In this report we describe the organization of the 3.3-kb genomic sequence of the nSMase 1 enclosing 10 exons. An nSMase 1-deficient mouse mutant with a deletion of exons II to VIII was constructed and characterized. The small nsmase 1 gene locus and the large deletion strongly reduced the recombination frequency (1:800).
The data reported here indicate that nSMase 1 deficiency does not affect the metabolism of SPM or of any lysophospholipid and causes no storage of SPM or of any other lipid. Furthermore, the nSMase 1 deficiency leaves the development and longevity of the nSMase 1-deficient mouse mutant unimpaired.
The determination of nSMase enzymatic activities in the different organs of the nsmase 1−/−
mouse clearly indicated that nSMase 1 is not responsible for the high nSMase activity in brain, in support of our previous immunoprecipitation experiments (35
) and other chromatographic studies on mouse brain sphingomyelinases (4
). nSMase 2 is the likeliest candidate responsible for the high nSMase activity in the central nervous system and the residual activity in the other organs.
The presence of additional nSMases could explain the unchanged lipid composition in organs of the nSMase 1-deficient mouse mutant. Another possibility would be limitation by the available detection methods of the minute amount of SPM.
However, the sensitive labeling experiments with EMFIs were also unable to detect any changes in lipid metabolism. Whether lipid analyses of aging animals and of subcellular fractions may reveal differences remains to be seen.
The presence of monoglycerols and of decreased levels of lyso-PC and/or lyso-PAF (data not shown and reference 28
) in highly nsmase 1
-overexpressing HEK cells led to the suggestion that the in vivo function of our cloned nSMase is that of a lysophospholipase C. The results of our experiments clearly demonstrate an identical metabolism of lyso-PC and lyso-PAF in the wild type and in nSMase 1-deficient EMFIs and make the proposed function of nSMase 1 as a lysophospholipase C unlikely.
nSMase 1 is an integral membrane protein of the ER membrane using SPM, a component of the bilayer as substrate. In the in vitro assay, hydrolysis of the lyso-PAF and lyso-PC, which possess detergent properties and are water-soluble subtrates, is minute. The cross-reactivity could be explained by their related structure around the phosphodiester bond, the free 3-hydroxy-group of the sphingosine moiety of SPM and the free 2-hydroxy group of the lysocompound.
At this stage, the biochemical and cell biological data derived from the comparison of the wild type and the nsmase 1−/−
mutant are insufficient to define the function of nSMase 1 in cellular metabolism. Therefore, we may only speculate on possible functions of nSMases, e.g., the regulation of the SPM/cholesterol ratio in membranes and their metabolism; nSMase 1 may contribute to the modification of local microdomains in the membrane organization (6
). Modification of the lipid bilayer by e.g., lysophosphatidic acid acyl transferase endophilin (30
), phospholipase D (10
), phospholipase C, and sphingomyelinase (26
), is a prerequisite for effective protein functions involved in vesicle formation, transport, and fusion (43
). nSMase 1, localized in the membranes of the endoplasmic reticulum, might be an additional modifying enzyme involved in these processes, including the disassembly of the Golgi membrane stacks during mitosis (25
The analysis of the nSMase 2 knockout (nsmase 2−/−) mouse might provide insights into the function of the nSMase enzyme family on the molecular level. Finally, the generation of an nSMase 1/nSMase 2 double-knockout mouse will answer the question whether the two nSMases cloned so far are the only ones in mammals or if additional isoforms contribute to the (sub)cellular SPM metabolism.