Maiken Lumby Henriksen

Heteromeric Complexes of Native Collectin Kidney 1 and Collectin Liver 1 Are Found in the Circulation with MASPs and Activate the Complement System

The complement system is an important part of the innate immune system. The complement cascade may be initiated downstream of the lectin activation pathway upon binding of mannan-binding lectin, ficolins, or collectin kidney 1 (CL-K1, alias CL-11) to suitable microbial patterns consisting of carbohydrates or acetylated molecules. During purification and characterization of native CL-K1 from plasma, we observed that collectin liver 1 (CL-L1) was copurified. Based on deglycosylation and nonreduced/reduced two-dimensional SDS-PAGE, we detected CL-K1 and CL-L1 in disulfide bridge-stabilized complexes. Heteromeric complex formation in plasma was further shown by ELISA and transient coexpression. Judging from the migration pattern on two-dimensional SDS-PAGE, the majority of plasma CL-K1 was found in complex with CL-L1. The ratio of this complex was in favor of CL-K1, suggesting that a heteromeric subunit is composed of one CL-L1 and two CL-K1 polypeptide chains. We found that the complex bound to mannan-binding lectin–associated serine proteases (MASPs) with affinities in the nM range in vitro and was associated with both MASP-1/-3 and MASP-2 in plasma. Upon binding to mannan or DNA in the presence of MASP-2, the CL-L1–CL-K1 complex mediated deposition of C4b. In favor of large oligomers, the activity of the complex was partly determined by the oligomeric size, which may be influenced by an alternatively spliced variant of CL-K1. The activity of the native heteromeric complexes was superior to that of recombinant CL-K1. We conclude that CL-K1 exists in circulation in the form of heteromeric complexes with CL-L1 that interact with MASPs and can mediate complement activation.

An enzyme-linked immunosorbent assay (ELISA) for quantification of human collectin 11 (CL-11, CL-K1)

Collectin 11 (CL-11), also referred to as collectin kidney 1 (CL-K1), is a pattern recognition molecule that belongs to the collectin group of proteins involved in innate immunity. It interacts with glycoconjugates on pathogen surfaces and has been found in complex with mannose-binding lectin-associated serine protease 1 (MASP-1) and/or MASP-3 in circulation. Mutation in the CL-11 gene was recently associated with the developmental syndrome 3MC. In the present study, we established and thoroughly validated a sandwich enzyme-linked immunosorbent assay (ELISA) based on two different monoclonal antibodies. The assay is highly sensitive, specific and shows excellent quantitative characteristics such as reproducibility, dilution linearity and recovery (97.7–104%). The working range is 0.15–34 ng/ml. The CL-11 concentration in two CL-11-deficient individuals affected by the 3MC syndrome was determined to be below 2.1 ng/ml. We measured the mean serum CL-11 concentration to 284 ng/ml in 100 Danish blood donors, with a 95% confidence interval of 269–299 ng/ml. There was no significant difference in the CL-11 concentration measured in matched serum and plasma samples. Storage of samples and repeated freezing and thawing to a certain extent did not influence the ELISA. This ELISA offers a convenient and reliable method for studying CL-11 levels in relation to a variety of human diseases and syndromes.

Characterization of the interaction between collectin 11 (CL-11, CL-K1) and nucleic acids.

Collectins are a group of innate immune proteins that contain collagen-like regions and globular C-type lectin domains. Via the lectin domains, collectins recognize and bind to various microbial carbohydrate patterns. Collectin 11 (CL-11) exists in complex with the complement activating MBL-associated proteases, MASPs. In the present work, we characterize the interaction between CL-11 and DNA, and show that CL-11 binds to DNA from a variety of origins in a calcium-independent manner. CL-11 binds also to apoptotic cells presenting extracellular DNA on their surface. The binding to DNA is sensitive to changes in ionic strength and pH. Competition studies show that CL-11 binds to nucleic acids and carbohydrates via separate binding-sites and oligomericity appears crucial for binding activity. Combined interaction with DNA and mannan strongly increases binding avidity. By surface plasmon resonance we estimate the dissociation constant for the binding between CL-11 and double stranded DNA oligonucleotides to K(D)=9-20 nM. In an in vitro assay we find that CL-11 binds to DNA coated surfaces, which leads to C4b deposition via MASP-2. We propose that CL-11, e.g. via complement, may play a role in response to particles and surfaces presenting extracellular DNA, such as apopototic cells, neutrophil extracellular traps and biofilms.

Genetic variation of COLEC10 and COLEC11 and association with serum levels of collectin liver 1 (CL-L1) and collectin kidney 1 (CL-K1)

Collectin liver 1 (CL-L1, alias CL-10) and collectin kidney 1 (CL-K1, alias CL-11), encoded by the COLEC10 and COLEC11 genes, respectively, are highly homologous soluble pattern recognition molecules in the lectin pathway of complement. These proteins may be involved in anti-microbial activity and in tissue development as mutations in COLEC11 are one of the causes of the developmental defect syndrome 3MC. We studied variations in COLEC10 and COLEC11, the impact on serum concentration and to what extent CL-L1 and CL-K1 serum concentrations are correlated. We sequenced the promoter regions, exons and exon-intron boundaries of COLEC10 and COLEC11 in samples from Danish Caucasians and measured the corresponding serum levels of CL-L1 and CL-K1. The median concentration of CL-L1 and CL-K1 was 1.87 μg/ml (1.00–4.14 μg/ml) and 0.32 μg/ml (0.11–0.69 μg/ml), respectively. The level of CL-L1 strongly correlated with CL-K1 (ρ = 0.7405, P <0.0001). Both genes were highly conserved with the majority of variations in the non-coding regions. Three non-synonymous variations were tested: COLEC10 Glu78Asp (rs150828850, minor allele frequency (MAF): 0.003), COLEC10 Arg125Trp (rs149331285, MAF: 0.007) and COLEC11 His219Arg (rs7567833, MAF: 0.033). Carriers of COLEC10 Arg125Trp had increased CL-L1 serum levels (P = 0.0478), whereas promoter polymorphism COLEC11-9570C>T (rs3820897) was associated with decreased levels of CL-K1 (P = 0.044). In conclusion, COLEC10 and COLEC11 are highly conserved, which may reflect biological importance of CL-L1 and CL-K1. Moreover, the strong inter individual correlation between the two proteins suggests that a major proportion are found as heterooligomers or subjected to the same regulatory mechanisms.

Collectin CL-LK Is a Novel Soluble Pattern Recognition Receptor for Mycobacterium tuberculosis

Understanding the molecular components of immune recognition of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, can help designing novel strategies to combat TB. Here, we identify collectin CL-LK as a novel soluble C-type lectin able to bind M. tuberculosis, and characterize mycobacterial mannose-capped lipoarabinomannan as a primary ligand for CL-LK. Mice deficient in CL-K1, one of the CL-LK subunits, do not display altered susceptibility to M. tuberculosis. However, we found that the amount of CL-LK in the serum of patients with active TB is reduced, compared to that in controls, and correlates inversely to the magnitude of the immune response to the pathogen. These findings indicate that CL-LK might be of interest for future diagnostic and treatment monitoring purposes.

COLEC10 is mutated in 3MC patients and regulates early craniofacial development

3MC syndrome is an autosomal recessive heterogeneous disorder with features linked to developmental abnormalities. The main features include facial dysmorphism, craniosynostosis and cleft lip/palate; skeletal structures derived from cranial neural crest cells (cNCC). We previously reported that lectin complement pathway genes COLEC11 and MASP1/3 are mutated in 3MC syndrome patients. Here we define a new gene, COLEC10, also mutated in 3MC families and present novel mutations in COLEC11 and MASP1/3 genes in a further five families. The protein products of COLEC11 and COLEC10, CL-K1 and CL-L1 respectively, form heteromeric complexes. We show COLEC10 is expressed in the base membrane of the palate during murine embryo development. We demonstrate how mutations in COLEC10 (c.25C>T; p.Arg9Ter, c.226delA; p.Gly77Glufs*66 and c.528C>G p.Cys176Trp) impair the expression and/or secretion of CL-L1 highlighting their pathogenicity. Together, these findings provide further evidence linking the lectin complement pathway and complement factors COLEC11 and COLEC10 to morphogenesis of craniofacial structures and 3MC etiology.

Calcium-sensitive immunoaffinity chromatography: Gentle and highly specific retrieval of a scarce plasma antigen, collectin-LK (CL-LK)

Immunoaffinity chromatography is a powerful fractionation technique that has become indispensable for protein purification and characterization. However, it is difficult to retrieve bound proteins without using harsh or denaturing elution conditions, and the purification of scarce antigens to homogeneity may be impossible due to contamination with abundant antigens. In this study, we purified the scarce, complement-associated plasma protein complex, collectin LK (CL-LK, complex of collectin liver 1 and kidney 1), by immunoaffinity chromatography using a calcium-sensitive anti-collectin-kidney-1 mAb. This antibody was characterized by binding to CL-LK at hypo- and physiological calcium concentrations and dissociated from CK-LK at hyperphysiological concentrations of calcium. We purified CL-LK from plasma to a purity of 41% and a yield of 38%, resulting in a purification factor of more than 88,000 in a single step. To evaluate the efficiency of this new purification scheme, we purified CL-LK using the same calcium-sensitive mAb in combination with acidic elution buffer and by using calcium-dependent anti-CL-K1 mAbs in combination with EDTA elution buffer. We found that calcium-sensitive immunoaffinity chromatography was superior to the traditional immunoaffinity chromatographies and resulted in a nine-fold improvement of the purification factor. The technique is applicable for the purification of proteins in complex mixtures by single-step fractionation without the denaturation of eluted antigens, and it allows for the purification of scarce proteins that would have otherwise been impossible to purify and, hence, to characterize. This technique may also potentially be applied for the purification of proteins that only interact with calcium ions at hyperphysiological concentrations.

Complement Defects in Patients with Chronic Rhinosinusitis

The complement system is an important part of our immune system, and complement defects lead generally to increased susceptibility to infections and autoimmune diseases. We have studied the role of complement activity in relation with chronic rhinosinusitis (CRS), and more specifically studied whether complement defects collectively predispose individuals for CRS or affect CRS severity. The participants comprised 87 CRS patients randomly selected from the general population, and a control group of 150 healthy blood donors. The CRS patients were diagnosed according to the European Position Paper on Rhinosinusitis and nasal Polyps criteria, and severity was evaluated by the Sino-nasal Outcome Test-22. Serum samples were analysed by ELISA for activity of the respective pathways of complement, and subsequently for serum levels of relevant components. We found that the frequency of complement defects was significantly higher among CRS patients than among healthy control subjects. A majority of Mannan-binding lectin deficient CRS patients was observed. The presence of complement defects had no influence on the severity of subjective symptoms. Our studies show that defects in the complement system collectively may play an immunological role related to the development of CRS. However, an association between severity of symptoms and presence of complement defects could not be demonstrated.

Surfactant proteins A, B, C and D in the human nasal airway: associated with mucosal glands and ciliated epithelium but absent in fluid-phase secretions and mucus.

Aims: To investigate the presence of surfactant protein (SP) A, B, C and D in nasal airways and to determine whether the proteins exert their main functions in nasal secretions or in the deeper layers of the nasal mucosa. Methods: Volunteers were recruited from the Department of ENT Head and Neck Surgery, Odense University Hospital, Denmark. The study included 39 subjects. Nasal mucosal biopsies were analyzed by immunohistochemistry, and bronchoalveolar and nasal lavages, nasal brush biopsies and nasal mucus were analyzed for SP-A, -B, -C and -D by SDS-PAGE and Western blotting. The presence of SP-A and SP-D in the first three samplings were also analyzed by enzyme-linked immunosorbent assay. Results: In nasal mucosal biopsies, SP-A, -B, -C and -D were all demonstrated in the serous acini of the submucosal glands and in the surface epithelium. SP-D was detected in nasal brush biopsies, whereas the other SPs were absent. Moreover, SP-A, -B, -C and -D were absent in nasal lavage and mucus. Conclusion: SP-A, -B, -C and -D exert their protective effect in the ductal epithelium of the submucosal glands rather than in nasal secretions and mucus. Further studies are required to clarify the functions of these proteins in nasal secretory pathways for understanding upper airway diseases. i 2014 S. Karger AG, Basel

CL-L1 and CL-K1 Exhibit Widespread Tissue Distribution With High and Co-Localized Expression in Secretory Epithelia and Mucosa

Collectin liver 1 (CL-L1, alias collectin 10) and collectin kidney 1 (CL-K1, alias collectin 11) are oligomeric pattern recognition molecules associated with the complement system, and mutations in either of their genes may lead to deficiency and developmental defects. The two collectins are reportedly localized and synthesized in the liver, kidneys, and adrenals, and can be found in the circulation as heteromeric complexes (CL-LK), which upon binding to microbial high mannose-like glycoconjugates activates the complement system via the lectin activation pathway. The tissue distribution of homo- vs. heteromeric CL-L1 and -K1 complexes, the mechanism of heteromeric complex formation and in which tissues this occurs, is hitherto incompletely described. We have by immunohistochemistry using monoclonal antibodies addressed the precise cellular localization of the two collectins in the main human tissues. We find that the two collectins have widespread and almost identical tissue distribution with a high expression in epithelial cells in endo-/exocrine secretory tissues and mucosa. There is also accordance between localization of mRNA transcripts and detection of proteins, showing that local synthesis likely is responsible for peripheral localization and eventual formation of the CL-LK complexes. The functional implications of the high expression in endo-/exocrine secretory tissue and mucosa is unknown but might be associated with the activity of MASP-3, which has a similar pattern of expression and is known to potentiate the activity of the alternative complement activation pathway.