β-Galactosidase Treatment Is a Common First-stage Modification of the Three Major Subtypes of Gc Protein to GcMAF

Materials and Methods

Materials. Lipopolysaccharide (LPS; from Escherichia coli O128:B12) and Sepharose CL-6B (cross-linked) were purchased from Sigma-Aldrich Japan Co. Ltd. (Tokyo, Japan). 25-Hydroxyvitamin D₃ [25(OH)D₃] and other biochemical-grade chemicals were purchased from WAKO Pure Chemical Industries Co. Ltd. (Osaka, Japan).

Preparation of human serum Gc_1s1s and Gc₂₂ protein. The Gc_1s1s and Gc₂₂ proteins were prepared as reported by Link et al. (21). Briefly, serum from healthy humans containing Gc_1s1s or Gc₂₂ proteins (each 7.0 ml) was diluted 1:1 with column buffer (50 mM Tris-HCl, 1.5 mM EDTA, 150 mM NaCl, 0.1% Triton X-100; pH 7.4) and applied to a 25(OH)D₃-Sepharose column which was prepared in our laboratory according to the method reported by Link et al. (21). The column was washed with 300 ml of column buffer. The protein remaining on the matrix was eluted with 6 M guanidine-HCl, and a 1-ml fraction was collected. Fractions of the protein peak encompassing the guanidine-eluted fraction were pooled and dialyzed with 10 mM sodium phosphate (pH 7.0). Subsequently, a hydroxyapatite column with a volume of 5 ml (Bio-Scale Mini CHT Type II Cartridge; Bio-Rad Laboratories, Tokyo, Japan) was equilibrated with 10 mM sodium phosphate (pH 7.0). The dialyzed sample obtained following 25(OH)D₃-Sepharose column chromatography was applied to the column. A linear gradient elution from 10 mM sodium phosphate to 200 mM sodium phosphate (pH 7.0) was used for column chromatography. Fractions were collected and concentrated with a centricon-concentrating unit (30,000 MWCO; Nihon Millipore Co. Ltd., Tokyo, Japan), and their protein concentrations were determined using the bicinchoninic acid (BCA) method (Pierce Chemical Co., Rockford, IL, USA). Bovine serum albumin (BSA) was used as a standard. The yields of purified Gc_1s1s and Gc₂₂ proteins were 2.294 mg and 1.867 mg, respectively.

Preparation of preGc_1s1sMAF and preGc₂₂MAF. The purified Gc_1s1s and Gc₂₂ proteins (200 μg) were incubated with 1000 mU β-galactosidase (grade III from bovine liver; Sigma-Aldrich Japan Co. Ltd., Osaka, Japan) in 100 mM sodium phosphate buffer (pH 7.0) in an Eppendorf tube at 37.5°C for 3 h. The reaction mixture was mixed with 25(OH)D₃-Sepharose beads and was stirred for 10 min at room temperature using a vortex mixer to separate the β-galactosidase. The solution was concentrated using a microcon-concentration unit (10,000 MWCO; Nihon Millipore Co. Ltd., Tokyo, Japan). The protein concentrations were determined using the BCA method, and the yields of preGc_1s1sMAF and preGc₂₂MAF were calculated as being 80.2 μg and 57.8 μg, respectively.

Western blotting. The Gc_1s1s and Gc₂₂ proteins and the preGc_1s1sMAF and preGc₂₂MAF subtypes were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and subsequently electroblotted onto a polyvinylidene fluoride (PVDF) membrane. Non-specific binding was blocked by overnight incubation in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 and 1% BSA at 4°C. The membranes were then probed with anti-human Gc globulin (DakoCytomation Co. Ltd., Kyoto, Japan) against the Gc_1s1s and Gc₂₂ proteins, and with Peanut Agglutinin (PNA) lectin (Arachis hypogaea, biotin-conjugated; Sigma-Aldrich Japan Co. Ltd., Osaka, Japan) and Helix pomatia (HPA) lectin (Helix pomatia, biotin-conjugated; Sigma-Aldrich Japan Co. Ltd., Osaka, Japan) for the Gc_1s1s and Gc₂₂ protein subtypes preGc_1s1sMAF and preGc₂₂MAF. After extensive washing, the blots were incubated with the secondary antibody [horseradish peroxidase (HRP)-labeled anti-rabbit IgG and electrochemiluminescence (ECL) streptavidin-HRP conjugate; GE Healthcare Japan Co., Tokyo, Japan]. The blots were developed using an ECL western blotting detection system (GE Healthcare Japan Co.). The visualization and quantification of the western blot bands was achieved using a LumiCube chemiluminescence analyzer and the JustTLC image analysis software (Liponics Inc., Tokyo, Japan).

Isolation and culture of mouse peritoneal macrophages. Resident mouse peritoneal macrophages were collected from 8-week-old female mice from the Institute for Cancer Research (ICR). After centrifugation at 1,000 rpm, at 4°C, for 15 min, macrophages were collected and cultured in 24-well plates at a density of 5×10⁵ cells/well in serum-free RPMI-1640 (Life Technologies Japan Ltd., Tokyo, Japan) for 1 h. The cultured cells were then washed three times with serum-free RPMI-1640 to separate adherent macrophages from non-adherent cells, such as T- and B-cells. The macrophages were cultured for 15 h, following which, the phagocytosis assay was performed as described below.

Phagocytosis assay for preGc_1s1sMAF and preGc₂₂MAF. Mouse peritoneal cells were layered onto coverslips in a 24-well plate. After 3 h of treatment (treated preGc_1s1sMAF and preGc₂₂MAF were pre-incubated with peritoneal fluid at 37°C for 1 h), the cultures were assayed for phagocytic activity. Sheep red blood cells (SRBCs; Nippon Bio-Supp. Center Co., Tokyo, Japan) were opsonized by rabbit hemolytic serum (anti-sheep red blood cells; Cosmo Bio Co., Tokyo, Japan). Opsonized SRBCs (0.5%) in serum-free RPMI-1640 were overlaid onto each macrophage-coated coverslip and were cultured for 1.5 h. The non-internalized erythrocytes were lysed by immersing the coverslip into a hypotonic solution (1/5 phosphate-buffered saline). The macrophages were fixed with methanol, air-dried, and stained with Giemsa stain. The number of phagocytosed erythrocytes per cell was determined microscopically; 250 macrophages were counted for each data point. The data are expressed in terms of the phagocytotic index (PI), which is defined as the percentage of macrophages with ingested erythrocytes multiplied by the average number of erythrocytes ingested per macrophage.

Statistical analysis. Data are expressed as the mean ±SD. The significance of the differences between the results of the independent experiments was analyzed using the Student's t-test. A p-value of <0.05 was considered significant.