Expression and binding assay of endoplasmic reticulum-Golgi intermediate compartment protein 53 (ERGIC-53, LMAN1)

Solubilization buffer: 50 mM of Tris-HCl, pH 8.0, containing 6 M guanidine, 1 mM of DTT, and 0.1 mM of ethylenediaminetetraacetic acid (EDTA)

Refolding buffer: 100 mM Tris-HCl, pH 7.5, containing 0.4 M L-arginine, 5 mM of reduced glutathione, 0.5 mM of oxidized glutathione, and 0.5 mM of phenylmethanesulfonyl fluoride

Dialysis buffer: 20 mM of Tris-HCl, pH 7.5, containing 25 mM of NaCl and 0.1 mM of EDTA

Isopropyl β-thiogalactopyranoside

Biotin ligase, BirA

R-phycoerythrin (PE)-labeled streptavidin

Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA)

HBS: 20 mM of HEPES-NaOH, pH 7.4, containing 150 mM of NaCl and 1 mM of EDTA

Propidium iodide (PI)

FACS Calibur (BD Biosciences, San Jose, CA)

CellQuest software (BD Biosciences)

Expression and preparation of soluble ERGIC-53 tetramer

a.

Construct plasmid encoding a soluble lectin domain of ERGIC-53 with an enzymatic biotinylation sequence (4) (Note 1).

b.

Transform E. coli cell with plasmid and induce expression by adding isopropyl β-thiogalactopyranoside.

c.

Recover expressed lectin domain as soluble proteins or inclusion bodies.

d.

Solubilize recovered inclusion bodies in solubilization buffer, diluted with refolding buffer to a protein concentration of 6 mM, and refolded in vitro by dialysis against dialysis buffer at 4°C for 24 h.

e.

After removal of insoluble material by centrifugation, apply the soluble fraction to anion-exchange chromatography and gel chromatography.

f.

Add biotin ligase BirA for biotinylation of soluble ERGIC-53 lectin domain.

g.

Mix soluble ERGIC-53 lectin domain with PE-labeled streptavidin to make PE-labeled soluble ERGIC-53 tetramer (Note 2).

Binding assay of soluble ERGIC-53 tetramer to cells by flow cytometry

a.

Harvest cultured mammalian cells and suspend in HBS at a concentration of 2 × 10⁷ cells/mL (Note 3).

b.

Add PE-labeled soluble ERGIC-53 tetramer at a concentration of 10–100 μg/mL to 10 μL of the cell suspension in a 96-well plate (Note 4).

c.

Allow it to stand at 25°C for 30 min.

d.

Wash the cells twice with HBS.

e.

Suspend in 200 μL of HBS containing 1 μg/mL PI.

f.

Measure the fluorescence intensity of PE-labeled ERGIC-53 tetramer at 575 nm by flow cytometry.

Other biochemical analysis of ERGIC-53 in mammalian cells

a.

Construct plasmid encoding ERGIC-53 with FLAG-tag at N-terminus (Note 5).

b.

Introduce plasmid into mammalian cells by lipofectamine 2000 according to manufacturer’s protocol.

c.

Culture transformed cells at 37°C for 24–48 h.

d.

Precipitate or stain ERGIC-53 using anti-FLAG antibody under appropriate conditions.

Enzymatic biotinylation sequence is as follows: GGGLNDIFEAQKIEWHE (4). Side chain of lysine is enzymatically biotinylated.

Partial purification of ERGIC-53 from cell lysates had been reported using mannose-immobilized beads (5). However, several mannose-binding lectins associated with quality control of glycoproteins are also present in the ER and Golgi apparatus. The interaction between these lectins and mannose is quite weak; thus, it is difficult to purify these proteins using conventional affinity chromatography methods.

To modify cell surface glycans, cultured cells are treated with castanospermine, deoxynojirimycin, deoxymannojirimycin, kifunensine, or swainsonine at concentrations of 1 mM, 1 mM, 2 μg/mL, or 10 μg/mL, respectively. Several lectin-resistant Chinese hamster ovary cell variants, such as Lec1, Lec2, or Lec8 with different N-glycan expressions are also available.

Calcium ions are required for sugar-binding of ERGIC-53, and EDTA inhibits its activity. Usually, 1 mM of calcium chloride is added to the buffer.

Because cytoplasmic tail of ERGIC-53 contains anterograde and retrograde transport signals in cells, the modification of C-terminus with tagged sequence may cause abnormal localization of the proteins.