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

Biochemistry and Biophysics Reports 5 (2016) 160–167 Contents lists available at ScienceDirect Biochemistry and Biophysics Reports journal homepage: www.elsevier.com/locate/bbrep Chemoenzymatically prepared konjac ceramide inhibits NGF-induced neurite outgrowth by a semaphorin 3A-like action Seigo Usuki a,n, Noriko Tamura b, Shota Sakai a, Tomohiro Tamura b, Katsuyuki Mukai c, Yasuyuki Igarashi a a 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 b National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Hokkaido, Japan c Healthcare Division, Daicel Corporation, Japan art ic l e i nf o a b s t r a c t Article history: Received 2 September 2015 Received in revised form 4 November 2015 Accepted 17 November 2015 Available online 19 November 2015 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. & 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Keywords: Ceramide Konjac NGF Semaphorin 3A Neurite outgrowth CRMP2 1. Introduction Epidermal neurotrophins play an important role for proliferation of structural skin cells such as keratinocytes, fibroblasts, and mast cells, and regulate peripheral innervation [1]. Cutaneous functions of nerve growth factor (NGF) have been well studied and known to maintain cutaneous innervation and cell survival by protecting sensory neurons via retrograde Abbreviations: NGF, nerve growth factor; Cer, ceramide; GlcCer, glucosylceramide; kCer, konjac ceramide; C2Cer, N-acetyl-D-erythro-sphingosine; C16Cer, Nhexadecanoyl-D-erythro-sphingosine; C18Cer, N-octadecanoyl-D-erythro-sphingosine; C24Cer, N-tetracosanoyl-D-erythro-sphingosine; EGCase I, endoglycoceramidase I; CRMP2, collapsin response mediator protein 2; pCRMP2, phospho-collapsin response mediator protein 2; LDH, lactate dehydrogenase; TBEA, trypan blue exclusion assay; CCK-8, cell counting kit 8; Sema 3A, semaphorin 3A; CBB, Coomassie Briliant Blue; BSA, bovine serum albumin; PBS, phosphate-buffered saline; DMEM, Dulbecco’s modified Eagle's medium n Corresponding author. E-mail address: (S. Usuki). neurotrophic signaling [2]. It is also well known that the cutaneous level of NGF is regulated as an autocrine and paracrine growth factor in all skin cells [3,4]. An excessive amount of NGF shows an additional function as a pain or itch sensitizer via the cutaneous innervation system. Epidermal NGF elevation stimulates skin cells, inducing overproduction of NGF, sensitizing the nociceptive nerve endings of sensory neurons, enhancing neurite outgrowth, and resulting in structural changes of the innervation system. Such extended neurites penetrate into the superficial layer of the skin and indicate hypersensitivity for external stimuli [5]. Thus, the undesired NGF causes extension of itch-causing nerve fibers and severe or long-lasting, itching skin, such as atopic eczema. To prevent external stimuli from inducing hypersensitivity of sensory neurons, epidermal ceramide is crucial for maintenance of homeostasis of an epidermal barrier. Epidermal ceramides are believed to be derived from pools of sphingomyelin and glucosylceramide (GlcCer) from differentiated keratinocytes via glucosylceramide synthase [6] and http://dx.doi.org/10.1016/j.bbrep.2015.11.016 2405-5808/& 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). S. Usuki et al. / Biochemistry and Biophysics Reports 5 (2016) 160–167 161 Fig. 1. (A). Chemical structure of typical ceramide species: C24Cer, one of the major species in human epidermis, and kCer, one of the major species in Amorphophallus konjac. (B) Time course of EGCase I reaction. The enzyme reaction was performed at 37 °C in sodium acetate buffer pH 5.0 with kGlcCer, 135 nmol; EGCase I, 70.3 mU. After the enzyme reaction, Bligh–Dyer extracts were prepared and were examined by development of TLC using chloroform:methanol:acetic acid (65:10:0.3, v/v) as solvent. TLC plates were sprayed with 10% cupric sulfate in 8% phosphoric acid, and heated at 180 °C. Plates were generally recorded using a photo scanner. The area density was quantitated using JustTLC software. (C) Bligh–Dyer extracts were subjected to medium pressure liquid chromatography on a Hi-Flash S column and programmed with a linear gradient elution from chloroform:methanol:acetic acid (99:2:0.4, v/v) to chloroform:methanol:acetic acid (99:4:0.4, v/v) for 10 min. Column fractions were analyzed by TLC as shown. (D) Molecular species of kCer and kGlcCer after EGCase I reaction were analyzed by LC–ESI–MS/MS. No differences in the relative amounts of kCer and kGlcCer were seen in the combinations of sphingoid bases (d18:2, t18:1) and hydroxyl fatty acids (C16:0-OH, C18:0-OH, C20:0-OH, C22:0-OH, C23:0-OH, C24:0-OH). sphingomyelin synthase [7], respectively, and function as an epidermal barrier of water permeability and external stimuli [8]. When this ceramide-enriched superficial structure is injured or acquires developmental dysfunctions, the external stimuli can penetrate into the barrier and irrita (...truncated)