Chemoenzymatically prepared konjac ceramide inhibits NGF-induced neurite outgrowth by a semaphorin 3A-like action

Seigo Usuki; Noriko Tamura; Shota Sakai; Tomohiro Tamura; Katsuyuki Mukai; Yasuyuki Igarashi

doi:10.1016/j.bbrep.2015.11.016

Chemoenzymatically prepared konjac ceramide inhibits NGF-induced neurite outgrowth by a semaphorin 3A-like action

Biochem Biophys Rep. 2015 Nov 19:5:160-167. doi: 10.1016/j.bbrep.2015.11.016. eCollection 2016 Mar.

Authors

Seigo Usuki¹, Noriko Tamura², Shota Sakai¹, Tomohiro Tamura², Katsuyuki Mukai³, Yasuyuki Igarashi¹

Affiliations

¹ Laboratory of Biomembrane and Biofunctional Chemistry, Graduate School of Advanced Life Science, Frontier Research Center for Post-Genome Science and Technology, Hokkaido University, Kita 21, Nishi 11, Kita Ward, Sapporo, Hokkaido 011-0021, Japan.
² National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Hokkaido, Japan.
³ Healthcare Division, Daicel Corporation, Japan.

Abstract

Dietary sphingolipids such as glucosylceramide (GlcCer) are potential nutritional factors associated with prevention of metabolic syndrome. Our current understanding is that dietary GlcCer is degraded to ceramide and further metabolized to sphingoid bases in the intestine. However, ceramide is only found in trace amounts in food plants and thus is frequently taken as GlcCer in a health supplement. In the present study, we successfully prepared konjac ceramide (kCer) using endoglycoceramidase I (EGCase I). Konjac, a plant tuber, is an enriched source of GlcCer (kGlcCer), and has been commercialized as a dietary supplement to improve dry skin and itching that are caused by a deficiency of epidermal ceramide. Nerve growth factor (NGF) produced by skin cells is one of the itch factors in the stratum corneum of the skin. Semaphorin 3A (Sema 3A) has been known to inhibit NGF-induced neurite outgrowth of epidermal nerve fibers. It is well known that the itch sensation is regulated by the balance between NGF and Sema 3A. In the present study, while kGlcCer did not show an in vitro inhibitory effect on NGF-induced neurite outgrowth of PC12 cells, kCer was demonstrated to inhibit a remarkable neurite outgrowth. In addition, the effect of kCer was similar to that of Sema 3A in cell morphological changes and neurite retractions, but different from C2-Ceramide. kCer showed a Sema 3A-like action, causing CRMP2 phosphorylation, which results in a collapse of neurite growth cones. Thus, it is expected that kCer is an advanced konjac ceramide material that may have neurite outgrowth-specific action to relieve uncontrolled and serious itching, in particular, from atopic eczema.

Keywords: BSA, bovine serum albumin; C16Cer, N-hexadecanoyl-D-erythro-sphingosine; C18Cer, N-octadecanoyl-D-erythro-sphingosine; C24Cer, N-tetracosanoyl-D-erythro-sphingosine; C2Cer, N-acetyl-D-erythro-sphingosine; CBB, Coomassie Briliant Blue; CCK-8, cell counting kit 8; CRMP2; CRMP2, collapsin response mediator protein 2; Cer, ceramide; Ceramide; DMEM, Dulbecco’s modified Eagle's medium; EGCase I, endoglycoceramidase I; GlcCer, glucosylceramide; Konjac; LDH, lactate dehydrogenase; NGF; NGF, nerve growth factor; Neurite outgrowth; PBS, phosphate-buffered saline; Sema 3A, semaphorin 3A; Semaphorin 3A; TBEA, trypan blue exclusion assay; kCer, konjac ceramide; pCRMP2, phospho-collapsin response mediator protein 2.