O-Glycosylation of Mucin-like Domain Retains the Neutral Ceramidase on the Plasma Membranes as a Type II Integral Membrane Protein

Abstract

Ceramidase is a key enzyme involved in regulating cellular levels of ceramide, sphingosine, and possibly sphigosine 1-phosphate and thus could modulate sphingolipid signaling. Here we report that O-glycosylation of the mucin-like domain of neutral ceramidases was required for localization to the surface of plasma membranes. The deduced amino acid sequences of the mammalian enzymes contain a serine-threonine-rich domain (mucin box), which follows the signal/anchor sequence, whereas those of bacterial and invertebrate enzymes completely lack a mucin box, suggesting that the specific domain has been acquired during evolution. In HEK293 cells overexpressing ceramidase, the enzyme was not only secreted into the medium after cleavage of the NH 2 -terminal signal/anchor sequence but also localized at the plasma membrane as a type II integral membrane protein. Lectin blot analysis using peanut agglutinin revealed that the mucin box of the enzyme is highly glycosylated with O-glycans. Interestingly, a mutant lacking the mucin box or possible O-glycosylation sites in the mucin box was secreted into the medium but not localized at the surface of the cells. Furthermore, a mucin box-fused chimera green fluorescent protein (GFP), but not GFP itself, with the signal/anchor sequence was distributed on the surface of the cells. These results suggest that O-glycosylation of the mucin box retains proteins on the plasma membranes. We also found that the 112-kDa membrane-bound enzyme from mouse kidney is O-glycosylated, whereas the 94-kDa soluble enzyme from liver is not. These results clearly indicate that post-translational modification of the enzyme with O-glycans is tissue-specific and helps the enzyme to localize at the surface of plasma membranes as a type II membrane protein.

Molecular Cloning and Functional Analysis of Zebrafish Neutral Ceramidase

“…The mucin box of the mammalian enzymes is highly glycosylated with O-glycans and retains the enzyme at the plasma membrane as a type II integral membrane protein, because a mucin box-deleted mutant CDase and an Ala-replacement mutant enzyme were found to be secreted into the culture medium when expressed in human embryonic kidney 293 cells (20). Furthermore, it was clarified that bacterial and invertebrate neutral CDases, lacking a mucin box, were released into the extracellular milieu (9,18).…”

“…The mammalian neutral CDases have a Ser/Thr-rich domain (mucin box) downstream of the N-terminal hydrophobic region, whereas bacterial and invertebrate enzymes do not (20). The mucin box of the mammalian enzymes is highly glycosylated with O-glycans and retains the enzyme at the plasma membrane as a type II integral membrane protein, because a mucin box-deleted mutant CDase and an Ala-replacement mutant enzyme were found to be secreted into the culture medium when expressed in human embryonic kidney 293 cells (20).…”

“…A deletion mutant CDase that lacks the mucin box and an Ala replacement mutant CDase in which all Ser or Thr residues in the mucin box are replaced by Ala were not retained in the cell membranes, indicating that Oglycosylation of the mucin box is required to keep the enzyme at the plasma membrane. The mammalian CDase was found to be expressed on plasma membranes as a type II integral protein in which the N-terminal hydrophobic sequence just before the mucin box serves as an anchor to the membranes, and on some occasions the enzyme was detached from the cell via processing of the anchor (20).…”

The Mucin Box and Signal/Anchor Sequence of Rat Neutral Ceramidase Recruit Bacterial Sphingomyelinase to the Plasma Membrane

“…This is the first report that a bacterial sphingolipid-degrading enzyme was recruited to plasma membranes in an active form, although this method has successfully been used to recruit a green fluorescent protein (GFP) to plasma membranes. 22) Rat neutral CDase was found mainly at the apical membranes of proximal tubules, distal tubules, and collecting ducts in the kidney. The enzyme was abundant in the DIM fractions (lipid rafts) with cholesterol and GM1a ganglioside.…”

“…1A). 22,23) O-Glycosylation of the mucin box was necessary to retain the neutral CDase on the plasma membranes as a type-II integral membrane protein, although the mechanism by which the mucin box retains CDase at the cell surface remains unclear.…”