Functional Analysis of the Glycosylation of Murine Acid Sphingomyelinase

Abstract

Glycosylation plays a crucial role in glycoprotein stability and its correct folding. Murine acid sphingomyelinase (ASM) is a lysosomal glycoprotein. We studied the functional role of its individual N-linked oligosaccharides needed to maintain enzymatic activity and protein stability. Mutagenized cDNA constructs were heterologously expressed. All six potential N-glycosylation sites were modified. Incomplete glycosylation of the most distant C-terminal site resulted in two isoforms. Oligosaccharides at N-84, N-173, and N-611 were found to be of minor importance for enzymatic activity. The glycosylation defect at N-333 or N-393 reduced the enzymatic activity to 40% and at N-518 to less than 20%. These mutations did not effect the K m value. Glycosylation at N-333 and N-393 mainly contributed to the enzyme stability and prevented degradation at lysosomal acidic pH, whereas the low residual enzymatic activity of mutant ASM deficient in glycosylation at N-518 was caused by protein misfolding. The mutant protein was also prone to proteolysis when trapped in the endoplasmic reticulum/cis-Golgi after brefeldin A application. Insufficiently glycosylated ASM formed a stable complex with BiP, an immunoglobulin heavy chain-binding protein, and thus remained in the endoplasmic reticulum.32 PO 4 labeling revealed that the glycosylation mutants of ASM were phosphorylated predominantly at mannose residues of oligosaccharides linked to N-84, N-333, and N-393.Acid sphingomyelinase (ASM, 1 E.C. 3.1.4.12) is an ubiquitous lysosomal hydrolase that cleaves sphingomyelin to ceramide and phosphocholine. In 1987, Quintern et al.(1) purified human acid sphingomyelinase from urine to homogeneity. Subsequent discovery of the primary structure enabled functional expression in an eucaryotic cell culture system (2).Deficient or reduced ASM activity causes sphingomyelin accumulation known in humans as Niemann-Pick disease sphingolipidosis. Depending on the amount of residual enzymatic activity, the neurovisceral type A or the less severe visceral type B of this autosomal recessive disease is observed (3).Recently, several mutations in the ASM gene of Niemann-Pick disease patients have been reported, leading to an enzyme of no or drastically reduced activity. Transgenic mice with a disrupted ASM gene exhibit a phenotype comparable to that of Niemann-Pick disease, type A, patients. The neurological symptoms of these mice are accompanied by the loss of Purkinje cells as revealed by histological studies (4,5).Besides the catabolic function of this lysosomal enzyme, involvement of ASM in the ceramide-dependent signal transduction pathway has been discussed controversially (6 -9). As a lysosomal hydrolase, ASM undergoes several posttranslational processing and targeting steps. The N-terminal signal sequence directs the primary translation product into the ER. The signal sequence, which has a length of approximately 45 amino acids for human as well as murine ASM according to the von Heijne prediction (10), is cleaved during this process. In the ER, o...

Caenorhabditis elegansContains Two Distinct Acid Sphingomyelinases

“…Elimination of any of these sites results in various degrees of decreased enzyme activity. A similar result is found in the murine ASM (11). An N-terminal region of the hASM (residues 89 -165) is found to be highly homologous to the sphingolipid activator protein saposin and may possess lipid-binding and/or sphingomyelinase activator properties (12).…”

Secretory sphingomyelinase in health and disease

“…Defects in N-glycosylation reduce enzyme stability and activity but do not affect K M values (Newrzella and Stoffel, 1996). Moreover, pH plays an important role in substrate binding but not catalytic velocity .…”

“…The two C-terminal N-glycosylation sites (N505 and N522) are essential for proper trafficking and affect S-ASM secretion and the enzymatic activity of L-ASM . Although presumably not glycosylated, removal of the fifth site reduces enzymatic activity to < 20% due to protein misfolding (Newrzella and Stoffel, 1996;Ferlinz et al, 1997).…”

Neutral Sphingomyelinase 1 Deficiency in the Mouse Causes No Lipid Storage Disease

“…The lysosomal aSMase (unigene nomenclature, SMPD1; SPM-phosphodiesterase 1) was the first sphingomyelinase to be cloned and characterized biochemically (16,17,20,21,31). In gene-targeting experiments by homologous recombination, asmase-null allelic mice were generated, which, like the human neurovisceral form (type A) of Niemann-Pick disease accumulate SPM in the reticuloendothelial system, predominantly in the liver, spleen, lung, bone marrow, and brain, leading to death in early childhood (9,19).…”