Expression of recombinant human acid sphingomyelinase in insect Sf21 cells: purification, processing and enzymatic characterization.
Biochemical and structural studies on human acid sphingomyelinase (haSMase) depend on the access to homogeneous biologically active enzyme. Due to the low abundance of native haSMase (n-haSMase) in human tissue, conventional purification strategies are not suitable for the isolation of preparative amounts of the enzyme. We describe a novel approach to the functional expression and purification of haSMase employing the baculovirus expression vector system. Infection of Spodoptera frugiperda 21 cells with recombinant baculovirus encoding haSMase leads to the expression of a glycosylated 75 kDa precursor protein, which is subsequently processed to an enzymatically active secreted 72 kDa haSMase. Variations in N-glycosylation and proteolytic maturation account for the difference in molecular mass between mature recombinant (72 kDa) and human placental haSMase (75 kDa). N-terminal amino acid sequencing of recombinant haSMase (r-haSMase) reveals a 23-residue N-terminal extension compared to the placental enzyme. The apparent K(m) and Vmax values for sphingomyelin degradation by r-haSMase in a micellar assay system are 32 microM and 0.56 mmol h-1 mg-1, respectively. In conclusion, the established baculovirus expression vector system provides an efficient tool for the expression and functional characterization of haSMase.
Kekulé-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universität Bonn, Germany.
O
O
Bartelsen
S
S
Lansmann
M
M
Nettersheim
T
T
Lemm
K
K
Ferlinz
K
K
Sandhoff
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010920:Placenta / Q000201:enzymology; D011134:Polysaccharides / Q000737:chemistry; D011498:Protein Precursors / Q000737:chemistry; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics*; D017421:Sequence Analysis; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry*; D013109:Sphingomyelins / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics
D006023:Glycoproteins; D009844:Oligosaccharides; D011134:Polysaccharides; D011498:Protein Precursors; D011994:Recombinant Proteins; D013109:Sphingomyelins; D013108:Sphingomyelin Phosphodiesterase