Expression of recombinant human acid sphingomyelinase in insect Sf21 cells: purification, processing and enzymatic characterization

Bartelsen, Oliver; Lansmann, Stephanie; Nettersheim, Michael; Lemm, Thorsten; Ferlinz, Klaus; Sandhoff, Konrad

doi:10.1016/s0168-1656(98)00070-4

Cited by 33 publications

(34 citation statements)

References 19 publications

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“…Identity of Mature L-SMase-Acid SMase has been purified from a variety of different sources including human urine (27)(28)(29), human brain (30), human placenta (7,31,32), overexpression in CHO cells (33), and Sf21 insect cells (34). Importantly, aSMase purified from extracellular sources (e.g.…”

Section: Discussionmentioning

confidence: 99%

A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

Jenkins

Idkowiak‐Baldys

Simbari

et al. 2011

Journal of Biological Chemistry

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Acid sphingomyelinase (aSMase) catalyzes the hydrolysis of sphingomyelin (SM) to form the bioactive lipid ceramide (Cer). Notably, aSMase exists in two forms: a zinc (Zn 2؉ )-independent lysosomal aSMase (L-SMase) and a Zn 2؉ -dependent secreted aSMase (S-SMase) that arise from alternative trafficking of a single protein precursor. Despite extensive investigation into the maturation and trafficking of aSMase, the exact identity of mature L-SMase has remained unclear. Here, we describe a novel mechanism of aSMase maturation involving C-terminal proteolytic processing within, or in close proximity to, endolysosomes. Using two different C-terminal-tagged constructs of aSMase (V5, DsRed), we demonstrate that aSMase is processed from a 75-kDa, Zn 2؉ -activated proenzyme to a mature 65 kDa, Zn 2؉ -independent L-SMase. L-SMase is recognized by a polyclonal Ab to aSMase, but not by anti-V5 or anti-DsRed antibodies, suggesting that the C-terminal tag is lost during maturation. Furthermore, indirect immunofluorescence staining demonstrated that mature L-SMase colocalized with the lysosomal marker LAMP1, whereas V5-aSMase localized to the Golgi secretory pathway. Moreover, V5-aSMase possessed Zn 2؉ -dependent activity suggesting it may represent the common protein precursor of S-SMase and L-SMase. Importantly, the 65-kDa L-SMase, but not V5-aSMase, was sensitive to the lysosomotropic inhibitor desipramine, co-fractionated with lysosomes, and migrated at the same M r as partially purified human aSMase. Finally, three aSMase mutants containing C-terminal Niemann-Pick mutations (R600H, R600P, ⌬R608) exhibited defective proteolytic maturation. Taken together, these results demonstrate that mature L-SMase arises from C-terminal proteolytic processing of pro-aSMase and suggest that impaired C-terminal proteolysis may lead to severe defects in L-SMase function.

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Section: Discussionmentioning

confidence: 99%

A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

Jenkins

Idkowiak‐Baldys

Simbari

et al. 2011

Journal of Biological Chemistry

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“…Furthermore, the overexpression of heterologous Sf hex results in a significantly higher fraction of secreted protein compared with endogenous ratios. If the intracellular sorting machinery is saturated, then the fraction of a protein that is secreted will increase, a situation that is amplified when proteins such as those targeted to the lysosome are overexpressed in insect cells using cDNA constructs such as human HexB (52), human GM2 (GalNAc␤1,4(NeuAc␣2,3)Gal␤1,4Glc␤1,1Ј-ceramide) activator protein (53), and human sphingomyelinase (54).…”

Section: Discussionmentioning

confidence: 99%

Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core

Tomiya

Narang

Park

et al. 2006

Journal of Biological Chemistry

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Paucimannosidic glycans are often predominant in N-glycans produced by insect cells. However, a ␤-N-acetylhexosaminidase responsible for the generation of paucimannosidic glycans in lepidopteran insect cells has not been identified. We report the purification of a ␤-N-acetylhexosaminidase from the culture medium of Spodoptera frugiperda Sf9 cells (Sfhex). The purified Sfhex protein showed 10 times higher activity for a terminal N-acetylglucosamine on the N-glycan core compared with tri-N-acetylchitotriose. Sfhex was found to be a homodimer of 110 kDa in solution, with a pH optimum of 5.5. With a biantennary N-glycan substrate, it exhibited a 5-fold preference for removal of the ␤(1,2)-linked N-acetylglucosamine from the Man␣(1,3) branch compared with the Man␣(1,6) branch. We isolated two corresponding cDNA clones for Sfhex that encode proteins with >99% amino acid identity. A phylogenetic analysis suggested that Sfhex is an ortholog of mammalian lysosomal ␤-N-acetylhexosaminidases. Recombinant Sfhex expressed in Sf9 cells exhibited the same substrate specificity and pH optimum as the purified enzyme. Although a larger amount of newly synthesized Sfhex was secreted into the culture medium by Sf9 cells, a significant amount of Sfhex was also found to be intracellular. Under a confocal microscope, cellular Sfhex exhibited punctate staining throughout the cytoplasm, but did not colocalize with a Golgi marker. Because secretory glycoproteins and Sfhex are cotransported through the same secretory pathway and because Sfhex is active at the pH of the secretory compartments, this study suggests that Sfhex may play a role as a processing ␤-Nacetylhexosaminidase acting on N-glycans from Sf9 cells.␤-N-Acetylhexosaminidase (hexosaminidase, EC 3.2.1.52) catalyzes the hydrolysis of nonreducing terminal N-acetyl-Dhexosamine residues in N-acetyl-␤-D-hexosaminides. Hexosaminidases belong to the glycosyl hydrolase (GH) 2 3, GH20, or GH84 family (1-4). Of these, family 20 hexosaminidases include mammalian lysosomal hexosaminidases, fungal exochitinases, bacterial chitobiases, and insect chitinolytic hexosaminidases.The hexosaminidase activity of insects and insect cells is of particular interest because of the role that the enzyme may play in altering the structures of N-glycans generated by these cells. The N-glycan synthesis pathway in insects differs from that in mammals in that insects and insect cells produce appreciable amounts of paucimannosidic glycans (reviewed in Ref. 5). The intracellular N-glycan processing pathway in the endoplasmic reticulum of insects has been observed to include the addition of a Glc 3 Man 9 GlcNAc 2 group to the acceptor Asn residue, followed by the subsequent trimming of the initial oligosaccharide to generate Man 5 GlcNAc 2 . Insect cells also contain significant levels of N-acetylglucosaminyltransferase I, which adds a GlcNAc residue to the Man␣(1,3) branch, followed by the removal of two Man residues to produce the GlcNAc␤(1,2)Man␣(1,3)(Man(1,6))-Man␤(1,4)GlcNAc␤(1,4)GlcNAc structure. Unlike ma...

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“…Similarly, deglycosylation treatment or expression of a dominant-negative sortilin variant results in trapping of the enzyme within the Golgi in its degradation-vulnerable form (Bartelsen et al, 1998;He et al, 1999;Ni and Morales, 2006).…”

Section: Unauthenticatedmentioning

confidence: 99%

“…MichaelisMenten kinetic analysis revealed roughly similar values of the Michaelis constant K m for SM affinity despite the use of differently labeled substrates: 77 μm for soluble ASM in the human epidermis (Bowser and Gray, 1978), 5-25 μm for ASM purified from placenta (Jones et al, 1981Sakuragawa, 1982), 65 μm for ASM from Bacillus cereus (Fujii et al, 2004), 47 μm for ASM from human fibroblasts (Sato et al, 1988), 25 μm for recombinant ASM from the conditioned medium of infected insect cells (Bartelsen et al, 1998), 20 μm for ASM from human CSF , 0.2-0.6 μm for ASM released from human fibroblasts and mouse L-cells, respectively (Weitz et al, 1983), 11 μm with a V MAX of 21 nmol/h/mg protein for HEK 293 whole cell lysates and 2 μm and a V MAX of 4.3 μmol/h/mg protein for ASM immunoprecipitated from Jurkat cells (Gulbins and Kolesnick, 2000). Specific activities ranged from 600 to 2500 μmol/h/mg (Sakuragawa, 1982;Yamanaka and Suzuki, 1982;Jones et al, 1983;Weitz et al, 1985;Lansmann et al, 1996).…”

Section: Enzyme Kineticsmentioning

confidence: 99%

Secretory sphingomyelinase in health and disease

Kornhuber

Rhein

Müller

et al. 2015

Biological Chemistry

122

138

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Acid sphingomyelinase (ASM), a key enzyme in sphingolipid metabolism, hydrolyzes sphingomyelin to ceramide and phosphorylcholine. In mammals, the expression of a single gene, SMPD1, results in two forms of the enzyme that differ in several characteristics. Lysosomal ASM (L-ASM) is located within the lysosome, requires no additional Zn 2+ ions for activation and is glycosylated mainly with high-mannose oligosaccharides. By contrast, the secretory ASM (S-ASM) is located extracellularly, requires Zn 2+ ions for activation, has a complex glycosylation pattern and has a longer in vivo half-life. In this review, we summarize current knowledge regarding the physiology and pathophysiology of S-ASM, including its sources and distribution, molecular and cellular mechanisms of generation and regulation and relevant in vitro and in vivo studies. Polymorphisms or mutations of SMPD1 lead to decreased S-ASM activity, as detected in patients with Niemann-Pick disease B. Thus, lower serum/ plasma activities of S-ASM are trait markers. No genetic causes of increased S-ASM activity have been identified. Instead, elevated activity is the result of enhanced release (e.g., induced by lipopolysaccharide and cytokine stimulation) or increased enzyme activation (e.g., induced by oxidative stress). Increased S-ASM activity in serum or plasma is a state marker of a wide range of diseases. In particular, high S-ASM activity occurs in inflammation of the endothelium and liver. Several studies have demonstrated a correlation between S-ASM activity and mortality induced by severe inflammatory diseases. Serial measurements of S-ASM reveal prolonged activation and, therefore, the measurement of this enzyme may also provide information on past inflammatory processes. Thus, S-ASM may be both a promising clinical chemistry marker and a therapeutic target.

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Expression of recombinant human acid sphingomyelinase in insect Sf21 cells: purification, processing and enzymatic characterization

Cited by 33 publications

References 19 publications

A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

Purification, Characterization, and Cloning of a Spodoptera frugiperda Sf9 β-N-Acetylhexosaminidase That Hydrolyzes Terminal N-Acetylglucosamine on the N-Glycan Core

Secretory sphingomyelinase in health and disease

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