Human Keratinocytes Synthesize Ultra Long‐Chain Sphingoid Bases (Up To C28) That Are Abundant in Skin Stratum Corneum

Abstract

Skin sphingolipids are unique and fulfill the important task of forming a hydrophobic barrier to limit water loss from the body but also to protect from allergen, bacterial and viral penetration through the skin. Skin sphingolipids are highly diverse in their combination of fatty acids and sphingoid bases, the presence of highly hydrophobic ω‐esterified ceramides (EOS‐CER), and the abundance of ultra long‐chain fatty acids (ULCFA, up to C36) within ceramide molecules. The chain length of sphingoid bases in ceramides is commonly attributed to C18 molecules but there are only a few reports demonstrating the presence of C20‐ and C22‐sphingoid bases in skin ceramides. As free sphingoid bases and ceramides are metabolically connected, it is assumed that similar to other tissues, C18‐sphingoid bases represent the majority of sphingoid bases within the total pool of free and ceramide‐linked molecules in the skin.Our sphingolipidomic work during the study of the mechanisms leading to the impairment of skin barrier function has revealed that ceramides with C18‐, C20‐, and C22‐ sphingoid bases are almost equally represented in human skin stratum corneum. Surprisingly, we found that free sphingoid bases in stratum corneum are highly abundant and composed primarily of ultra long‐chain (C24‐ and C26‐) dihydrosphingosines. This finding prompted us to investigate in details the ability of skin keratinocytes to synthesize ultra long‐chain length sphingoid bases. Using undifferentiated and Ca2+‐differentiated primary human keratinocytes, [U‐13C,15N]‐serine, and stable isotope pulse‐labeling approach in vitro, we have confirmed that all ceramides and free sphingoid base molecules do incorporate the labeled serine into their structure. Next, we explored the ability of serine palmitoyltransferase (SPT), the enzyme that initiates sphingolipid biosynthesis, to synthesize ultra long‐chain sphingoid bases in vitro. By comparing the activity of SPT in total cell lysates prepared from undifferentiated and Ca2+‐differentiated human keratinocytes, 16:0–24:0‐Coenzyme A molecules and [U‐13C,15N]‐serine, we confirmed the ability of SPT expressed in human keratinocytes to produce, albeit at low capacity, C24‐ and C26‐3‐keto‐dihydrosphingosines. As keratinocytes and skin stratum corneum do not have measurable amounts of ceramides with C24‐C26‐dihydrosphingosines or C24‐C26‐sphingosines, we conclude that keratinocyte ceramide synthases are unable to utilize dihydrosphingosines with carbon chain length more than C22, thus providing a foundation for the accumulation of C24‐C26‐dihydrosphingosine in human stratum corneum. The question of the possible special biological function of long‐chain dihydrosphingosines in human skin is currently under investigation.