Sphingolipid Activator Proteins Are Required for Epidermal Permeability Barrier Formation

Abstract

The epidermal permeability barrier is maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the major components of these multilayered membranes, derive in large part from hydrolysis of glucosylceramides mediated by stratum corneum ␤-glucocerebrosidase (␤-GlcCerase). Prosaposin (pSAP) is a large precursor protein that is proteolytically cleaved to form four distinct sphingolipid activator proteins, which stimulate enzymatic hydrolysis of sphingolipids, including glucosylceramide. Recently, pSAP has been eliminated in a mouse model using targeted deletion and homologous recombination. In addition to the extracutaneous findings noted previously, our present data indicate that pSAP deficiency in the epidermis has significant consequences including: 1) an accumulation of epidermal glucosylceramides together with below normal levels of ceramides; 2) alterations in lipids that are bound by ester linkages to proteins of the cornified cell envelope; 3) a thickened stratum lucidum with evidence of scaling; and 4) a striking abnormality in lamellar membrane maturation within the interstices of the stratum corneum. Together, these results demonstrate that the production of pSAP, and presumably mature sphingolipid activator protein generation, is required for normal epidermal barrier formation and function. Moreover, detection of significant amounts of covalently bound -OH-GlcCer in pSAP-deficient epidermis suggests that deglucosylation to -OH-Cer is not a requisite step prior to covalent attachment of lipid to cornified envelope proteins.

Accumulation of protein‐bound epidermal glucosylceramides in β‐glucocerebrosidase deficient type 2 Gaucher mice

“…1a). RecNci I mice mainly stored GlcCer(EOS) and GlcCer a/b in contrast to the previously described sphingolipid activator protein de¢cient pSAP knockout mice which mainly accumulated the rather hydrophobic GlcCer(EOS) within their epidermis [11]. Apparently its enzymatic hydrolysis is most dependent on the presence of SAP-C.…”

“…Most importantly, Cer(EOS), the product of GlcCer(EOS) hydrolysis, was nearly absent in RecNci I mice. The levels of Cer(NS), (NP) and (C24,26-AS), which previously have been shown to comprise the lipid backbones of GlcCer a/b [11], also were reduced to less than 40% of normal control. Interestingly, Cer(C16-AS) levels remained una¡ected indicating a direct de novo synthetic route not requiring subsequent glucosylation/deglucosylation steps for this most polar Cer species.…”

“…Recently, we have identi¢ed a novel component of the lipid bound envelope, g-hydroxylated GlcCer, suggesting that deglucosylation is not a required step for the covalent attachment of lipids to the corni¢ed cell envelope [11]. In the present work we analyzed the levels of free and covalently bound glucosylceramides and their metabolic products in the epidermis of RecNci I mice with special regard to g-hydroxylated glucosylceramides.…”

“…Unbound lipids were extracted and separated as described previously [11]. For separation of glucosylceramides, the thin-layer silica gel 60 plates (Merck Darmstadt, Germany) were developed with chloroformmethanol-water (70:30:5, vol/vol/vol).…”

“…Ceramides were resolved twice using chloroform-methanol-acetic acid (190:9:1, vol/vol/vol) as developing solvent. Bound lipids were recovered and analyzed as described previously [11]. Brie£y, covalently bound lipids were released by incubation of preextracted samples with 1 M KOH in 95% methanol for 2 h at 60³C, recovered by extraction into chloroform and separated by TLC using twice chloroform-methanol-acetic acid (190:9:1, vol/vol/ vol) as developing solvent.…”