Purification, Measurement of Concentration, and Functional Complement Assay of Human Ficolins

Matsushita, Misao; Kilpatrick, David C.; Shiraki, Hiroshi; Liu, Yu; Tateishi, Koichiro; Tsujimura, Mitsushi; Endo, Yuichi; Fujita, Teizo

doi:10.1007/978-1-62703-724-2_12

Cited by 7 publications

(10 citation statements)

References 33 publications

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“…Ficolin‐2 concentrations were measured according to the procedure (enzyme‐linked immunosorbent assay) described by Matsushita et al , 53 with our modification (time‐resolved immunofluorometric assay). Briefly, plates (Optiplate‐384 HB, Perkin Elmer) were coated with murine anti‐ficolin‐2 monoclonal antibodies (clone GN1; concentration 1.8 μg ml −1 in phosphate buffered saline (pH 7.4); 20 μl per well) and incubated overnight at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

Lectin pathway factors in patients suffering from juvenile idiopathic arthritis

et al. 2017

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Both complement activation and certain infections (including those with Yersinia sp.) may contribute to the pathogenesis of juvenile idiopathic arthritis (JIA). We investigated factors specific for the lectin pathway of complement: mannose-binding lectin (MBL), ficolins and MBL-associated serine protease-2 (MASP-2), in 144 patients and 98 controls. One hundred and six patients had oligoarticular disease and 38 had polyarticular disease. In 51 patients (out of 133 tested), Yersinia-reactive antibodies were found (JIA Ye group). MBL deficiency was significantly more frequent in the JIA Ye group than in patients without Yersinia-reactive antibodies or in controls. Median serum ficolin-2 level was significantly lower (and proportion of values deemed ficolin-2 insufficient greater) in JIA patients irrespective of their Yersinia antibody status. The minority (C) allele at -64 of the FCN2 gene was less frequent among JIA patients than among control subjects. No differences were found in the frequency of FCN3 gene +1637delC or MASP2 +359 A>G mutations nor for median values of serum ficolin-1, ficolin-3 or MASP-2. However, high levels of serum ficolin-3 were under-represented in patients, in contrast to MBL. MBL, ficolin-1, ficolin-2, ficolin-3 and MASP-2 were also readily detectable in synovial fluid samples but at a considerably lower level than in serum. Our findings suggest a possible role for the lectin pathway in the pathogenesis of JIA, perhaps secondary to a role in host defence, and indicate that investigations on the specificity of lectin pathway recognition molecules towards specific infectious agents in JIA might be fruitful.

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Section: Methodsmentioning

confidence: 99%

Lectin pathway factors in patients suffering from juvenile idiopathic arthritis

et al. 2017

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“…Also, some bacteria and their specific serotypes (like Salmonella enterica O:6,7 serotype) can have mannose (tetramannose repeats) in their lipopolysaccharides, although this is not common . Ficolins have their own specificity repertoires . Structures that usually are recognized contain acetylated monosaccharides.…”

Section: Recognition Of Targets By the Classical And Lectin Pathwaysmentioning

confidence: 99%

Self‐nonself discrimination by the complement system

Meri

2016

FEBS Letters

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The alternative pathway (AP) of complement can recognize nonself structures by only two molecules, C3b and factor H. The AP deposits C3b covalently on nonself structures via an amplification system. The actual discrimination is performed by factor H, which has binding sites for polyanions (sialic acids, glycosaminoglycans, phospholipids). This robust recognition of ‘self’ protects our own intact viable cells and tissues, while activating structures are recognized by default. Foreign targets are opsonized for phagocytosis or killed. Mutations in factor H predispose to severe diseases. In hemolytic uremic syndrome, they promote complement attack against blood cells and vascular endothelial cells and lead, for example, to kidney and brain damage. Even pathogens can exploit factor H. In fact, the ability to bind factor H discriminates most pathogenic microbes from nonpathogenic ones.

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“…GlcNAc has been reported to elute ficolin-2 from GlcNAc- or CysNAc-agarose or -sepharose (e.g., refs. (Krarup et al, 2004; Faro et al, 2008; Lacroix et al, 2009; Matsushita et al, 2014)). In our system, GlcNAc in concentrations up to 450 mM resulted in diffuse elution, with low concentrations of ficolin-2 across many fractions rather than most eluted material being confined to a few fractions (as in Figure 2); thus, CysNAc is the more efficient eluent in our protocol.…”

Section: Discussionmentioning

confidence: 99%

“…Studies of ficolin-2 function require purified ficolin-2, and several protocols are described to purify ficolin-2 from human serum or plasma using agarose or sepharose beads conjugated to GlcNAc or CysNAc (Krarup et al, 2004; Faro et al, 2008; Matsushita et al, 2014). However, the reported protocols are apparently inefficient, with multiple steps of purification and low reported yields.…”

Section: Introductionmentioning

confidence: 99%

Rapid and efficient purification of ficolin-2 using a disposable CELLine bioreactor

Geno

Spencer

Nahm

2015

Journal of Immunological Methods

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The human opsonin ficolin-2 (L-ficolin) is an innate pattern-recognizing molecule that binds to acetylated moieties. Upon binding, ficolin-2 activates complement through the lectin pathway, opsonizing the target to promote phagocytic clearance. Ficolin-2 has been found to interact with a growing number of pathogenic bacteria, fungi, and viruses. Ficolin-2 also has proposed roles in host homeostasis, including the clearance of apoptotic cells. Consequently, there is an increased interest in studying ficolin-2, and access to purified ficolin-2 is necessary for these studies. Ficolin-2 purified from serum, plasma, or cell culture supernatants has been a useful tool in the characterization of ficolin-2 function; however, available protocols are laborious and inefficient, requiring additional processing of starting materials (e.g., polyethylene glycol precipitation or dialysis) and multiple steps of purification. Here, we investigated a simple solution to the problem: use of a simple, disposable bioreactor requiring only standard tissue culture equipment. Using this system, we generated cell culture supernatants containing high concentrations of recombinant ficolin-2, which permitted rapid purification of high-purity recombinant ficolin-2 without processing the supernatants. Purified recombinant ficolin-2 retained its binding capacity and supported complement activation in vitro. Bioreactor cultivation will likely be generally useful in the production of other recombinant proteins in the study of the complement system.

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Purification, Measurement of Concentration, and Functional Complement Assay of Human Ficolins

Cited by 7 publications

References 33 publications

Lectin pathway factors in patients suffering from juvenile idiopathic arthritis

Lectin pathway factors in patients suffering from juvenile idiopathic arthritis

Self‐nonself discrimination by the complement system

Rapid and efficient purification of ficolin-2 using a disposable CELLine bioreactor

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