Glycosylation of ovine prolactin during cell-free biosynthesis

Recently, a glycosylated form of ovine PRL (oPRL) was isolated from a crude pituitary preparation. As glycosylation of PRL was unexpected and because the composition of the oligosaccharide-containing peptide indicated the carbohydrate portion to be extensively degraded, studies of the glycosylation of oPRL during cell-free biosynthesis were initiated. Two glycosylated forms of oPRL can be recognized when biosynthesis occurs in ovine pituitary microsomes. Both forms are converted to mature PRL by digestion with endoglycosidase H and, thus, appear to contain only asparagine-linked, high mannose-type carbohydrate moieties. In contrast, immunoprecipitates from bovine pituitary microsomes consist of the expected (and nonglycosylated) pre-PRL and PRL. The results are consistent with the absence of a sequence segment in the bovine hormone which permits glycosylation (Asn-X-Ser- or Thr-) and the presence of the segment Asn31-Leu-Ser- in the ovine hormone. The occurrence of two glycosylated forms of oPRL is not understood; it may result from an additional site in oPRL capable of glycosylation.     

Critical function for the Ras-GTPase activating protein RASA3 in vertebrate erythropoiesis and megakaryopoiesis

Phenotype-driven approaches to gene discovery using inbred mice have been instrumental in identifying genetic determinants of inherited blood dyscrasias. The recessive mutant scat (severe combined anemia and thrombocytopenia) alternates between crisis and remission episodes, indicating an aberrant regulatory feedback mechanism common to erythrocyte and platelet formation. Here, we identify a missense mutation (G125V) in the scat Rasa3 gene, encoding a Ras GTPase activating protein (RasGAP), and elucidate the mechanism producing crisis episodes. The mutation causes mislocalization of RASA3 to the cytosol in scat red cells where it is inactive, leading to increased GTP-bound Ras. Erythropoiesis is severely blocked in scat crisis mice, and ~94% succumb during the second crisis (~30 d of age) from catastrophic hematopoietic failure in the spleen and bone marrow. Megakaryopoiesis is also defective during crisis. Notably, the scat phenotype is recapitulated in zebrafish when rasa3 is silenced. These results highlight a critical, conserved, and nonredundant role for RASA3 in vertebrate hematopoiesis.