Binding of human secretory leukocyte protease inhibitor in uterine cervical mucus to immunoglobulins: pathophysiology in immunologic infertility and local.

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Binding of human secretory leukocyte protease inhibitor in uterine cervical mucus to immunoglobulins: pathophysiology in immunologic infertility and local immune defense Masashi Hirano, M.D., Masaharu Kamada, M.D., Ph.D., Masahiko Maegawa, Ph.D., Hironori Gima, Ph.D., Toshihiro Aono, Ph.D. Fertility and Sterility Volume 71, Issue 6, Pages 1108-1114 (June 1999) DOI: 10.1016/S0015-0282(99)00142-9

FIGURE 1 Western blot analysis of human cervical mucus proteins fractionated by SDS-PAGE. Note that all three samples (lanes a–c) contain a 15-kd protein band that binds human IgG (biotinylated IgG [b-IgG]). Seminal plasma (lane d) contains a 16-kd protein designated Ig-binding factor that is absent from cervical mucus. Biotinylated alkaline phosphatase (b-AP) as a negative control did not bind with the 15-kd protein of the cervical mucus sample (lane a) or with the 16-kd protein of the seminal plasma (lane d). The molecular weight (MW) markers are 106, 80, 49.5, 32.5, 27.5, and 18.5 kd from top to bottom. Fertility and Sterility 1999 71, 1108-1114DOI: (10.1016/S0015-0282(99)00142-9)

FIGURE 2 Western blot analysis of partially purified proteins of human cervical mucus fractionated by SDS-PAGE. Note positive staining of the 15-kd protein with human IgG (lane a), IgM (lane b), IgA (lane c), IgG1 (lane d), IgG2 (lane e), IgG3 (lane f), and IgG4 (lane g). The molecular weight (MW) markers are 106, 80, 49.5, 32.5, 27.5, and 18.5 kd from top to bottom. Fertility and Sterility 1999 71, 1108-1114DOI: (10.1016/S0015-0282(99)00142-9)

FIGURE 3 Top: Amino acid sequence of the amino terminus of purified Ig-binding protein of human cervical mucus. The initial 21 amino acid residues are identical to those of SLPI. Bottom: SDS-PAGE (left) and Western blot analysis (right) of partially purified Ig-binding protein from human cervical mucus and SLPI. Note that the water-insoluble fraction (right, lane a) contains a protein that migrates as a 15-kd band that binds human IgG (left, lane a), whereas the water-soluble fraction lacks the 15-kd band (right, lane b). Recombinant SLPI migrates as a 15-kd band (right, lane c) and interacts with human IgG (left, lane c). The molecular (MW) markers are 208, 115, 79.5, 49.5, 34.8, 28.3, 20.4, and 7.2 kd from top to bottom. Fertility and Sterility 1999 71, 1108-1114DOI: (10.1016/S0015-0282(99)00142-9)

FIGURE 4 Interaction of recombinant SLPI with human IgG fragments determined by Western blot analysis. Note that SLPI binds the Fc (lane b) and Fab (lane c) fragments as well as IgG (lane a), but not bovine serum albumin (lane d). Fertility and Sterility 1999 71, 1108-1114DOI: (10.1016/S0015-0282(99)00142-9)

FIGURE 5 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (left) and Western blot analysis (right) of Ig-binding protein and recombinant SLPI under reducing and nonreducing conditions. Partially purified Ig-binding protein of cervical mucus (lane a) and two separately prepared samples of recombinant SLPI (lanes b and c) bind human IgG under reducing conditions. Under nonreducing conditions, both recombinant SLPIs migrate as broad bands and do not interact with human IgG (lanes d and e). The original cervical mucus sample (lanes f and g) contains a 15-kd band that binds human IgG under nonreducing (lane g) and reducing (lane f) conditions. The molecular weight (MW) markers are 106, 80, 49.5, 32.5, 27.5, and 18.5 kd from top to bottom. Fertility and Sterility 1999 71, 1108-1114DOI: (10.1016/S0015-0282(99)00142-9)

FIGURE 6 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (left) and Western blot analysis (right) of recombinant SLPI treated with varying concentrations of reduced glutathione. The concentration of reduced glutathione is 1 mM in lane a, 5 mM in lane b, and 10 mM in lane c. Note the positive staining on treatment with the 10-mM concentration (lane c). The molecular weight (MW) markers are 208, 115, 79.5, 49.5, 34.8, 28.3, 20.4, and 7.2 kd from top to bottom. Fertility and Sterility 1999 71, 1108-1114DOI: (10.1016/S0015-0282(99)00142-9)