Occurrence of two molecular forms of human acid sphingomyelinase

Human acid sphingomyelinase (ASM) hydrolyses sphingomyelin to ceramide and phosphocholine. Metabolic studies on COS-1 cells transfected with ASM cDNA revealed the occurrence of an enzymically inactive precursor which is differentially processed to two predominant native glycoprotein forms: a 70 kDa polypeptide corresponding to human urinary protein and a 57 kDa form. Formation of these potentially active forms was shown to be restricted to distinct compartments. Maturation of the ASM precursor to a predominant 70 kDa form occurs exclusively inside acidic organelles, whereas variable amounts of 57 kDa ASM are detectable immediately after biosynthesis. Metabolic labelling of transfected COS-1 cells with [32P]Pi further suggests that this form obviously does not carry oligomannosylphosphate residues, in contrast with the mature lysosomal ASM. In order to verify that this early form of active ASM results from co-post-translational proteolysis of the ASM precursor and not from the use of different translation-initiation sites on the ASM mRNA, appropriate 5'-mutagenized cDNA constructs were transiently expressed. These results clearly indicate that the first potential in-frame AUG is exclusively used for translation initiation in vivo and that deletion of the proposed signal sequence for endoplasmic reticulum import completely eliminates the ability of the translation product to enter the vacuolar apparatus. As there are two different subcellular sites of maturation of the ASM precursor, and intracellular targeting of the two processed forms appears to be different, the two ASM proteins may contribute to distinct physiological functions

Functional Implications of Novel Human Acid Sphingomyelinase Splice Variants

BACKGROUND: Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity. METHODOLOGY/PRINCIPAL FINDINGS: We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models. CONCLUSIONS/SIGNIFICANCE: These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity

An Introduction to Sphingolipid Metabolism and Analysis by New Technologies

Sphingolipids (SP) are a complex class of molecules found in essentially all eukaryotes and some prokaryotes and viruses where they influence membrane structure, intracellular signaling, and interactions with the extracellular environment. Because of the combinatorial nature of their biosynthesis, there are thousands of SP subspecies varying in the lipid backbones and complex phospho- and glycoheadgroups. Therefore, comprehensive or “sphingolipidomic” analyses (structure-specific, quantitative analyses of all SP, or at least all members of a critical subset) are needed to know which and how much of these subspecies are present in a system as a step toward understanding their functions. Mass spectrometry and related novel techniques are able to quantify a small fraction, but nonetheless a substantial number, of SP and are beginning to provide information about their localization. This review summarizes the basic metabolism of SP and state-of-art mass spectrometric techniques that are producing insights into SP structure, metabolism, functions, and some of the dysfunctions of relevance to neuromedicine

Occurrence of two molecular forms of human acid sphingomyelinase.

Human acid sphingomyelinase (ASM) hydrolyses sphingomyelin to ceramide and phosphocholine. Metabolic studies on COS-1 cells transfected with ASM cDNA revealed the occurrence of an enzymically inactive precursor which is differentially processed to two predominant native glycoprotein forms: a 70 kDa polypeptide corresponding to human urinary protein and a 57 kDa form. Formation of these potentially active forms was shown to be restricted to distinct compartments. Maturation of the ASM precursor to a predominant 70 kDa form occurs exclusively inside acidic organelles, whereas variable amounts of 57 kDa ASM are detectable immediately after biosynthesis. Metabolic labelling of transfected COS-1 cells with [32P]Pi further suggests that this form obviously does not carry oligomannosylphosphate residues, in contrast with the mature lysosomal ASM. In order to verify that this early form of active ASM results from co-post-translational proteolysis of the ASM precursor and not from the use of different translation-initiation sites on the ASM mRNA, appropriate 5'-mutagenized cDNA constructs were transiently expressed. These results clearly indicate that the first potential in-frame AUG is exclusively used for translation initiation in vivo and that deletion of the proposed signal sequence for endoplasmic reticulum import completely eliminates the ability of the translation product to enter the vacuolar apparatus. As there are two different subcellular sites of maturation of the ASM precursor, and intracellular targeting of the two processed forms appears to be different, the two ASM proteins may contribute to distinct physiological functions

Molecular analysis of the acid sphingomyelinase deficiency in a family with an intermediate form of Niemann-Pick disease.

A novel point mutation in the lysosomal acid sphingomyelinase gene has been identified in the recently reported Serbian family with a clinically and biochemically atypical intermediate form of Niemann-Pick disease. The mutation was a T1171-->G transversion resulting in substitution of glycine for normal tryptophan at amino acid residue 391. The coding sequence was otherwise normal. All of the five affected individuals were almost certainly homoallelic, and both of the two obligate heterozygotes studied also carried the same mutation. This mutation is therefore likely to be directly associated with the atypical phenotype of these patients. Expression in COS-1 cells suggested a higher residual activity than that in cultured fibroblasts. A recently developed high-affinity rabbit antihuman sphingomyelinase antibody allowed us to study for the first time the biosynthesis, processing, and targeting of a mutant sphingomyelinase by metabolic labeling of cultured fibroblasts. The mutant enzyme protein was normally synthesized, processed, and routed to the lysosome but was apparently unstable and degraded rapidly once it reached the lysosome. Together with the finding of the relatively high residual activity in COS-1 cells, we interpret our observations to mean that instability and rapid breakdown of the mature mutant enzyme protein, due to the mutation rather than direct inactivation of the catalytic activity, is the primary mechanism for the deficiency of sphingomyelinase activity in these patients. A high prevalence of this mutation in the Serbian population is likely, since the family pedigree indicates that members from four reportedly unrelated families must have contributed the same mutation

Cell

AbstractInvasion of human mucosal cells by N. gonorrhoeae via the binding to heparansulfate proteoglycan receptors is considered a crucial event of the infection. Using different human epithelial cells and primary fibroblasts, we show here an activation of the phosphatidylcholine-specific phospholipase C (PC-PLC) and acidic sphingomyelinase (ASM) by N. gonorrhoeae, resulting in the release of diacylglycerol and ceramide. Genetic and/or pharmacological blockade of ASM and PC-PLC cause inhibition of cellular invasion by N. gonorrhoeae. Complementation of ASM-deficient fibroblasts from Niemann-Pick disease patients restored N. gonorrhoeae–induced signaling and entry processes. The activation of PC-PLC and ASM, therefore, is an essential requirement for the entry of N. gonorrhoeae into distinct nonphagocytic human cell types including several epithelial cells and primary fibroblasts