Olfactory marker protein contains a leucine-rich domain in the Ω-loop important for nuclear export

Abstract

Olfactory marker protein (OMP) is a cytosolic protein expressed in mature olfactory receptor neurons (ORNs). OMP modulates cAMP signalling and regulates olfactory sensation and axonal targeting. OMP is a small soluble protein, and passive diffusion between nucleus and cytoplasm is expected. However, OMP is mostly situated in the cytosol and is only sparsely detected in the nuclei of a subset of ORNs, hypothalamic neurons and heterologously OMP-expressing cultured cells. OMP can enter the nucleus in association with transcription factors. However, how OMP is retained in the cytosol at rest is unclear. Because OMP is proposed to affect cell differentiation, it is important to understand how OMP is distributed between cytoplasm and nucleus. To elucidate the structural profile of OMP, we applied several bioinformatics methods to a multiple sequence alignment (MSA) of OMP protein sequences and ranked the evolutionarily conserved residues. In addition to the previously reported cAMP-binding domain, we identified a leucine-rich domain in the Ω-loop of OMP. We introduced mutations into the leucine-rich region and heterologously expressed the mutant OMP in HEK293T cells. Mutations into alanine increased the nuclear distribution of OMP quantified by immunocytochemistry and western blotting. Therefore, we concluded that OMP contains a leucine-rich domain important for nuclear transport.

Keywords: Bioinformatics; Hypothalamic neurons; Leucine-rich sequence; Multiple sequence alignment (MSA); Nuclear export signals (NES); Nuclear transport; Olfactory marker protein (OMP).

Fig. 1

OMP occasionally enters the nucleus in neurons and cultured cells. a, b OMP-IR in the mouse olfactory epithelium (OE). OMP-IR was detected in the cytosol from dendritic knob to somas. OMP-IR was usually not detected in the nuclei of ORNs (a), but some ORNs exhibited OMP-IR inside nuclei. c, d Closed views of ORNs with OMP (c) exclusively in the cytosol or occasionally (d) in the nucleus. Note that OMP-IR even in the nucleus was excluded from dense nuclear foci in diamidino-2-phenylindole (DAPI) staining (white arrow). e OMP-IR was detected in neurons in the paraventricular nucleus (PV) of the hypothalamus. f, g Close views of neurons in the paraventricular neurons with OMP-IR f exclusively in the cytosol or g in both the cytosol and the nucleus. 3V, third ventricle. h, i OMP-IR in HEK293T cells. OMP-IR was detected either h exclusively in the cytosol or i in both the cytosol and the nucleus. The cells with OMP-IR in the cytosol or in the nucleus are labelled with an open star or N, respectively, in a–i

Fig. 2

OMP contains a leucine-rich sequence in the Ω-loop. a Spectral views of the MSA of OMP proteins from various vertebrate phylogenies. The amino acid number was expanded to the comprehensive serial number. Highly scored residues by b, c around the leucine-rich sequence and cyclic nucleotide-binding domain (CNBD) are highlighted. B–d Spectral views for the b entropy, c LIST-S2 metric and d PKV. In d, the dotted lines were added for visibility of the similarly scored distant residues. e Crystal structure of OMP. A highly conserved leucine-rich sequence region was detected in the Ω-loop of OMP. CNBD, cyclic nucleotide-binding domain. f Overlay of different conformations of OMP depicting that the Ω-loop is flexible

Fig. 3

Mutations in the leucine-rich sequence alter the nuclear export efficacy of OMP. a, b Representative pictures of OMP-IR in HEK293T cells expressing a OMP^WT or b OMP^A. c Quantification of the cell numbers (%) showing nuclear OMP-IR with apparent signal voids surrounding dense nuclear foci among all OMP-IR cells transfected with OMP^WT (filled blue circles) and OMP^A (filled red squares). n = 5 experiments each. One-tailed Student’s T test: OMP^WT vs. OMP^A, *P = 7.36 × 10^–10. d The OMP-IR ratio of the nucleus/cytoplasm (N/C ratio). n = 100 cells for OMP^WT and 102 cells for OMP^A. One-tailed Student’s T test: OMP^WT vs. OMP^A, **P = 6.97 × 10^–34. e OMP-IR from cytoplasmic (C) and nuclear (N) fractions extracted from HEK293T cells expressing OMP^WT or OMP^A. f N/C ratio for the cell-fractional OMP-IR detected by western blotting in e. One-tailed Student’s T test: OMP^WT vs. OMP^A, ^#P = 0.0089. g Hydrophobicity of OMP^WT (blue) and OMP^A (red) around the Ω-loop. +, mutated residues