Characterization of Acid Sphingomyelinase Activity in Human Cerebrospinal Fluid

Christiane Mühle,Fabio Canneva,Johannes Kornhuber,Hagen B Huttner,Silke Walter,Erich Gulbins,Martin Reichel,Piotr Lewczuk,Leah J Siskind

doi:10.1371/journal.pone.0062912

Christiane Mühle, Fabio Canneva + Show 7 more

Open Access

https://doi.org/10.1371/journal.pone.0062912

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Abstract

BackgroundAs a key enzyme in sphingolipid metabolism, acid sphingomyelinase (ASM) is involved in the regulation of cell fate and signaling via hydrolysis of sphingomyelin to form ceramide. While increased activity of the lysosomal form has been associated with various pathological conditions, there are few studies on secretory ASM limited only to cell models, plasma or serum.MethodsAn optimized assay based on a fluorescent substrate was applied to measure the ASM activity in cerebrospinal fluid (CSF) collected from mice and from 42 patients who were classified as controls based on normal routine CSF values.ResultsWe have detected ASM activity in human CSF, established a sensitive quantitative assay and characterized the enzyme’s properties. The enzyme resembles plasmatic ASM including protein stability and Zn2+-dependence but the assays differ considerably in the optimal detergent concentration. Significantly increased activities in the CSF of ASM transgenic mice and undetectable levels in ASM knock-out mice prove that the measured ASM activity originates from the ASM-encoding gene SMPD1. CSF localized ASM activities were comparable to corresponding serum ASM levels at their respective optimal reaction conditions, but no correlation was observed. The large variance in ASM activity was independent of sex, age or analyzed routine CSF parameters.ConclusionsHuman and mouse CSF contain detectable levels of secretory ASM, which are unrelated to serum ASM activities. Further investigations in humans and in animal models will help to elucidate the role of this enzyme in human disease and to assess its value as a potential biomarker for disease type, severity, progress or therapeutic success.

Highlights

Enzymes involved in sphingolipid metabolism influence the concentration of bioactive lipids such as ceramide and sphingosine1-phosphate, thereby controlling diverse cellular processes including proliferation, differentiation, migration, adhesion, growth arrest and apoptosis [1]
We provide evidence that S-acid sphingomyelinase (ASM) activity is present in cerebrospinal fluid (CSF) and may be quantified by a sensitive enzyme assay based on a fluorescent substrate
BODIPY-C12-SM to BODIPY-C12-ceramide was detectable within several days of incubation of CSF samples under reaction conditions utilized for plasmatic secretory ASM (S-ASM) (Fig. 1A)

Summary

Introduction

Enzymes involved in sphingolipid metabolism influence the concentration of bioactive lipids such as ceramide and sphingosine1-phosphate, thereby controlling diverse cellular processes including proliferation, differentiation, migration, adhesion, growth arrest and apoptosis [1]. Among these enzymes, acid sphingomyelinase (ASM, EC 3.1.4.12) is a lysosomal glycoprotein that catalyzes the hydrolysis of the major eukaryotic membrane constituent sphingomyelin (SM) to yield phosphorylcholine and ceramide. As a key enzyme in sphingolipid metabolism, acid sphingomyelinase (ASM) is involved in the regulation of cell fate and signaling via hydrolysis of sphingomyelin to form ceramide. While increased activity of the lysosomal form has been associated with various pathological conditions, there are few studies on secretory ASM limited only to cell models, plasma or serum

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Conclusion