Lipid Raft Composition Modulates Sphingomyelinase Activity and Ceramide-Induced Membrane Physical Alterations

Abstract

Lipid rafts and ceramide (Cer)-platforms are membrane domains that play an important role in several biological processes. Cer-platforms are commonly formed in the plasma membrane by the action of sphingomyelinase (SMase) upon hydrolysis of sphingomyelin (SM) within lipid rafts. The interplay between SMase activity, initial membrane properties (i.e., phase behaviour and lipid lateral organization) and lipid composition, amount of product (Cer) generated, and how it modulates membrane properties was studied using fluorescence methodologies in model membranes. The activity of SMase was evaluated by following the hydrolysis of radioactive SM. It was observed that: i) enzyme activity and extent of hydrolysis is strongly dependent on membrane physical properties but not on substrate content, being higher in raft-like mixtures, i.e., those mixtures with ld-lo (liquid disordered-liquid ordered) phase separation, and ii) Cer-induced alterations are also dependent on membrane composition, namely on cholesterol (Chol) content. In the lowest-Chol range, Cer segregates together with SM into small, ∼8.5 nm, Cer/SM-gel domains. Upon increasing Chol, the ability of Cer to recruit SM and to form gel domains strongly decreases. In the high Chol range, a Chol-enriched/SM-depleted lo phase predominates. Together, these data suggest that in biological membranes Chol in particular, and raft domains in general, play an important role in the modulation of SMase activity and in the regulation of membrane physical properties by restraining Cer-induced alterations.