Have we helped solve the forty-year-old mystery of the “OBP” function?

Published on: 12/16/2021
Author/s : Jean-François Picimbon / Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100, P.R. China.
Odorant Binding Proteins (OBPs, https://en.wikipedia.org/wiki/Odorant-binding_protein) are small soluble “bowl-like” proteins composed of 6 Cys, six-seven α-helices and a central hydrophobic pocket. This structure is largely described in insects and strongly believed to play a key role in the transport of hydrophobic chains such as odorant/pheromone molecules. Following their discovery in the antennal branches of moths, they have been named pheromone-binding proteins (PBPs) and general odorant-binding proteins (GOBPs) to distinguish between functions entirely, exclusively and strictly tuned to the detection of specific odors, i.e. sex pheromones and kairomones released by host-plants. After Vogt and Riddiford (1981), the concept of OBP, PBP and GOBP, strictly tuned to olfaction came from extremely high protein concentration in the sensillum lymph surrounding the olfactory receptors to odor molecule fully integrated in the binding pocket of the protein (X-ray view, Sandler et al., 2000).
However, the physiological view was poorly analyzed compared to structural and binding properties. After Picimbon (2003), PBPs comprise a single gene cluster that arose through early gene duplication as shown in an evolutionary analysis of moth’s (Picimbon and Gadenne, 2002; Abraham et al., 2005). OBPs, including PBPs and GOBPs, may have split at a very early stage over the course of evolution, i.e. back to the birth, emergence and diversification of prokaryotic cells such as fungi and bacteria, ~ 2 Bya (Picimbon, 2019). Such an early set of evolution and gene expression in bacteria seriously conflict with a function narrowly tuned to the olfactory system. How could an OBP from bacteria express for odors?
The PBP from geometrids that use polyenic hydrocarbons as pheromones is very similar to noctuid’s. Closely related moth species have similar PBPs even when they evolve different pheromone systems (Abraham, 2002; Picimbon and Gadenne, 2002; Picimbon, 2003; Abraham et al., 2005).
When PBPs and GOBPs were first identified, a major criterion of interest was their antennal specificity; a protein so specific to the (male) antennae would certainly drive a function to pheromone recognition. However, a protein that is largely expressed in the antennae but not exclusively expressed in antennae does not necessarily imply olfactory functions. This is the case of PBPs and GOBPs, known to have given the name “OBPs” to the whole protein family on the assumption of olfactory function in insects. Gene expression solely in the antennae refers to antennal-specific protein and strongly suggests odorant-binding in olfactory function. After a complete survey of gene expression in multiple tissues across the various developmental stages of the silkworm moth Bombyx mori, it turns out that PBPs and GOBPs are NOT specific to the antennae. They are expressed in high amounts in the antennae and legs (in aging females) but they are also expressed in high amounts in the thorax, gut, fat body and other metabolic tissues, particularly in response to sublethal insecticide stress exposure (Guo et al., 2021). This is not a tissue expression profiling we would expect for an olfactory binding protein (Figure 1).
Similarly, focus on the functional binding site and binding experiments using only one single category of ligands is not enough to assess potential mechanism and true physiological relevance of the protein. Analyzing protein structure, additional information is required to understand the physiological context in which the protein activity is used. Where the protein located, and what are the regulatory elements. When combined with the tissue expression profiling of the gene in eggs, larvae and nymph stages, information concerning the potential role of “OBP” in developmental processes can be determined (Figure 1). When PBPs and GOBPs are effective part of changes in biochemical responses required for physiological adaptations of the moth exposed to chemical stress, even more information concerning the potential role of “OBP” in metabolic processes can be provided (Guo et al., 2021).
Guo et al. (2021) report about a very rigorous study in examining spatio/temporal expression for OBPs, in particular in response to insecticide exposure. The extremely broad expression of PBPs and GOBPs, from the antennae and head to thorax, gut and fat body (Figure 1), expression in eggs, embryo and ageing (elderly) insects, strongly agree with the structural data also reported in Guo et al. (2021). For the past 40 years, most of the studies in insects (and vertebrates) have considered only one hypothesis “OBP for olfaction”. This reference exclusively tuned to olfaction also applies to all functional analyses and binding studies conducted on OBPs. Based upon our extensive physiological study in the silkworm, PBP/GOBP in metabolic processes for growth and immunity becomes a stronger research hypothesis.
“OBPs” such as PBP and GOBP are detected in the head (brain) and the early embryo, perhaps suggesting a function in neuroplasticity and/or neurogenesis for these proteins. The presence of OBPs in the silk gland raises more questions regarding the paradigm of their functionality tuned to nerves. To solve the OBP mystery, we have carried out the work around the theme “physiological regulation”, and we investigated OBP using multiple experiments, analyzing this gene at different developmental stages, in males and females, and using biochemical, molecular and structural approaches in Guo et al. (2021). When the studies of OBPs over 40 years are focusing only on interaction with odors and pheromones, the induction of PBPs and GOBPs in metabolic tissues in response to insecticide exposure adds new profound interest to the OBP protein gene family that appears to be much more versatile than believed so far. In Guo et al. (2021), age-, development- and tissue-dependent expression of OBPs have been studied in moths, with a protein structure in relation with physiology, something expected to solve the true function of the protein. The work refers to a high amount of tissues covered as well as specific physiological conditions (insecticide stress and biochemical changes in metabolic tissues). The work is rather descriptive, but the description argues strongly for non-olfactory functions of OBPs. The role of PBPs and GOBPs was tuned to pheromone. Using docking, we report that vitamin could be selective and potent ligand for PBP/GOBP (see Figures 2-4). Vitamin binding is in very good agreement with the ubiquitous expression of PBPs and GOBPs revealed in Guo et al. (2021). Vitamins act as both catalysts (coenzymes or co-pilots that increase the rate of metabolic reactions) and key participants in the chemical reaction. In a variety of building blocks, they assemble and pair up with multiple enzymes in order to help fulfill multiple functions. Accordingly, we posit a new hypothesis in which OBPs are pleiotropic carrier proteins that function in sensory systems, CNS, brain cognitive functions, silk and pheromone production, development, metabolism, insect defense, disease prevention and adaptation, for which micronutrient intake is essential or even vital.
Have we helped solve the forty-year-old mystery of the “OBP” function? - Image 1
Figure 1. Tissue expression analysis of “odorant“-binding proteins. PBP1: “pheromone”-binding protein type 1, PBP2: “pheromone”-binding protein type 2, GOBP1: “general odorant”-binding protein type 1, GOBP2: “general odorant”-binding protein type 2. PBPs interact with pheromones, while GOBPs are turned to plant flower odor detection. Tissue distribution of PBPs and GOBPs does not agree with this assumption. Moth/Ant: Antennae, Lg: Legs, Th: Thorax, G: Gut, FB: Fat body, Wg: Wing, Ep:Epidermis, HPL: Hair pencils (male aphrodisiac). The triangles (oriented down) show down-regulation. Larger circles indicate up-regulation upon exposure to insecticide chemical (dose: 4.2 ppm). Metabolic regulation occurs at both PBP and GOBP levels in thorax and fat body. Developmental distribution of PBPs and GOBPs also does not agree with the assumption “OBP/PBP/GOBP tuned to odor detection”. PBPs and GOBPs are expressed in many various tissues from embryo, larva, nymph and mature adult.
Have we helped solve the forty-year-old mystery of the “OBP” function? - Image 1
Figure 2. PBP/GOBP-vitamin K docking results. PBP1 and GOBP2 interact with vitamin K1 and vitamin K2, respectively. The relative binding affinities of OBP to vitamin compounds is much higher than those measured for the odorant ligand molecule (bombykol). G: -11.8/-11.1 vs -7.4-4.3.
Have we helped solve the forty-year-old mystery of the “OBP” function? - Image 2
Figure 3. Overlap of odorant, vitamin K1 and protein binding sites within Bombyx mori PBP1. The same side-chain overlap (interaction) is found for bombykol (sex pheromone) and vitamin K1 (micronutrient, essential for the proper functioning of the metabolism). In the same pose, the affinity of PBP1 to K1 is significantly much higher than the affinity measured for the odorant molecule, providing structural information for high affinity binding of non-semiochemical ligand to PBP1 protein. Not only the aliphatic side-chain, but also the naphtoquinone ring points in the central cavity of PBP1.
Have we helped solve the forty-year-old mystery of the “OBP” function? - Image 3
Figure 4. Mapping of vitamin-binding cavity in the “PBP1” of the silkworm moth Bombyx mori.

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