Truncated acid sphingomyelinase induces substantially accelerated LDL aggregation in vitro.

High levels of low-density lipoprotein cholesterol (LDL-C) in circulation are linked to an elevated risk of atherosclerotic cardiovascular diseases (ASCVD). LDL contributes to ASCVD by forming plaques under oxidative and lipolytic conditions in the arterial wall. One of the enzymes involved in LDL lipolysis is acid sphingomyelinase (ASMase) found in atherosclerotic plaques. The susceptibility of LDL to aggregate in vitro upon treatment by recombinant human ASMase (rhASMase) has been shown to associate with ASCVD events in patients having established atherosclerosis. We produced three rhASMase variants and compared their activities toward sphingomyelin and LDL particles. The produced rhASMases represent two C-terminally truncated ASMases with His-tags on the opposite termini and a N-terminally tagged full-length version. The truncated rhASMases showed 3.5-fold higher activity on average in all tested conditions than the full-length version, which is consistent with the previous studies on C-terminal cysteine loss. The truncated rhASMases also induced two-fold faster LDL aggregation and showed improved general stability compared to the full-length rhASMase. A novel method for nanoparticle characterization, optofluidic force induction (OF2i), was applied for continuous measurement of the early onset of the LDL aggregate formation, not previously available by alternative methods. Molecular dynamics simulations on the rhASMases exploring potential differences in their lipid surface binding revealed more dynamic nature of the tag-free N-terminus. We also show associations between LDL aggregation susceptibility, sphingomyelin content and hydrolysis rate, highlighting the role of LDL lipid composition in association with the development of atherosclerosis.