Natural resistance to intracellular infections: Nramp1 encodes a membrane phosphoglycoprotein absent in macrophages from susceptible (Nramp1 D169) mouse strains.

摘要: The mouse Nramp1 gene (Bcg/Ity/Lsh) controls innate defense to infection with intracellular parasites such as Mycobacterium, Salmonella, and Leishmania. Sequence analysis of predicts a hydrophobic, membrane-associated protein expressed exclusively in monocyte/macrophage lineages. A single G169D substitution within the fourth predicted transmembrane domain is associated susceptibility (Bcg) inbred strains. To initiate biochemical characterization analyze molecular basis Nramp1(D169) allele, oligopeptides derived from two fusion proteins containing first 54 last 35 residues were used raise specific anti-Nramp1 antisera. In addition, c-Myc epitope (EQKLISEEDL) was introduced in-frame at C terminus follow its expression yeast heterologous system. Western crude membrane fractions demonstrated that indeed an integral (resistant urea extraction). resident Bcg(r) (129sv, Nramp1(G169)) macrophages, one antisera identified by immunoprecipitation 90,000 100,000 apparent m.w. absent macrophages knockout mice bearing null mutation Nramp1. could be metabolically labeled orthophosphate sensitive glycosylase treatment, verifying phosphoglycoprotein. Parallel macrophage extracts Bcg(s) strains (C57BL6/J BALB/c, Nramp1(D169)) failed detect mature Nrampl these cells, suggesting prevents proper maturation or integration protein, resulting rapid degradation.