A New Ligand-Based Method for Purifying Active Human Plasma-Derived Ficolin-3 Complexes Supports the Phenomenon of Crosstalk between Pattern-Recognition Molecules and Immunoglobulins

被引：4

作者：

Man-Kupisinska, Aleksandra ^{[1
]}

Michalski, Mateusz ^{[2
,3
]}

Maciejewska, Anna ^{[1
]}

Swierzko, Anna S. ^{[2
]}

Cedzynski, Maciej ^{[2
]}

Lugowski, Czeslaw ^{[1
]}

Lukasiewicz, Jolanta ^{[1
]}

机构：

[1] Polish Acad Sci, Hirszfeld Inst Immunol & Expt Therapy, Dept Immunochem, Wroclaw, Poland

[2] Polish Acad Sci, Inst Med Biol, Lab Immunobiol Infect, Lodz, Poland

[3] Univ Lodz, Inst Microbiol Immunol & Biotechnol, PL-90131 Lodz, Poland

来源：

PLOS ONE | 2016年 / 11卷 / 05期

关键词：

MANNAN-BINDING LECTIN; H-FICOLIN; COMPLEMENT ACTIVATION; PATHWAY ACTIVATION; HAFNIA-ALVEI; SERINE-PROTEASE; HAKATA ANTIGEN; CORE OLIGOSACCHARIDE; HUMAN SERUM; PURIFICATION;

D O I：

10.1371/journal.pone.0156691

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

Despite recombinant protein technology development, proteins isolated from natural sources remain important for structure and activity determination. Ficolins represent a class of proteins that are difficult to isolate. To date, three methods for purifying ficolin-3 from plasma/serum have been proposed, defined by most critical step: (i) hydroxyapatite absorption chromatography (ii) N-acetylated human serum albumin affinity chromatography and (iii) anti-ficolin-3 monoclonal antibody-based affinity chromatography. We present a new protocol for purifying ficolin-3 complexes from human plasma that is based on an exclusive ligand: the O-specific polysaccharide of Hafnia alvei PCM 1200 LPS (O-PS 1200). The protocol includes (i) poly(ethylene glycol) precipitation; (ii) yeast and L-fucose incubation, for depletion of mannose-binding lectin; (iii) affinity chromatography using O-PS 1200-Sepharose; (iv) size-exclusion chromatography. Application of this protocol yielded average 2.2 mg of ficolin-3 preparation free of mannose-binding lectin (MBL), ficolin-1 and -2 from 500 ml of plasma. The protein was complexed with MBL-associated serine proteases (MASPs) and was able to activate the complement in vitro. In-process monitoring of MBL, ficolins, and total protein content revealed the presence of difficult-to-remove immunoglobulin G, M and A, in some extent in agreement with recent findings suggesting crosstalk between IgG and ficolin-3. We demonstrated that recombinant ficolin-3 interacts with IgG and IgM in a concentration-dependent manner. Although this association does not appear to influence ficolin-3-ligand interactions in vitro, it may have numerous consequences in vivo. Thus our purification procedure provides Ig-ficolin-3/MASP complexes that might be useful for gaining further insight into the crosstalk and biological activity of ficolin-3.

引用

页数：19