Nrp1 is Activated by Konjac Ceramide Binding-Induced Structural Rigidification of the a1a2 Domain.

Seigo Usuki,Tomohiro Tamura,Kunikazu Tanji,Kohei Yuyama,Kenji Monde,Daisuke Mikami,Takashi Saitoh,Yoshiaki Yasutake,Yuta Murai,Yasuyuki Igarashi,Noriko Tamura,Katsuyuki Mukai

doi:10.3390/cells9020517

Abstract

Konjac ceramide (kCer) is a plant-type ceramide composed of various long-chain bases and α-hydroxyl fatty acids. The presence of d4t,8t-sphingadienine is essential for semaphorin 3A (Sema3A)-like activity. Herein, we examined the three neuropilin 1 (Nrp1) domains (a1a2, b1b2, or c), and found that a1a2 binds to d4t,8t-kCer and possesses Sema3A-like activity. kCer binds to Nrp1 with a weak affinity of μM dissociation constant (Kd). We wondered whether bovine serum albumin could influence the ligand–receptor interaction that a1a2 has with a single high affinity binding site for kCer (Kd in nM range). In the present study we demonstrated the influence of bovine serum albumin. Thermal denaturation indicates that the a1a2 domain may include intrinsically disordered region (IDR)-like flexibility. A potential interaction site on the a1 module was explored by molecular docking, which revealed a possible Nrp1 activation mechanism, in which kCer binds to Site A close to the Sema3A-binding region of the a1a2 domain. The a1 module then accesses a2 as the IDR-like flexibility becomes ordered via kCer-induced protein rigidity of a1a2. This induces intramolecular interaction between a1 and a2 through a slight change in protein secondary structure.

Highlights

Semaphorin 3A (Sema3A) is a secretory signaling protein, belonging to the large semaphorin family, that acts as a guidance signal for growth cone collapse and nerve repulsion in axonal outgrowth [1], and as a guidance signal for intracellular repulsion for axonal trafficking [2] through activation of semaphorin 3A (Sema3A) signaling, via the Sema3A receptor neuropilin 1 (Nrp1)
Alkaline phosphatase (AP)-Sema3A in Co-IP samples was increased to 2−4 pmol for Nrp1 and 3−5.5 pmol for a1a2
Ceramides are composed of sphingosine and fatty acids, and frequently contain sphingolipids such as glycosphingolipids, sphingomyelins, and free ceramides on the mammalian cell membrane [31]

Summary

Introduction

Semaphorin 3A (Sema3A) is a secretory signaling protein, belonging to the large semaphorin family, that acts as a guidance signal for growth cone collapse and nerve repulsion in axonal outgrowth [1], and as a guidance signal for intracellular repulsion for axonal trafficking [2] through activation of Sema3A signaling, via the Sema3A receptor neuropilin 1 (Nrp). Sema3A is associated with various physiological and pathophysiological processes, such as cell migration, immune responses, angiogenesis, and cancer [3]. Sema3A is often up-regulated in some cancers [4,5,6] but down-regulated in others [7,8]. These conflicting effects indicate that Nrp may be a dual receptor for vascular endothelial growth factor (VEGF) and Sema3A. Sema3A may appear to be an ambiguous target for the development of cancer therapies.

Methods

Results

Conclusion