The secondary structural characteristics of one of the Robinia pseudoacacia lectins (RPA3) have been investigated by FTIR spectroscopy and have been established from absorption measurements in the amide I,I' frequency range and from the quantitative estimation of the rate of NH----N2H exchange. In an anhydrous state the protein structure consists mainly of antiparallel and parallel beta-structures, which represent 60% of the overall secondary structure of RPA3. Data obtained in different polar media (KBr, 2-chloroethanol, 2H2O, NaCl-2H2O and/or DPPC) reveal that RPA3 is a highly flexible protein. In pure 2H2O a rapid solvation of free peptide units and weak peripheral hydrogen bonds occurs, followed by the solvation of more internal parts of the lectin. The protein precipitates before total unfolding is reached. Increasing the ionic strength modifies the rate of NH----N2H exchange. NaCl concentrations of less than or equal to 0.15 M stabilize RPA3 in a structure close to that of the lyophilized lectin and diminish the rate of exchange, whereas higher NaCl concentrations partially disrupt the original secondary structure and increase the rate of exchange. Furthermore RPA3 was shown to interact with DPPC through polar interactions between the polar heads of the phospholipid and specific peptide units. These interactions appear to favor the NH----N2H exchange.

We have examined the passage of maternal cells into the fetus during the gestation and postpartum in mice. Using enhanced green fluorescent protein (EGFP)-transgenic females, we showed that maternal cells frequently gain access to the fetus, mostly in syngeneic pregnancies, but also in allogeneic and outbred crosses. EGFP-transgenic cells, including B, T, and natural killer cells, can persist until adulthood, primarily in bone marrow and thymus. We then asked whether maternal cells, bearing antigens not inherited by the fetus, influence the development of fetal and neonatal B lymphocytes. We have used the B cell receptor 3-83 mu/delta transgenic mouse model, whose B cells recognize the major histocompatibility complex class I molecules H-2Kk and H-2Kb, with a high or moderate affinity, respectively. The fate of transgenic B cells in animals exposed to noninherited H-2Kk or H-2Kb maternal antigens (NIMA) during gestation and lactation was compared with those of nonexposed controls. In H-2Kk-exposed fetuses, NIMA-specific transgenic B cells are partially deleted during late gestation. Nondeleted cells have downmodulated their B cell receptor. In contrast, in NIMA H-2Kb-exposed neonates, transgenic B cells present an activated phenotype, including proliferation, upregulation of surface CD69, and preferential localization in the T cell zone of splenic follicles. This state of activation is still clearly detectable up to 3 wk of age. Thus, we show that fetal and neonatal B cell development is affected by maternal cells bearing antigens noninherited by the fetus and that this phenomenon is highly dependent on the affinity of the B cell receptor for the NIMA.

We have produced transgenic mice using the mouse placental lactogen type II promoter to force and restrict the expression of the mouse major histocompatibility complex (MHC) class I molecule, H-2K(b), to the placenta. We show that the transgenic MHC antigen H-2K(b) is expressed exclusively in trophoblast giant cells from Day 10.5 until the end of gestation. This expression affects neither the fetal development nor the maternal tolerance to the fetus in histoincompatible mothers. We have used the 3.83 B cell receptor (BcR) transgenic mouse line to follow the fate of H-2K(b)-specific maternal B cells in mothers bearing H-2K(b)-positive placentas. Our results suggest that transgenic H-2K(b) molecules on trophoblast giant cells are recognized by 3.83 BcR-transgenic B cells in the bone marrow of pregnant females. This antigen recognition triggers the deletion of a bone marrow B cell subpopulation, including immature and transitional B cells. Their percentage decreases during the second half of gestation and is down to 8% on Day 17.5, compared to 22% in the (3.83 Tg female x Fvb) control group. This deletion might contribute to the process of maternal tolerance of the conceptus.

Two lectins, RPA 1 and RPA 3, were purified from Robinia pseudoacacia seeds. These two lectins differ in their physicochemical and biological properties. By analytical ultracentrifugation the Mr values of RPA 1 and RPA 3 were estimated to be 59,000 and 105,000 respectively. From SDS/polyacrylamide-gel-electrophoresis data it was estimated that RPA 1 consisted of two subunits of Mr 34,000, and RPA 3 of two types of subunits (Mr 30,500 and 29,000). RPA 1 and RPA 3 were found to be glycoproteins of comparable amino acid composition. RPA 1 was the more highly glycosylated molecule (11.6% versus 4.3%). The carbohydrate-specificity of RPA 1 appears to be complex. RPA 3 was inhibited by N-acetyl-D-galactosamine and human alpha-glycoproteins. Both lectins exerted a mitogenic effect on human peripheral-blood lymphocytes. Concentrations between 0.5 and 1 microgram of RPA 3/ml gave optimal proliferative responses, whereas for RPA 1 concentrations higher than 10 micrograms/ml were needed for these responses.