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.

Collectin 11 (CL-11, CL-K1) is a MASP-1/3-associated plasma collectin with microbial-binding activity.

Collectins play important roles in the innate immune defense against microorganisms. Recently, a new collectin, collectin 11 (CL-11 or CL-K1), was identified via database searches. In present work, we characterize the structural and functional properties of CL-11. Under nonreducing conditions, in gel permeation chromatography recombinant CL-11 forms disulfide-linked oligomers of 100 and 200 kDa. A mAb-based ELISA estimates the concentration of CL-11 in plasma to be 2.1 µg/ml, and the presence of CL-11 in plasma was further verified by Western blotting and mass spectrometry. Mannan-binding lectin-associated serine protease 1 (MASP-1) copurified with CL-11 and the interaction in plasma with MASP-1 and/or MASP-3 was further demonstrated using ELISA. We identified the adrenal glands, the kidneys, and the liver as primary sites of expression. CL-11 lectin activity was demonstrated by ELISA and showed that CL-11 has preference for l-fucose and d-mannose. We finally show that CL-11 binds to intact bacteria, fungi, and viruses and that CL-11 decreases influenza A virus infectivity and forms complexes with DNA. On the basis of the significant concentration of CL-11 in circulation and CL-11's interaction with various microorganisms and MASP-1 and/or MASP-3, it is conceivable that CL-11 plays a role in activation of the complement system and in the defense against invading microorganisms.

Evolutionary conservation of mannan-binding lectin (MBL) in bony fish: identification, characterization and expression analysis of three bona fide collectin homologues of MBL in the rainbow trout (Onchorhynchus mykiss).

The complement system of fish is generally as complex as in mammals, and in addition Teleost fish often possess several genes encoding different subtypes of a given complement component, such as C3-1, C3-3 and C3-4. Initiators of both the classical (C1) and alternative pathway (factor B) have been characterized in the rainbow trout but so far no molecules of the lectin pathway have been identified. Based on the generally accepted idea of complement evolution, which predicts that the alternative pathway predates the two other pathways, and that the lectin pathway developed before the classical, we set out to characterize members of the lectin pathway in fish. We identified and characterized three homologues of mannan-binding lectin (MBL) with a bona fide collectin structure. By means of RT-PCR and immunohistochemistry using monoclonal antibodies we found that they were synthesized in the spleen, the anterior intestine and the liver. In the liver, we saw co-expression with mannan-binding lectin associated serine protease (MASP). The MBL homologues 2 and 3 (MBL-H2,3) were also found in the vascular system of the rainbow trout. By means of gel size exclusion chromatography of serum we found that MBL-H2,3 oligomerized heterogeneously from monomers to tetramers of a trimeric collagenous subunit. Sequence comparison and phylogenetic studies showed that the homologues were more related with MBL than any other collectins, and that two previously characterized trout proteins, designated MBL1 and MBL2, should be reconsidered as MBL candidates.

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.

Generation of a C3c specific monoclonal antibody and assessment of C3c as a putative inflammatory marker derived from complement factor C3.

There is a general need for markers of systemic inflammation in acute or chronic diseases, where complement activation is involved. Available methods to monitor complement activation are elaborate and of low sensitivity; they include haemolytic assays (CH50), quantification of fluid phase terminal complex (C5b-C9) and quantification of complement split products by precipitation-in-gel techniques (e.g. C3d). We have developed a mouse monoclonal antibody (mAb) that is able to detect fluid phase C3c without interference from other products generated from the complement component C3. The C3c specific mAb was tested in different ELISA combinations with various types of in vitro activated sera and with plasma or serum samples from factor I deficient patients. The specificity of the mAb was evaluated in immunoprecipitation techniques and by analysis of eluted fragments of C3 after immunoaffinity chromatography. The C3c mAb was confirmed to be C3c specific, as it showed no cross-reactivity with native (un-cleaved) C3, with C3b, iC3b, or with C3d. Also, no significant reaction was observed with C3 fragments in factor I deficient sera or plasma. This antibody forms the basis for the generation of a robust ELISA that allows for a quick and reliable evaluation of complement activation and consumption as a marker for inflammatory processes. We established the C3c plasma range in 100 healthy Danish blood donors with a mean of 3.47 µg/ml and a range of 2.12-4.92 µg/ml. We believe that such an antibody might be of potential value in the assessment of in vivo complement activity during the inflammatory processes.

High prevalence of human anti-bovine IgG antibodies as the major cause of false positive reactions in two-site immunoassays based on monoclonal antibodies.

A sandwich ELISA for quantification of the endometrial protein PP14 revealed false positive reactions in 81% of male sera (n = 54). The PP14 ELISA was based on two monoclonal antibodies (Mabs) with different epitope specificities--a catcher and a biotinylated indicator. The monoclonal antibodies were purified by protein G affinity chromatography from culture supernatant containing 10% (v/v) fetal calf serum (FCS). Human anti-animal IgG (bovine, mouse, horse, and swine) antibodies and human anti-bovine serum albumin antibodies were measured using an ELISA design, with direct bridging of the solid phase and biotinylated antigens. The false positive reactions were abolished by addition of 1% (v/v) bovine serum to the dilution buffer (DB). Human anti-bovine IgG antibodies (HABIA) were detected in 99 out of 104 sera from blood donors (50 females; 54 males). HABIA levels in male sera (n = 54) were positively correlated to the false positive signals in the PP14 ELISA (r = 0.923; p < 0.0001). Antibodies to IgG from other mammalian species (mouse, horse, and swine) were also detected in the donor sera, but levels and frequencies were lower compared to that of HABIA. Furthermore, HABIA were positively correlated to human anti-bovine serum albumin antibodies in the donor sera (r = 0.639; p < 0.0001; n = 103). HABIA (prevalence 95%) cause false positive reactions due to crossbinding of contaminating bovine IgG and/or crossreaction with mouse IgG in two-site immunoassays. The apparent presence of human anti-mouse IgG antibodies (HAMA), described to create false positive results, may be due to a crossreacting fraction of the polyclonal circulating antibodies against bovine IgG.

Extracellular matrix building marked by the N-terminal propeptide of procollagen type I reflect aggressiveness of recurrent breast cancer.

The purpose of our study was to examine the association between extracellular matrix homeostasis and aggressive breast cancer as reflected by the synthesis of type I collagen marked by circulating concentration of the aminoterminal propeptide of type I procollagen (PINP). Pre-therapeutic serum PINP concentrations were measured in 154 healthy women and 100 patients referred with their first metastatic manifestation of breast cancer and correlated to the metastatic pattern, response to therapy, time to progression and survival with a minimal follow-up of 5 years. Fifty-four percent of the patients had serum PINP concentrations greater than the 95th percentile of the healthy controls and 38% were high PINP level patients with values clearly outside normal range (>125 ng/ml). Patients with high PINP levels were more sick (p = 0.002), had a higher tumor burden (p = 0.013) and revealed a lower responsiveness to anthracycline-based therapy (p = 0.0002) as well as an accelerated time to disease progression (p = 0.00001) and death (p = 0.0006). Median survival in the high serum PINP level group was less than half of that in the group with low PINP level (14.5 vs. 32 months). The lowest PINP levels were seen when the cancer was restricted to the lymph node and skin and increasing PINP levels were found if the cancer had spread to the lungs, the bones, the bone marrow and the liver. High PINP level at recurrence and lack of estrogen receptors (ER) independently reflected aggressive tumor behavior after recurrence with an equal great impact on time to progression and survival. Patients with a high PINP level and primarily ER-negative tumors survived a median of only 6 months with no one alive after 22 months. By contrast patients with a low PINP level and ER-positive tumors had a median survival of 37 months and 23% were still alive after 5 years. Aggressive breast cancer induces a strong fibroproliferative response with synthesis of type I collagen. Serum PINP levels may be a diagnostic and prognostic tool that indicate breast cancer activity, aggressiveness, expansion and metastasis and a predictor of outcome after anthracycline-based chemotherapy.

Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen.

This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious advantages using this assay, are that it can be performed directly on culture supernatants in the early phase of monoclonal antibody production, and also works for antigens with repetitive epitopes. Moreover, the bonus effect, i.e., a signal in excess of the reference signal when sets of monoclonal antibodies with different epitope specificity are compared, gives a relative measure of affinity.