Recombinant sugar binding proteins and their functional analysis: Siglec-7

Authors：

Category

Sugar binding proteins

Protocol Name

Authors

Yamaji, Toshiyuki *
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases

Hashimoto, Yasuhiro
Department of Biochemistry, School of Medicine, Fukushima Medical University

*To whom correspondence should be addressed.

KeyWords

Siglec-7 Sialic acid Recombinant protein Flow cytometry

Reagents

●	Expression vectors that contain Siglec-7 (wild type)-Fc and Siglec-7 (R124K mutant)-Fc.: Expression plasmids, pCXN₂-Siglec-7-Fc and pCXN₂-R124K mutant-Fc, which lose binding ability to sialic acids, were prepared previously (Miyazaki K et al. (2004) Cancer Res., 64(13): 4498–4505). The human Fc used was derived from pEF-Fc vector constructed by Dr. Yoshihara (RIKEN BSI, Wako, Japan).
●	CHO cells
●	Ham’s F-10 or F-12 10% fetal calf serum (FCS)
●	Geneticin (G418): for selection
●	HRP- and fluorescence (Alexa 488 etc.) -conjugated anti-human IgG: Alexa Fluor 488-labeled goat anti-human IgG (Invitrogen/Life Technologies, Inc., Carlsbad, CA)
●	ASF104 non-serum medium (Ajinomoto Healthy Supply, Inc., Tokyo, Japan)
●	Sodium butylate
●	Protein G Sepharose 4 Fast Flow (GE Healthcare, Little Chalfont, UK)
●	Disposable polypropylene column
●	Millex-HV 0.45um-pore filter unit (Merck Millipore, Billerica, MA)
●	Phosphate-buffered saline (PBS)
●	0.1M Glycine-HCl, pH2.8
●	1M Tris-HCl, pH8.0
●	Centricon (Merck Millipore): Ultracel YM-30 etc.
●	Bovine serum albumin (BSA)

Instruments

●	Rotating mixer
●	Fluorescence flow cytometer

Methods

Preparation of recombinant Siglec-7-Fc and its mutant (R124K)-Fc fusion proteins (see Note 1)

1)	Transfect plasmids containing Siglec-7-Fc and R124K mutant-Fc cDNAs into CHO cells and culture for 2 days.	Comment 0

2)	Dilute the transfected cells into 96-well culture plates and add 800 μg/mL G418 to prepare stable transfectants.	Comment 0

3)	Perform dot plot analysis to select the clone that secretes the recombinant proteins at the highest level (use anti-Siglec-7 or HRP-anti human IgG).	Comment 0

4)	Adapt the cells in ASF104 non-serum medium.	Comment 0

5)	Culture the sub-confluent cells in ASF medium with 5 mM sodium butylate for 1 week.	Comment 0

6)	Collect the medium and filter with a 0.45 μm-pore filter unit to remove debris.	Comment 0

7)	Add Protein G-Sepharose beads to the filtrate and rotate overnight at 4˚C.	Comment 0

8)	Pack the beads in an empty column and wash with at least 5 column volumes of cold PBS.	Comment 0

9)	Elute the bound proteins with cold 0.1 M Glycine-HCl, pH 2.8, and immediately neutralize with 1/10 volume of 1M Tris-HCl, pH 8.0.	Comment 0

10)

Replace the buffer with PBS or an appropriate buffer by Centricon.

Comment 0

Binding of recombinant Siglec-7-Fc to the cell surface (see Note 2)

1)	Wash cells with 1% BSA/PBS once by centrifugation (2000 rpm for 5 min at 4˚C).	Comment 0

2)	Incubate the cells with Siglec-7-Fc or R124K mutant-Fc (4–10 μg/mL in 1%BSA/PBS) for 40–60 min on ice.	Comment 1

3)	Centrifuge and wash with 1% BSA/PBS once.	Comment 0

4)	Incubate the cells with fluorescent (Alexa-488 etc.) anti-human IgG (4–10 μg/mL in 1%BSA/PBS) for 40–60 min on ice.	Comment 0

5)	Wash with 1% BSA/PBS once and re-suspend the cells in the buffer.	Comment 0

Analyze with a fluorescence flow cytometer.

Comment 0

Notes

About 1mg recombinant Siglec-7-Fc proteins was prepared from 150 mL culture supernatant.
As another method, the preincubated complex of recombinant Siglec-7-Fc and anti-human IgG can also be used for cell staining (Miyazaki K et al. (2004) Cancer Res., 64(13): 4498–4505). The binding amount of Siglec-7-Fc may vary with the staining protocol because the binding of Siglec-7 is affected by its multi-valency. Note that the binding of recombinant Siglec-7-Fc to the cell surface does not mean that the cells express functional Siglec-7 ligands, because the sugar-binding site of Siglec-7 on the cell surface is usually masked by endogenous sialoconjugates; therefore, as a next step, cell-cell interactions through Siglec-7 and ligand candidates should be examined.

Discussion

Interpretation

Recombinant Siglec-7-Fc proteins bound to KST cells more than KV cells, suggesting that disialo-epitopes on O-glycans are more preferable ligand candidates for Siglec-7. These bindings are sialic-acid dependent because R124K mutant-Fc bound less to both cells.

Figure & Legends

Fig. 1. Preferential binding of Siglec-7-Fc to disialo-glycans on the cell surface of K562 transfectants.

K562 transfectants that express disialo-epitopes on O-glycans by introducing ST8SiaVI sialyltransferase (named KST cells) and mock cells (KV cells) were stained with Siglec-7-Fc and R124K mutant-Fc as described above. Fluorescent intensity was measured by flow cytometry.

Copyrights

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Date of registration：2014-11-06 14:15:20