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Isolation and detection of human lactoferrin binding protein in human amniotic membrane
Otsuki, K; Yoda, A; Mitsuhashi, Y; Shimizu, Y; Saito, H; Yanaihara, T; Makino, Y
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It is known that lactoferrin (LF) has many biological functions. From our previous reports about intra-uterine infections, the concentrations of amniotic LF and interleukin-6 (IL-6) with intra-uterine infection are higher than those without infection. LF inhibits the production of IL-6 induced by lipopolysaccharides in cultured amniotic cells. Although various studies were reported about the binding proteins for LF in regard to the iron absorption and protection from infection in various tissues, no information is available about human LF binding proteins in human reproductive tissues.
In the present study, an attempt was made to isolate the binding protein for human LF amniotic membrane using affinity column chromatography. Amniotic membranes were obtained at the time of the elective term Caesarean section. The membranes were minced and homogenized in ice cold 10mM potassium-phosphate buffer (pH 7.5) containing 0.15M NaCl, 5mM PMSF, 0.02% NaN3 and 2% Triton X-100 with polytron homogenizer for 10 min. Supernatant was taken after centrifugation at 43,000 x g for 90 min. For the preparation of affinity column chromatography, iron saturated human LF (Sigma, Co., St. Louis, MO) was stirred gently with CNBr-activated Sepharose 4B column (Pharmacia Biotech, Tokyo, Japan) at 4 degrees C over night, and following incubation was performed with the supernatant for 2 h. After the LF immobilized gel was packed in the column, LF binding protein was eluted with 0.2 M NaCOOH3 buffer (pH 3.7) containing 0.15 M NaCl and 0.1% CHAPS. The elute was immediately adjusted to pH 7.0 with NaOH.
The binding activity of LF binding protein was determined by using ELISA. The saturation curve of LF binding to LF binding proteins was almost linear. These results show, that the binding of human LF to LF binding protein in amniotic membrane has specific binding to human LE The existence of LF binding protein was firstly investigated in amniotic membrane. With our previous studies, we suggest that localization of LF binding protein in amniotic membrane may have a key mechanism for intra-uterine infection.
amnion; amniotic membrane; binding protein; lactoferrin; receptor
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