Journal: Proceedings of the National Academy of Sciences of the United States of America
Article Title: Cloning and characterization of the mammalian brain-specific, Mg2+-dependent neutral sphingomyelinase
Figure Lengend Snippet: Sequence analysis of nSMase2. ( A ) Neighbor-joining dendrogram for representative members of the phosphohydrolase superfamily. All bifurcations are supported by more than 400 of 1,000 bootstrap samples, except for the one labeled “?”. The letters on the right represent the subfamilies, as described in Discussion . Species abbreviations are as follows: HS, human; MM, mouse; SC, Saccharomyces cerevisiae ; SA , Staphylococcus aureus ; BC, Bacillus cereus ; LI, Leptospira interrogans ; XL, Xenopus laevis ; DM, Drosophila melanogaster ; AH, Aeromonas hydrophila ; SS, Synechocystis sp.; MP , Mycoplasma pulmonis ; BS, Bacillus subtilis ; DD, Dictyostelium discoideum ; EC, Escherichia coli ; BM, Bombyx mori ; TB, Trypanosoma brucei ; HD, Haemophilus ducreyi ; and CJ, Campylobacter jejuni . In cases where no systematic gene nomenclature is available, the following abbreviations were used: SC SMase, yeast sphingomyelinase Yer019w; HLb, β-hemolysin; RRP1, recombination repair protein 1; Exo3, exonuclease III; LINE-1, LINE-1 reverse transcriptase; TX1, transposon TX1 ORF2; RTAmy, reverse transcriptase of Amy transposon; NucH, extracellular nuclease H; MNU, membrane nuclease A; CtdB, cytolethal distending toxin B-subunit. ( B ) Alignment of human and murine nSMase2 with representative other SMases. Sequence numbering is relative to unprocessed protein. The Mg 2+ -complexing glutamic acid (▿), the asparagine involved in substrate binding (arrow), and the general base histidine (●) are indicated above the sequence. The two transmembrane regions of nSMase2 are boxed. ( C ) Schematic diagram of SMase domain structure. Open boxes labeled “T” or “S” represent putative transmembrane or signal sequences, respectively.
Article Snippet: A truncated murine nSMase (mnSMase)2-cDNA construct coding for amino acids 310–655 was fused to a C-terminal 6xHis-tag derived from the plasmid vector pcDNA3.1/Myc-His (Invitrogen) and expressed in Escherichia coli [BL21(DE3)pLysS] using the pET-expression system (Novagen).
Techniques: Sequencing, Labeling, Binding Assay