contrast, the number of C4b bound per C4BP remained constant (3.79±0.60) when the C4b density on EMan was increased. The data also show that C4BP regulates assembly and decay of the lectin pathway C3/C5 convertase more stringently than the classical pathway C3/C5 convertase because of a ∼7- to 13-fold greater affinity for C4b deposited via the lectin pathway than the classical pathway. C4BP thus regulates efficiently the four times greater potential of the lectin pathway than the classical pathway in generating the C3/C5 convertase and hence production of pro-inflammatory products, which are required to fight infections but occasionally cause pathological inflammatory reactions.

A Novel Inhibitor of the Alternative Pathway of Complement Attenuates Intestinal Ischemia/Reperfusion-Induced Injury

Complement activation has been demonstrated to contribute significantly to the expression of IR-induced tissue damage. Each of the three complement pathways, classic, alternative, and lectin, has been implicated in the instigation of tissue pathology. In this study, we used a selective inhibitor of the alternative pathway, that is, a soluble form of complement receptor of the immunoglobulin superfamily (CRIg-Fc) to determine whether it can prevent IR tissue injury. We demonstrate that treatment of C57B1/6 mice prior to mesenteric IR prevents local (intestinal) and remote (lung) injury by limiting deposition of complement and entry of polymorphonuclear cells to the sites of injury. Our results show that CRIg-Fc represents a candidate to limit IR injury as it occurs in various clinical conditions.

Ficolin 2 (FCN2) functional polymorphisms and the risk of rheumatic fever and rheumatic heart disease

Ficolins are pattern-recognition proteins involved in innate immunity, which upon binding to their specific pathogen-associated molecular patterns on the microbial surfaces trigger the immune response either by binding to collectin cellular receptors or by initiating the complement lectin pathway. In humans, three ficolin genes have been identified, which encode ficolin-1 (M-ficolin), ficolin-2 (L-ficolin) and ficolin-3 (H-ficolin or Hakata antigen). Ficolin-2 was shown to bind to lipoteichoic acid, a cell wall constituent in all Gram-positive bacteria such as Streptococcus pyogenes, which is the aetiological agent of rheumatic fever (RF) and its most severe sequelae, chronic rheumatic heart disease (CRHD). Here we investigated polymorphisms in the promoter region of the FCN2 gene (at positions -986/-602 and +4) in 122 patients with RF and CRHD and in 210 healthy subjects from the same geographic region and socioeconomic background. The haplotype -986/-602/-4 G/G/A, which is related to low levels of L-ficolin, was observed more frequently in the CRHD group when compared to the healthy subjects [99/162, 61.1% versus 211/420, 50.2%, odds ratio (OR) 1.6, confidence interval (CI) 95% 1.1-2.3, P = 0.021]. The haplotype -986/-602/-4 A/G/A was observed more frequently in the healthy group when compared to the affected (RF plus CRHD) subjects (31/420, 7.4% versus 6/244, 2.5%, OR 3.2, CI 95% 0.13-0.77, P = 0.008). Based on those findings, one can conclude that polymorphisms associated with low levels of L-ficolin level may predispose an individual to recurrent and/or more severe streptococcal infection.

Molecular and expression analysis of complement component C5 in the nurse shark ( Ginglymostoma cirratum) and its predicted functional role

We present the complete cDNA sequence of shark ( Ginglymostoma cirratum) pro-C5 and its molecular characterization with a descriptive analysis of the structural elements necessary for its potential functional role as a potent mediator of inflammation (fragment C5a) and initiator molecule (fragment C5b) for the assembly of the membrane attack complex (MAC) upon activation by C5 convertase. In mammals the three complement activation cascades, the classical, alternative and lectin pathways, converge at the activation of C3, a pivotal complement protein. It is, however, the subsequent activation of the next complement component, C5, which is the focal point at which the initiation of the terminal lytic pathway takes place and involves the stepwise assembly of the MAC. The effector cytolytic function of complement occurs with the insertion of MAC into target membranes causing dough-nut like holes and cell leakage. The lytic activity of shark complement results in structurally similar holes in target membranes suggesting the assembly of a shark MAC that likely involves a functional analogue of C5. The composition of shark MAC remains unresolved and to date conclusive evidence has been lacking for shark C5. The gene has not been cloned nor has the serum protein been characterized for any elasmobranch species. This report is the first to confirm the presence of C5 homologue in the shark. GcC5 is remarkably similar to human C5 in overall structure and domain arrangement. The GcC5 cDNA measured 5160-bp with 5′ and 3′ UTRs of 35 bp and 79 bp, respectively. Structural analysis of the derived protein sequence predicts a molecule that is a two-chain structure which lacks a thiolester bond and contains a C5 convertase cleavage site indicating that activation will generate two peptides, akin to C5b and C5a. The putative GcC5 molecule also contains the C-terminal C345C/Netrin module that characterizes C3, C4 and C5. Multiple alignment of deduced amino acid sequences shows that GcC5 shares more amino acid identities/similarities with mammals than that with bony fish. We conclude that at the time of emergence of sharks the elaborate mosaic structure of C5 had already evolved.

Differential proteomics analysis of the surface heterogeneity of dextran iron oxide nanoparticles and the implications for their in vivo clearance

In order to understand the role of plasma proteins in the rapid liver clearance of dextran-coated superparamagnetic iron oxide (SPIO) in vivo, we analyzed the full repertoire of SPIO-binding blood proteins using novel two-dimensional differential mass spectrometry approach. The identified proteins showed specificity for surface domains of the nanoparticles: mannan-binding lectins bound to the dextran coating, histidine-rich glycoprotein and kininogen bound to the iron oxide part, and the complement lectin and contact clotting factors were secondary binders. Nanoparticle clearance studies in knockout mice suggested that these proteins, as well as several previously identified opsonins, do not play a significant role in the SPIO clearance. However, both the dextran coat and the iron oxide core remained accessible to specific probes after incubation of SPIO in plasma, suggesting that the nanoparticle surface could be available for recognition by macrophages, regardless of protein coating. These data provide guidance to rational design of bioinert, long-circulating nanoparticles.

New insights on the structural/functional properties of recombinant human mannan-binding lectin and its variants

Inefficient activation of complement lectin pathway in individuals with variant mannan-binding lectin (MBL) genotypes has been attributed to poor formation of higher order oligomers by MBL. But recent studies have shown the presence of large oligomers of MBL (∼450 kDa) in serum of individuals with variant MBL alleles. The recombinant forms of MBL (rMBL) variants except MBL/B that assemble into higher order oligomers have not yet been reported. In the present study, structural/functional properties of recombinant forms of wild type MBL (rMBL/A) and its three structural variants, rMBL/B, C, and D generated in insect cells were examined. Western blot analysis indicated covalently linked monomers to hexamers while gel filtration chromatography exhibited non-covalently linked higher order oligomers in addition to prevalent low oligomeric forms. Mannan binding determined by ELISA showed rMBL/A but not the structural variants bind to mannan. Apparent avidity of monoclonal antibody used was found to be about 18- to 52-fold weaker for rMBL structural variants than rMBL/A. Complement activation varied with maximum impairment apparent in rMBL/C followed by rMBL/B, but rMBL/D was functional to the same extent as rMBL/A. Comparison of rMBL/A to MBL purified from plasma (pMBL/A) indicated 8- and 24-fold weaker binding to mannan by BIAcore analysis and ELISA and about 5-fold lesser efficiency in activating complement. The findings provide new insights on the structural/functional properties of rMBL variants and imply that lectin pathway activation may be impaired in individuals, homozygous for the mutant alleles, MBL/C and to a lesser extent MBL/B but not MBL/D.

Local Inflammation Induces Complement Crosstalk Which Amplifies the Antimicrobial Response

By eliciting inflammatory responses, the human immunosurveillance system notably combats invading pathogens, during which acute phase proteins (CRP and cytokines) are elevated markedly. However, the Pseudomonas aeruginosa is a persistent opportunistic pathogen prevalent at the site of local inflammation, and its acquisition of multiple antibiotic-resistance factors poses grave challenges to patient healthcare management. Using blood samples from infected patients, we demonstrate that P. aeruginosa is effectively killed in the plasma under defined local infection-inflammation condition, where slight acidosis and reduced calcium levels (pH 6.5, 2 mM calcium) typically prevail. We showed that this powerful antimicrobial activity is provoked by crosstalk between two plasma proteins; CRP∶L-ficolin interaction led to communication between the complement classical and lectin pathways from which two amplification events emerged. Assays for C4 deposition, phagocytosis, and protein competition consistently proved the functional significance of the amplification pathways in boosting complement-mediated antimicrobial activity. The infection-inflammation condition induced a 100-fold increase in CRP∶L-ficolin interaction in a pH- and calcium-sensitive manner. We conclude that the infection-induced local inflammatory conditions trigger a strong interaction between CRP∶L-ficolin, eliciting complement-amplification pathways which are autonomous and which co-exist with and reinforce the classical and lectin pathways. Our findings provide new insights into the host immune response to P. aeruginosa infection under pathological conditions and the potential development of new therapeutic strategies against bacterial infection.

Two factors of the lectin pathway of complement, l-ficolin and mannan-binding lectin, and their associations with prematurity, low birthweight and infections in a large cohort of Polish neonates

Ficolins and one collectin, mannan-binding lectin (MBL), are the only factors known to activate the lectin pathway (LP) of complement. There is considerable circumstantial evidence that MBL insufficiency can increase susceptibility to various infections and influence the course of several non-infectious diseases complicated by infections. Much less information is available concerning l-ficolin. We report the results of a prospective study to investigate any association between either MBL deficiency or l-ficolin deficiency with prematurity, low birthweight or perinatal infections in a large cohort of Polish neonates, representing an ethnically homogenous population (n=1832). Cord blood samples were analysed to determine mbl-2 gene variants, MBL concentrations and MBL-MASP-2 complex activities (MBL-dependent lectin pathway activity) as well as l-ficolin levels. Median concentrations of l-ficolin and MBL were 2500 and 1124ng/ml, respectively, while median LP activity was 272mU/ml. After genotyping, 60.6% of babies were mbl-2 A/A, 35.4% were A/O and 4% were O/O genotypes. We found relative l-ficolin deficiency to be associated with prematurity, low birthweight and infections. l-Ficolin concentration correlated with gestational age and with birthweight, independently of gestational age. Preterm deliveries (<38 weeks) occurred more frequently among neonates with low LP activity but not with those having low serum MBL levels. Similarly, no association of serum MBL deficiency with low birthweight was found, but there was a correlation between LP activity and birthweight. Genotypes conferring very low serum MBL concentrations were associated with perinatal infections, and high-MBL-conferring genotypes were associated with prematurity. Our findings suggest that l-ficolin participates in host defence during the perinatal period and constitute the first evidence that relative l-ficolin deficiency may contribute to the adverse consequences of prematurity. Some similar trends were found with facets of MBL deficiency, but the observed relationships were weaker and less consistent.

Manno(Rhamno)Biose-Containing Capsular Polysaccharides of Klebsiella pneumoniae Enhance Opsono-Stimulation of Human Polymorphonuclear Leukocytes

We tested the relationship between the capsular and the O-antigen structures and the ability of bacteria to trigger respiratory burst in human polymorphonuclear leukocytes (PMNL).Capsulated and non-capsulated variants as well as capsule-switched derivatives of Klebsiella serotypes bearing or lacking manno(rhamno)biose repeats in their capsular polysaccharides and expressing either mannose-rich or mannose-poor O antigens were tested for their ability to induce respiratory burst and survive in human PMNL. Luminol-enhanced chemiluminescence (CL) was measured to quantify respiratory burst. Intracellular survival was quantified by determining the viable counts of intracellular bacteria. K serotypes and the capsule-switched derivative lacking manno(rhamno)biose induced significantly lower CL than those expressing manno(rhamno)biose. Manno(rhamno)biose-lacking serotypes survived in the cells significantly better than serotypes expressing these repeats. C1q depletion did not affect CL induced by the manno(rhamno)biose-containing serotype, whereas factor B depletion revealed a significantly reduced CL. Likewise, EGTA in the presence of Mg²⁺ significantly decreased CL, but the values were higher than those induced by the bacterium opsonized with factor B-depleted serum. In the presence of EGTA, Mg²⁺-treated factor B-depleted serum revealed a significant reduction in the CL response compared with the responses induced by opsonization with factor B-depleted serum alone. These results indicate, in addition to the alternative pathway, a manno(rhamno)biose pattern recognition of Klebsiella by PMNL probably by the complement lectin pathway.

Impact of the complement lectin pathway on cytomegalovirus disease early after kidney transplantation

This study retrospectively investigated the association between pre-transplant levels of mannose-binding lectin (MBL) plus the associated serine protease (MASP)-2 and the occurrence of cytomegalovirus (CMV) infection and symptomatic CMV disease during the first 12 weeks after kidney transplantation. Materials and methods. Altogether 159 consecutive single kidney transplant recipients were included. The patients were screened for CMV pp65 antigenaemia every second week. No CMV prophylaxis or pre-emptive treatment was given. MBL and MASP-2 were measured in samples taken at transplantation and 10 weeks later. CMV infection, defined as at least one positive test, was found in 95 patients (59.8%). MBL and MASP-2 measured at transplantation were similar in patients with and without CMV infection. The incidence of CMV infection was also similar in 36 patients (58.3%) with pre-transplant MBL levels below the reference level (500 microg/L) and in patients with higher MBL levels (60.2%). Symptomatic CMV disease was diagnosed in 35 patients (22%), and MASP-2 levels at transplantation in the lower quartile range (<or=148 microg/L) was significantly associated with CMV disease during the first 12 weeks, P = 0.028. MBL levels decreased significantly from transplantation to 10 weeks later, and median (interquartile range) fell from 2597 (526-4939) microug/L to 1520 (270-3069) microg/L (P < 0.001). In contrast, MASP-2 levels increased significantly from 252 (148-382) microg/L to 380 (302-492) microg/L (P < 0.001). Pre-transplant MBL levels do not influence the incidence of any CMV infection or symptomatic CMV disease during the first 12 weeks after kidney transplantation. However, low MASP-2 levels may play a role in the development of symptomatic CMV disease.

The Complement System in Schizophrenia

Several lines of evidence suggest that immunological factors contribute to schizophrenia. Since 1989, the role of complement, a major effector of innate immunity and an adjuvant of adaptive immunity, has been explored in schizophrenia. Increased activity of C1, C3, C4 complement components in schizophrenia has been reported by two or more groups. Two studies on different subject cohorts showed increased MBL-MASP-2 activity in patients versus controls. More then one report indicated a significant high frequency of FB*F allotype and low prevalence of the FS phenotype of complement factor B in schizophrenia. From the data reported, it is likely that the disorder is accompanied by alterations of the complement classical and lectin pathways, which undergo dynamic changes, depending on the illness course and the state of neuro-immune crosstalk. Recent findings, implicating complement in neurogenesis, synapse remodeling and pruning during brain development, suggest a reexamination of the potential role of complement in neurodevelopmental processes contributing to schizophrenia susceptibility. It is plausible that the multicomponent complement system has more than one dimensional association with schizophrenia susceptibility, pathopsychology and illness course, understanding of which will bring a new perspective for possible immunomodulation and immunocorrection of the disease.

Interaction between complement lectin L-ficolin and hepatitis C virus envelope glycoproteins

Interaction between complement lectin L-ficolin and hepatitis C virus envelope glycoproteins

Identification of the Site of Human Mannan-Binding Lectin Involved in the Interaction with Its Partner Serine Proteases: The Essential Role of Lys55

Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys(55)Ala abolished interaction with the MASPs and MAp19 and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys(55)Gln and Lys(55)Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys(55)Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3. Mutation Arg(47)Glu inhibited interaction with MAp19 and decreased the ability of MBL to trigger the lectin pathway. Mutant Arg(47)Lys showed no interaction with the MASPs or MAp19, likely resulting from a defect in oligomerization. In contrast, mutation Arg(47)Ala had no impact on the interaction with the MASPs and MAp19, nor on the ability of MBL to trigger the lectin pathway. Mutation Pro(53)Ala only had a slight effect on the interaction with MASP-1 and -3, whereas mutations at residues Leu(49) and Leu(56) were ineffective. In conclusion, the MASP binding site of MBL involves a sequence stretch centered on residue Lys(55), which may form an ionic bond representing the major component of the MBL-MASP interaction. The binding sites for MASP-2/MAp19 and MASP-1/3 have common features but are not strictly identical.

A Novel Fluorochrome Linked Immunosorbent Assay (FLISA) for the complete analysis of the mannose binding lectin (MBL) complement pathway.

A Novel Fluorochrome Linked Immunosorbent Assay (FLISA) for the complete analysis of the mannose binding lectin (MBL) complement pathway.

Activation of the Lectin Pathway by Natural IgM in a Model of Ischemia/Reperfusion Injury

Reperfusion of ischemic tissues elicits an acute inflammatory response involving serum complement, which is activated by circulating natural IgM specific to self-Ags exposed by ischemia. Recent reports demonstrating a role for the lectin pathway raise a question regarding the initial events in complement activation. To dissect the individual roles of natural IgM and lectin in activation of complement, mice bearing genetic deficiency in early complement, IgM, or mannan-binding lectin were characterized in a mesenteric model of ischemia reperfusion injury. The results reveal that IgM binds initially to ischemic Ag providing a binding site for mannan-binding lectin which subsequently leads to activation of complement and injury.

Deficiency of Mannose-Binding Lectin Greatly Increases Susceptibility to Postburn Infection withPseudomonas aeruginosa

Burn injury disrupts the mechanical and biological barrier that the skin presents against infection by symbionts like the Pseudomonas aeruginosa, a Gram-negative bacteria. A combination of local factors, antimicrobial peptides, and resident effector cells form the initial response to mechanical injury of the skin. This activity is followed by an inflammatory response that includes influx of phagocytes and serum factors, such as complement and mannose-binding lectin (MBL), which is a broad-spectrum pattern recognition molecule that plays a key role in innate immunity. A growing consensus from studies in humans and mice suggests that lack of MBL together with other comorbid factors predisposes the host to infection. In this study we examined whether MBL deficiency increases the risk of P. aeruginosa infection in a burned host. We found that both wild-type and MBL null mice were resistant to a 5% total body surface area burn alone or s.c. infection with P. aeruginosa alone. However, when mice were burned then inoculated s.c. with P. aeruginosa at the burn site, all MBL null mice died by 42 h from septicemia, whereas only one-third of wild-type mice succumbed (p = 0.0005). This result indicates that MBL plays a key role in containing and preventing a systemic spread of P. aeruginosa infection following burn injury and suggests that MBL deficiency in humans maybe a premorbid variable in the predisposition to infection in burn victims.

Molecular cloning and characterization of novel ficolins from Xenopus laevis

Ficolins are proteins characterized by the presence of collagen- and fibrinogen-like domains. Two of three human ficolins, L-ficolin and H-ficolin, are serum lectins and are thought to play crucial roles in host defense through opsonization and complement activation. To elucidate the evolution of ficolins and the primordial complement lectin pathway, we cloned four ficolin cDNAs from Xenopus laevis, termed Xenopus ficolin (XeFCN) 1, 2, 3 and 4. The deduced amino acid sequences of the four ficolins revealed the conserved collagen- and fibrinogen-like domains. The full sequences of the four ficolins showed a 42-56% identity to human ficolins, and 60-83% between one another. Northern blots showed that XeFCN1 was expressed mainly in liver, spleen and heart, and XeFCN2 and XeFCN4 mainly in peripheral blood leukocytes, lung and spleen. We isolated ficolin proteins from Xenopus serum by affinity chromatography on N-acetylglucosamine-agarose, followed by ion-exchange chromatography. The final eluate showed polymeric bands composed of two components of 37 and 40 kDa. The N-terminal amino acid sequences and treatment with endoglycosidase F showed that the two bands are the same XeFCN1 protein with different masses of N-linked sugar. The polymeric form of the two types of XeFCN1 specifically recognized GlcNAc and GalNAc residues. These results suggest that like human L-ficolin, XeFCN1 functions in the circulation through its lectin activity.