Isolation of mannan-binding protein from human serum (plasma)

Katsuaki Higashi; Motohiro Nonaka; Nobuko Kawasaki; Toshisuke Kawasaki

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Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-.

Isolation of mannan-binding protein from human serum (plasma)

Katsuaki Higashi, Master

Kyoto University

Email: pj.ca.u-otoyk.ts@h31.ikaustak.ihsagih

Motohiro Nonaka, Dr.

Kyoto University

Email: pj.ca.u-otoyk@r4.orihotom.akanon

Corresponding author.

Nobuko Kawasaki, Dr.

Ritsumeikan University

Email: pj.ca.iemustir.tsg@84000v41

Toshisuke Kawasaki, Dr.

Ritsumeikan University

Email: pj.ca.iemustir.cf@kasawakt

Corresponding author.

Created: September 1, 2021; Last Revision: May 24, 2022.

Introduction

Mannan (mannose)-binding protein (MBP), also called mannan-binding lectin (MBL), is a C-type mammalian lectin that affects innate immunity (1–7). MBP recognizes glycans on the surfaces of bacteria and viruses, activating complement or directly opsonizing them (4–7). MBP also recognizes the distinct structures of N-glycans expressed in the colorectal cancers (8–11). To experimentally elucidate the immunological function of MBP, such as the relationship between MBP ligand expression and tumor progression, large amounts of high quality of MBP are needed. The following methods demonstrate an efficient and reproducible MBP purification protocol from human plasma or serum samples.

Protocol

Methods 1 and 2 start with human serum and plasma, respectively. Pretreatment is required where human plasma is used as a starting material, as described in method 2. Method 1 is composed of mannan column affinity chromatography and Sepharose CL-6B gel filtration. Method 2 is composed of two affinity column purification steps: a mannan column and a heparin column. The eluates from the columns are analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Materials

1.

Purification of MBP from human serum

a.: Pooled normal human serum (obtained from fresh blood by natural coagulation)
b.: Mannan from Saccharomyces cerevisiae (Sigma-Aldrich, St. Louis, MO)
c.: CNBr-activated Sepharose 4B (GE-Healthcare, Little Chalfont, UK)
d.: Sepharose 4B-mannan (yeast mannan-coupled with CNBr-activated Sepharose 4B, 5 mg mannan/mL gel)
e.: 2× Loading buffer for Sepharose 4B-mannan affinity column chromatography: 0.08 M imidazole-HCl buffer, pH 7.8 containing 2.5 M NaCl and 0.04 M CaCl₂
f.: Loading buffer for Sepharose 4B-mannan affinity column chromatography: 0.04 M imidazole-HCl buffer, pH 7.8 containing 1.25 M NaCl and 0.02 M CaCl₂
g.: Elution buffer for Sepharose 4B-mannan affinity column chromatography: 0.02 M imidazole-HCl buffer, pH 7.8 containing 1.25 M NaCl and 2 mM of EDTA
h.: Sepharose CL-6B column (0.9 × 105 cm) (Pharmacia, Uppsala, Sweden)
i.: Gel filtration medium for Sepharose CL-6B: 0.02 M imidazole-HCl buffer, pH 7.8 containing 0.5 M NaCl, 1 mM of EDTA, and 0.1% (v/v) Triton X-100
j.: Coomassie brilliant blue G250 (Sigma-Aldrich)

2.

Purification of MBP from human plasma

a.: Polyethylene glycol 4,000
b.: Heparin-agarose (AF–Heparin HC- 650M: Tosoh Corp., Tokyo, Japan)
c.: Loading buffer for heparin-agarose affinity column chromatography: 20 mM of phosphate buffer, pH 7.0 and 1 mM of EDTA
d.: Washing buffer for heparin-agarose affinity column chromatography: 20 mM of phosphate buffer, pH 7.0, 0.1 M NaCl, and 1 mM of EDTA
e.: Elution buffer for heparin-agarose affinity column chromatography: 20 mM of phosphate buffer, pH 7.0, 0.2 M NaCl, and 1 mM of EDTA

3.

Radioimmunoassay

a.: Carrier free Na¹²⁵I (Radiochemical Centre, Amersham, England)
b.: Chloramine T (Sigma-Aldrich)

Instruments

1.: Columns for open-column chromatography (column I, 2.6 × 30 cm; column II, 1.0 × 20 cm; and column III, 1.0 × 10 cm)
2.: 0.22 μm membrane filter (Millipore, MA)
3.: Amicon Ultrafree-MC,10 kDa cut-off (Millipore, MA)
4.: Chromoscan 200, Joyce-Loebl densitometer (Joyce-Loebl, England)

Methods

1.

Purification of MBP from human serum

a.: Add an equal volume of 2× loading buffer to human serum (batch of 500 mL).
b.: Wash a Sepharose 4B-mannan column (column I, gel volume: 100 mL) with three column volumes of elution buffer.
c.: Equilibrate with one column volume of loading buffer.
d.: Apply human serum prepared in step (a) to the column I.
e.: Wash the column with loading buffer until the absorbance at 280 nm becomes less than 0.05 cm⁻¹ (5 times the column volume) at a flow rate of 50 mL/h (Note 1).
f.: Elute the protein bound to the column with approximately four column volumes of elution buffer at a flow rate of 15 mL/h.
g.: Add 1 M CaCl₂ to the eluate to a final concentration of 20 mM of CaCl₂.
h.: Wash a smaller second column (column II, gel volume: 15 mL) with three column volumes of elution buffer.
i.: Equilibrate with one column volume of loading buffer.
j.: Apply eluate prepared in step (g) to the column II.
k.: Wash the column with loading buffer.
l.: Elute the protein bound to the column with elution buffer.
m.: Add 1 M CaCl₂ to the eluate to a final concentration of 20 mM of CaCl₂.
n.: Wash a smaller third column (column III, gel volume: 5 mL) with three column volumes of elution buffer.
o.: Equilibrate with one column volume of loading buffer.
p.: Apply eluate prepared in step (m) to the column III.
q.: Wash the column with loading buffer.
r.: Elute the bound proteins with three column volumes of elution buffer.
s.: Concentrate eluate prepared in step (r) and dialyze against gel filtration medium.
t.: Apply nondiffusible material to Sepharose CL-6B column.
u.: Eluate the column with gel filtration medium (Note 2).
v.: Protein determination using the method of Lowry et al. (12) with a minor modification to prevent interference by Triton X-100 (13) and MBP binding activity by enzyme-linked immunosorbent assay (ELISA) method described separately (refer to the chapter “Assay method for the lectin activity of mannan-binding protein”) or radioimmunoassay described below for each tube (Figure 1).
w.: Subject an aliquot of the fractions to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (10% or 12.5% acrylamide gel) under reducing conditions and pool the fractions comprising the single 31 kDa band as purified MBP (Figure 2).

2.

Purification of MBP from human plasma

a.: Prepare Cohn’s alcohol fractionation (14) of plasma proteins, Fr. II and Fr. III. The fraction can be maintained as a frozen cake at −80°C.
b.: Suspend the 120 g of thawed cake with 1 L of distilled water by stirring for 1–2 h at 4°C.
c.: Adjust the pH of the suspension to 5.25 with 0.5 M HCl.
d.: Extract the MBP-containing fraction derived from the suspension by vigorous stirring for 1 h at 4°C.
e.: Centrifuge the mixture at 18,000 ×g for 30 min at 4°C and discard the precipitate.
f.: Add NaCl to final concentration of 0.15 M.
g.: Adjust the pH of the supernatant to 7.0 with 1 M NaOH or 1M HCl.
h.: Add polyethylene glycol 4,000 to final concentrations of 4% (w/v).
i.: Stir the suspension vigorously at 4°C for 1 h, and place it for 40 h at 4°C.
j.: Centrifuge the suspension at 18,000 ×g for 30 min at 4°C and discard the precipitated residue.
k.: To the clear supernatant (ca. 1.1 L), add NaCl, 1 M CaCl₂, and 1 M imidazole buffer (pH 7.8) to final concentrations of 1 M, 20 mM, and 10 mM, respectively.
l.: Filter the solution through a 0.22 μm membrane filter.
m.: Apply the filtrate to a mannan column as described in Method 1 step1 (a) followed by Method 1 steps 1 (b)–(r).
n.: Concentrate the pooled eluate fraction, and change the buffer to heparin column loading buffer by centrifugation in 10 kDa cut-off Amicon Ultra, or briefly dialyze the concentrated fraction against heparin column loading buffer (Note 3).
o.: Apply the mannan afﬁnity-puriﬁed protein dissolved in the heparin column loading buffer to a heparin-agarose column (gel volume: 10 mL).
p.: Wash each column with 50 mL of heparin column loading buffer and heparin column washing buffer.
q.: Elute the bound proteins with heparin column elution buffer (Note 2).
r.: Concentrate the eluate with a centrifugal ﬁlter device and store as purified MBP (Figure 3) (Note 4).
s.: Add NaCl to a final concentration of 0.5–1.0 M to the eluate from the heparin column to keep the MBP stable (Note 5).

3.

Radioimmunoassay

a.: Radioimmunoassay of MBP is performed using ¹²⁵I-mannan according to the method as described for hepatic asialoglycoprotein receptor using ¹²⁵I-asialo-orosomucoid (refer to the chapter “Isolation and determination of hepatic asialoglycoprotein receptor”).
b.: Mannan (100–300 µg) is iodinated with 1–2 mCi of carrier free Na¹²⁵I using chloramine T (Sigma-Aldrich).

Notes

1.

Immunoglobulins (mainly IgG), which may be natural antibodies against glycans, also bind to a mannan-Sepharose 4B column. The affinity of this type of immunoglobulin to a mannan column is lower than that of MBP. Thus, an amount of immunoglobulins contaminating the MBP in the eluate from the 1st mannan affinity column decreases with washing of the column with an excess amount of loading buffer (5 column volumes of loading buffer for step [f]). The immunoglobulins are eluted in the retarded fractions, whereas MBP is recovered in the eluted fraction from a mannan column. Check the eluted fractions in step [s] by SDS–PAGE under reducing conditions and pool the fractions comprising the 31 kDa band.

2.

Trace amounts of serine proteases, termed MASPs (MBP-associated serine proteases), which are involved in the complement activation via the lectin pathway, sometimes coexist in the MBP preparations obtained using these methods. When it is necessary to remove these esterase activities, the method described in Matsushita M. & Fujita T. 1992 may be applied to obtain an MBP preparation free of MASPs (15).

3.

Heparin-agarose is used as a common affinity column for various proteins through ionic interactions. MBP binds nucleic acid ligands (16) and might be captured by heparin-agarose.

4.

Through these procedures, ~1 mg of purified MBP is routinely obtained from 1 L of human serum with recovery of ~30%, with 15,000–20,000-fold purification.

5.

MBP storage condition

a.: MBP easily becomes aggregated and denatured with a low salt concentration of the heparin column loading buffer. Therefore, the dialysis or the buffer-exchange with membrane filtration of the MBP-containing solution after mannan column chromatography must be performed in a short time (within 1–2 h).
b.: The purified MBP dissolved with a high salt concentration of more than 0.5 M NaCl can be stored at 4°C for at least 6 months. Do not freeze the MBP solution. Note that MBP is easily inactivated at acidic pH below pH 6.

References

1.: Kawasaki T, Etoh R, Yamashina I. Isolation and characterization of mannan-binding protein from rabbit liver. Biochem Biophys Res Commun. 1978 Apr 14;81(3):1018–24. [PubMed: 666781] [CrossRef]
2.: Kozutsumi Y, Kawasaki T, Yamashina I. Isolation and characterization of a mannan-binding protein from rabbit serum. Biochem Biophys Res Commun. 1980 Jul 31;95(2):658–64. [PubMed: 6774714] [CrossRef]
3.: Kawasaki N, Kawasaki T, Yamashina I. Isolation and characterization of a mannan-binding protein from human serum. J Biochem. 1983 Sep;94(3):937–47. [PubMed: 6643429] [CrossRef]
4.: Kawasaki T. Structure and biology of mannan-binding protein, MBP, an important component of innate immunity. Biochim Biophys Acta. 1999 Dec 6;1473(1):186–95. [PubMed: 10580138] [CrossRef]
5.: Ikeda K, Sannof T, Kawasaki N, Kawasaki T, Yamashina I. Serum lectin with known structure activates complement through the classical pathway. J Biol Chem. 1987 Jun 5;262(16):7451–4. [PubMed: 3584121] [CrossRef]
6.: Lu J, Thiel S, Wiedemann H, Timpl R, Reid K. Binding of the pentamer/hexamer forms of mannan-binding protein to zymosan activates the proenzyme C1r2C1s2 complex, of the classical pathway of complement, without involvement of C1q. J Immunol. 1990 Mar 15;144(6):2287–94. [PubMed: 2313094]
7.: Yokota Y, Arai T, Kawasaki T. Oligomeric structures required for complement activation of serum mannan-binding proteins. J Biochem. 1995 Feb;117(2):414–9. [PubMed: 7608132] [CrossRef]
8.: Ma Y, Uemura K, Oka S, Kozutsumi Y, Kawasaki N, Kawasaki T. Antitumor activity of mannan-binding protein in vivo as revealed by a virus expression system: mannan-binding protein dependent cell-mediated cytotoxicity. Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):371–5. [PMC free article: PMC15143] [PubMed: 9892640] [CrossRef]
9.: Terada M, Khoo KH, Inoue R, Chen CI, Yamada K, Sakaguchi H, Kadowaki N, Ma BY, Oka S, Kawasaki T, Kawasaki N. Characterization of oligosaccharide ligands expressed on SW1116 cells recognized by mannan-binding protein. J Biol Chem. 2005 Mar 25;280(12):10897–913. [PubMed: 15634673] [CrossRef]
10.: Kawasaki N, Lin CW, Inoue R, Khoo KH, Kawasaki N, Ma BY, Oka S, Ishiguro M, Sawada T, Ishida H, Hashimoto T, Kawasaki T. Highly fucosylated N-glycan ligands for mannan-binding protein expressed specifically on CD26 (DPPVI) isolated from a human colorectal carcinoma cell line, SW1116. Glycobiology. 2009 Apr;19(4):437–50. [PubMed: 19129245] [CrossRef]
11.: Nonaka M, Imaeda H, Matsumoto S, Ma BY, Kawasaki N, Mekata E, Andoh A, Saito Y, Tani T, Fujiyama Y, Kawasaki T. Mannan-binding protein, a C-type serum lectin, recognizes primary colorectal carcinomas through tumor-associated Lewis glycans. J Immunol. 2014 Feb 1;192(3):1294–301. [PubMed: 24391218] [CrossRef]
12.: Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–75. [PubMed: 14907713]
13.: Hudgin RL, Pricer WE Jr, Ashwell G, Stockert RJ. The isolation and properties of a rabbit liver binding protein specific for asialoglycoproteins. J Biol Chem. 1974 Sep 10;249(17):5536–43. [PubMed: 4370480]
14.: Cohn EJ, Strong LE, Hughes WL, Mulford DJ, Ashworth JN, Melin M, Taylor HL. Preparation and properties of serum and plasma proteins; a system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids. J Am Chem Soc. 1946 Mar;68:459–75. [PubMed: 21015743] [CrossRef]
15.: Matsushita M, Fujita T. Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease. J Exp Med. 1992 Dec 1;176(6):1497–1502. [PMC free article: PMC2119445] [PubMed: 1460414] [CrossRef]
16.: Nakamura N, Nonaka M, Ma BY, Matsumoto S, Kawasaki N, Asano S, Kawasaki T. Characterization of the interaction between serum mannan-binding protein and nucleic acid ligands. J Leukoc Biol. 2009;86:737–748. [PubMed: 19465640] [CrossRef]
17.: Weber K, Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–12. [PubMed: 5806584]

Footnotes

: The authors declare no competing or financial interests.

Figures

Figure 1:

Chromatography of partially purified mannan-binding protein (MBP) from human serum on a Sepharose CL-6B column. (This figure is transferred from Reference no 3). The eluate from the third affinity chromatography on a Sepharose 4B-mannan column is applied to a column of Sepharose CL-6B. Portions of each fraction are used for the determination of protein (●) and binding activity (○) based on which the specific activity (▲) was calculated. Two pooled fractions are collected as indicated by bars. The arrows denote the elution positions of marker proteins (molecular weights given in parentheses): 1, blue dextran; 2, IgM (900,000); 3, β-galactosidase (520,000); 4, ferritin (443,000); 5, aldolase (160,000); 6, bovine serum albumin (67,000); 7, ovalbumin (43,500); 8, cytochrome c (11,700); and 9, phenol red.

Figure 2:

Polyacrylamide gel electrophoresis (PAGE) of the purified binding protein from human serum. (This figure is transferred from Reference no. 3). The purified binding protein (G-I, 10 µg) in gel filtration medium is analyzed by electrophoresis on SDS/10% polyacrylarnide gels according to the procedure of Weber and Osborn (17). Protein bands are stained with Coomassie brilliant blue G250. Scanning of the gels was performed using a Joyce-Loebl densitometer, Chromoscan 200, at 575 nm. Arrows denote the migration of the marker dye. The numbers at the right indicate the migration positions corresponding to molecular weights (× 10³) of standard proteins. The purified MBP preparation gives a single band corresponding to 31 kDa (subunit size) under reducing conditions (~92% purity) and some more than 100 kDa bands (3–6 oligomers of the structural unit) (3 × subunits), the whole molecular mass being approximately 300–600 kDa under non-reducing conditions on SDS–PAGE.

$Figure 3: . Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of MBP-containing fractions in the purification steps from human plasma.$

Figure 3:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of MBP-containing fractions in the purification steps from human plasma. Lane 1, before application to a mannan column; lane 2, the eluate from mannan column; lanes 3–5, the eluate from heparin column. Lanes 2–4, under reducing conditions; lanes 1 and 5, under nonreducing conditions. Lanes 3 and 4 are the same sample with different concentrations. SDS–PAGE was performed on a 12.5% acrylamide gel. The proteins are detected using colloidal Coomassie brilliant blue G-250 staining.

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Bookshelf ID: NBK593883PMID: 37590627

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