Human Pregnancy Specific Beta-1-Glycoprotein 1 (PSG1) Has a Potential Role in Placental Vascular Morphogenesis

Ha, Cam T.; Wu, Julie A.; Irmak, Ster; Lisboa, Felipe A.; Dizon, Anne M.; Warren, James W.; Ergun, Suleyman; Dveksler, Gabriela S.

The availability of the crystal structure of the murine CEArelated cell adhesion molecule CEACAM1 has made it possible to model the structure of other members of the CEA family [41] . The N-terminal domain of most human PSGs, with the exception of PSG1, PSG4, and PSG8, contain an Arg-Gly-Asp (RGD) motif [18] . This motif is on the FG loop, which is solvent exposed and considered to be a biologically important element in CEACAM1. PSG1 has the sequence GDD rather than RGD, but the importance of this region for receptor binding and activity for all PSGs has been frequently cited [19, 20] . To investigate the possible importance of this region of the PSG1 protein, we mutated the G at position 93 and the D at position 95 to S and L, respectively. We selected the amino acids S and L based on their presence in the corresponding position in human CEACAM3. These changes are not expected to affect the folding of the protein while being substantially different from the amino acids present in wild-type PSG1. Like wild-type PSG1-Fc, the resulting protein has an approximate molecular mass of 70 kDa (Fig. 7A) . The PSG1GDD!SDL mutant was compared to the wild-type protein for its ability to induce TGFB1 in HTR-8/SVneo, monocytes, and HEEC. Our In C, HTR-8/SVneo cells were pre-treated with 10 lg/ml of anti-TGFB1 antibody or isotype-matched control antibody (IM) 1 h before addition of 50 lg/ml of PSG1-Fc or FLAG-Fc. After 24 h, VEGFA in the supernatants was analyzed by ELISA. All cells were treated in triplicate, and data are representative of at least three independent experiments. For A, B, D, and E, statistical significance was determined by one-way ANOVA, and the Pvalue between doses was less than 0.01. For C, statistical significance between the PSG1-Fc-treated and FLAG-Fc-treated samples was determined by two-tailed Student t-test (*P , 0.05). Error bars represent the SEM. results show that the PSG1GDD!SDL mutant induced TGFB1 over control protein (Fig. 7B) .