**Retina, Retinol, Retinal and the Natural History of Vitamin A as a Light Sensor**

#### **Ming Zhong, Riki Kawaguchi, Miki Kassai and Hui Sun**

**Abstract:** Light is both the ultimate energy source for most organisms and a rich information source. Vitamin A-based chromophore was initially used in harvesting light energy, but has become the most widely used light sensor throughout evolution from unicellular to multicellular organisms. Vitamin A-based photoreceptor proteins are called opsins and have been used for billions of years for sensing light for vision or the equivalent of vision. All vitamin A-based light sensors for vision in the animal kingdom are G-protein coupled receptors, while those in unicellular organisms are light-gated channels. This first major switch in evolution was followed by two other major changes: the switch from bistable to monostable pigments for vision and the expansion of vitamin A's biological functions. Vitamin A's new functions such as regulating cell growth and differentiation from embryogenesis to adult are associated with increased toxicity with its random diffusion. In contrast to bistable pigments which can be regenerated by light, monostable pigments depend on complex enzymatic cycles for regeneration after every photoisomerization event. Here we discuss vitamin A functions and transport in the context of the natural history of vitamin A-based light sensors and propose that the expanding functions of vitamin A and the choice of monostable pigments are the likely evolutionary driving forces for precise, efficient, and sustained vitamin A transport.

Reprinted from *Nutrients*. Cite as: Zhong, M.; Kawaguchi, R.; Kassai, M.; Sun, H. Retina, Retinol, Retinal and the Natural History of Vitamin A as a Light Sensor. *Nutrients* **2012**, *4*, 2069-2096.

#### **1. Sunlight and Vitamin A**

The prevalent light source throughout evolution has been sunlight shining on the surface of the earth. For billions of years, vitamin A biology has been tightly linked to sunlight. Living organisms use sunlight primarily as a source of energy, the source of information for vision, and an indicator of time. Remarkably, vitamin A-based chromophore has evolved as the light sensors for all three usages (Table 1). Archaebacteria use vitamin A-based light-driven pumps to harvest light energy (e.g., by creating the electrochemical gradient of protons to drive ATP synthase). This is an alternative mechanism to chlorophyll-based phototrophy. For adjusting the biological clock, vitamin A-based photoreceptor proteins are used as the light sensors, although flavin-based photoreceptor proteins have also been used for this purpose. However, for vision or the equivalent of vision, the vast majority of species use vitamin A-based chromophore as the light sensor. Vitamin A-based chromophore is the exclusive choice for vision in multicellular organisms.

Even vitamin A's name is tightly linked to vision. The scientific name for vitamin A derivatives is retinoid, which is derived from the word "retina". Retinoids include retinol (the alcohol form), retinal (the aldedyde form, also called retinaldehyde or retinene) and retinoic acid (the acid form). Although vitamin A existed as a chemical before it functioned as a vitamin and retinal existed as a light sensor before there was a retina, we still use these names to refer to these chemicals.

What makes vitamin A so special for vision (the perception of light)? Why was vitamin A repeatedly chosen by evolution as the sensor for sunlight? What determined the region of the electromagnetic spectrum that is visible to the human eye? There are two important factors that provide likely answers for these related questions. First, the conjugation of the aldehyde end of retinal to photoreceptor proteins causes a red shift in its absorbance to the visible range (from the perspective of human vision). Visible light (visible due to vitamin A-based light sensors) generally matches the peak irradiance of sunlight on the earth's surface [1,2]. In contrast, most other light sensors absorb primarily in the UV range (e.g., flavin-based light sensors). Second, the large light-induced conformational change of vitamin A-based chromophore makes it ideal as a ligand for membrane receptors. The large conformational change likely makes it easier for the photoreceptor protein to distinguish the silent state (in the dark) and the activated state (in the light).

#### *1.1. The First Major Switch in the Evolution of Vitamin A-Based Light Sensors*

All vitamin A-based photoreceptor proteins are called opsins. All opsins in the animal kingdom that sense light for vision (visual pigments) are G-protein coupled receptors. Visual pigments sense light for daytime and nighttime vision and encode wavelength information of light for color vision [3]. All opsins in the animal kingdom are homologous to visual pigments. In contrast, opsins in unicellular organisms are light-gated channels (for light sensing) or light-driven pumps (for harvesting light energy) [4]. The switch from ion channel and pumps to G-protein coupled receptors is the first major event in the evolution of vitamin A-based light sensors (Table 1). Opsins that are light-gated ion channels or pumps use all-*trans* retinal as the chromophore, while opsins that are G-protein coupled receptors all use 11-*cis* retinal as the chromophore (Table 1 and Figure 1). The vitamin A-based light sensors listed in Table 1 are not meant to be all inclusive because some species contain surprisingly large numbers of opsins, and the functions of some opsins are still not well understood. Using humans and mice as an example, the human retina has long-, medium- and short-wave visual pigments in cone photoreceptor cells [5,6], rhodopsin in rod photoreceptor cells [7–10], melanopsin in ganglion cells [11–17], peropsin in the apical microvilli of the retinal pigment epithelium (RPE) [18] and RGR in the intracellular membranes of the RPE [19–22] (Figure 2). The mouse retina does not express the long-wave cone pigment [23], but has an additional opsin called neuropsin, which is mostly localized to the amacrine and ganglion cell layers [24,25].

**45**

Although there is tremendous diversity in the absorption maxima of cone visual pigments, the peak absorbance of rhodopsin, the dim light receptor, in many species is 500 nm. Why not 450 nm or 550 nm? One likely explanation is that the wavelength of the peak irradiance of sunlight on earth surface is 500 nm. Moonlight, the dominant light in natural world at night, is reflected sunlight and thus has same peak irradiance. The peak absorbance of rhodopsin matches this peak irradiance to achieve maximum sensitivity to available light. In contrast, maximum sensitivity is less important for cone visual pigments, which diversify their absorbance maxima for color vision. An extreme example of how available light determines the absorption spectra of visual pigments is the color vision of coelacanth, which lives at a depth about 200 m. To detect color and light in deep ocean where available light spans a very narrow range around 480 nm, coelacanth has only two visual pigments with absorption maxima of 478 nm and 485 nm, respectively [26]. This is in sharp contrast to another extreme example of a fish that has vision both above and below water (*Anableps anableps*). This fish has ten different opsins to adapt to vision both above and below water [27]. Visual pigments can achieve the exact absorption maximum that meets the organism's biological need through several mechanisms of spectral tuning. The common mechanism of spectral tuning is to change the protein environment that surrounds the chromophore [28–35]. Generally, opsin environments that encourage ʌ-electron delocalization of retinal cause a red shift in the absorption maximum. Another mechanism is by changing the structure of the chromophore itself. Although retinal is the universal chromophore for all vitamin A-based light sensors, the exact isomer of retinal can be different between species (Figure 1). For example, aquatic animals are known to shift absorption maxima of visual pigments by using the A1 (11-*cis* retinal) or A2 (11-*cis*-3,4 dehydroretinal) chromophore [36–40]. There are also examples of terrestrial vertebrates using vitamin A2-based visual pigments, which belong to the most red-shifted visual pigments (e.g., absorption maximum of 625 nm) [41]. A2 pigments absorb longer wavelengths of light compared to the A1 version because of the extension of the conjugated chain of the chromophore.


**Table 1.** Evolution of vitamin A-based light sensors (opsins) from bacteria to humans. The symbol # denotes the sensing of light by visual


**Table 1.** *Cont.* 


**Figure 1.** Examples of structural divergence of biologically active retinoids. For simplicity, only representative biologically active endogenous retinoids are shown.

**Figure 2.** Schematic diagram of the localization of various opsins in human and mouse retina and retinal pigment epithelium (RPE). Only cells or cellular structures that express opsins are shown and are color-coded. There are species variations. Human, but not mouse, has the long-wave cone pigment. Neuropsin is expressed in the mouse retina, but not in the human retina.

*1.2. The Second Major Switch in the Evolution of Vitamin A-Based Light Sensors* 

The second major change in the evolution of vitamin A-based light sensors is the emergence of monostable pigments (Table 2). All opsins before vertebrate visual pigments (from opsins of unicellular organisms to invertebrate opsins) are bistable pigments, which can be regenerated by light after photobleaching (Table 2). All vertebrate visual pigments are monostable pigments, which release the chromophore after every photoisomerzation event and depend on an enzymatic cycles called the visual cycle to regenerate [42–46]. To compete with bleached rhodopsin for chromophore in daylight, cone visual pigments have their unique regeneration pathway that is different from the visual cycle that regenerates rhodopsin [47–51]. The isomerase in this cone-specific pathway has now been identified [52]. The mechanisms of chromophore release by bleached vertebrate rhodopsin and cone pigments have been studied recently [53,54]. Compared to the regeneration of the bistable pigments by light, the regeneration of monostable pigments requires much more complex mechanisms involving many enzymes and transport proteins (Table 3). The regaining of the 11-*cis* retinal by the monostable pigment after light-induced release is an important factor affecting the dark adaptation of photoreceptor cells [44]. Without the chromophore, the opsin apoprotein itself can activate signal transduction [55,56].


**Table 2.** Convergent and divergent events in the evolution of vitamin A-based light sensors.

Vision is known to optimize energy use [57,58]. For chromophore regeneration, vertebrate photoreceptor cells took the seemingly paradoxical evolutionary choice of using the much more energy-inefficient monostable pigments. In contrast, bistable pigments are regenerated by light after photobleaching without any cellular energy. Comparing the energy efficiency of the two mechanisms is analogous to comparing heating a building using fossil fuel versus solar energy. To make it even more "wasteful", we need to constantly consume cellular energy to regenerate bleached rhodopsin in daylight, even when rod photoreceptor cells are completely saturated and do not contribute to visual perception. In contrast, bistable pigments only need enzymatic regeneration when the photoreceptor protein is degraded, not when it is bleached [59]. This regeneration is important during nutritional deficiency.

To convert the released free all-*trans* retinal back to 11-*cis* retinal for monostable pigments, evolution produced many proteins dedicated to the visual cycle. All these proteins are potential causes of blinding diseases. An example is ABCA4 (ABCR) [60,61], whose surprising existence is a testament to the sophistication of the visual cycle. ABCA4 is an ATP-dependent membrane transport protein in photoreceptor disc membranes, and its function is to accelerate the transport of retinal conjugate across photoreceptor disc membranes [62–68]. Loss of ABCA4 function leads to the accumulation of A2E, a toxic bis-retinoid adduct and delayed dark adaptation.


**Table 3.** Comparison of bistable pigments and monostable pigments.

ABCA4 mutations are associated with several blinding diseases in humans, including Stargardt macular dystrophy [69], cone rod dystrophy [70] and retinitis pigmentosa [70,71]. Since ABCA4 functions to accelerate the regeneration of monostable pigments and species that do not have monostable pigments naturally lack ABCA4, human diseases associated with ABCA4 ultimately originated from the choice of monostable pigments during evolution. There may still be other unknown components of the visual cycle. For example, ABCA4 is expressed in the disc membranes of photoreceptor cells where it can play no role in retinal transport between the RPE and photoreceptor cells.

Although vertebrates exclusively use monostable pigments for vision, theydo have endogenous bistable pigments in the inner retina including melanopsin [72–75] and neuropsin [25,76]. Exogenously expressed bistable pigments from unicellular organisms even function well both in the vertebrate retina [77] and the brain (as employed by the technique optogenetics) [78]. Bistable pigments can be repeatedly stimulated by light in vertebrate neurons that have no access to the visual cycle [78]. Why did evolution come up with monostable pigments, which require much more "maintenance"? Despite the many advantages of bistable pigments, there have to be very good reasons for monostable pigments to exist as the universal pigments for vertebrate vision. The first likely reason is survival in the dark. Vision at night can offer tremendous survival advantages for both predators (e.g., to find more prey) and prey (e.g., to avoid predators). Unlike bistable pigments, monostable pigments can regenerate in complete darkness and therefore are likely more suitable for continuous night vision. Bistable pigments can be formed in the dark only in the initial formation of the bistable pigment [79]. Another possible advantage is color vision. Monostable pigments may be more precise in discriminating different wavelengths of light (the basis of color vision) because the response of a bistable pigment to light is confounded by its two absorption maxima (one for activation and one for regeneration). There may be other reasons to justify the choice of this highly energy-consuming and disease-prone regeneration mechanism for visual pigments.

#### **2. Broadening of the Biological Functions of Vitamin A**

#### *2.1. Expanding Biological Functions of Vitamin A*

If vitamin A is taken in for vision, why not use it for something else? That's exactly what happened in evolution (Figure 1 and Table 2). Vertebrates broaden the use of vitamin A to many other essential biological functions, including its essential roles in embryonic development, maturation of the immune system, maintenance of epithelial integrity, and in the adult brain for learning and memory and neurogenesis [80–87]. This is the third major change in the biology of vitamin A. Most of these new functions are mediated by the acid form of vitamin A (retinoic acid) [88,89]. Since this functional diversification in evolution, vitamin A deficiency would no longer be limited to effects on vision, and vitamin A became an essential nutrient for almost all vertebrate organs.

Vitamin A deficiency affects many vertebrate organs [90,91]. The most well known effects of vitamin A deficiency in humans are night blindness [92] and increased childhood mortality and morbidity [93]. In adults, vitamin A deficiency can lead to profound impairment of hippocampal long-term potentiation and long-term depression [94] and impairment in learning and memory [95]. Vitamin A deficiency can also lead to pathological changes in the lung [96,97], the skin [98], the thyroid [99] and the male and female reproductive systems [90,100]. It was recently discovered that retinol, but not retinoic acid, prevents the differentiation and promotes the feeder-independent culture of embryonic stem cells [101]; retinal inhibits adipogenesis [102]; and retinoic acid regulates protein translation in neurons independent of its roles in regulating gene transcription [103,104]. Given its numerous biological functions, retinoid plays positive or negative roles in a wide-range of human diseases, such as visual disorders [45], cancer [105,106], infectious diseases [82], diabetes [107,108], teratogenicity [109], and skin diseases [110].

#### *2.2. Retinoid Toxicity Associated with the Evolution of Vitamin A Functions*

Broadened biological activity of vitamin A is a double-edged sword that also leads to broader toxicity caused by excessive vitamin A or its derivatives (Table 4). Retinoid toxicity can be caused by physical properties of retinoid (e.g., acting like a detergent at sufficient concentrations), chemical reactivity of retinoid (e.g., modification of random proteins by free retinal), or inappropriate biological activities (e.g., retinoic acid activating or suppressing gene expression at the wrong cell type or at the wrong time) (Table 4). Excessive vitamin A uptake can lead to severe toxicity in humans [109,111–113]. Water-miscible, emulsified, and solid forms of retinol are much more toxic than oil-based retinol preparations [114]. Excessive retinoic acid is even more toxic than retinol, consistent with the fact that retinoic acid is more biologically active [115]. Retinoid therapy for human diseases is often associated with side effects such as terotogenicity [109,115,116]. Chronic exposure to clinical doses of 13-*cis* retinoic acid suppresses hippocampal neurogenesis and disrupts hippocampal-dependent memory [117]. In addition, 13-*cis* retinoic acid intake causes night blindness [118].

Retinal is the vitamin A derivative that is most toxic, due to its chemical reactivity. Even when vitamin A is used only for light sensing, retinal can be toxic [119] due to its chemical toxicity in randomly modifying proteins through Schiff base formation. Retinal toxicity becomes more severe for organisms using monostable pigments, which constantly release free retinal in daylight. As a protein that interacts with retinal, ABCA4 in photoreceptor cells is sensitive to retinal-mediated photooxidative damage [120]. A photoreceptor cell culture study revealed that retinal is much more toxic than retinol in mediating photooxidative damage [121]. Photooxidation caused by all-*trans* retinal released from monostable pigments has been observed in single vertebrate photoreceptor cells [122]. Knocking out both ABCA4 and RDH8, two genes that function to reduce retinal toxicity, causes severe retina degeneration [123]. The constant release of free retinal in daylight by the monostable pigments also paves the way for the generation of a toxic chemical derived from retinal called A2E, a unique vitamin A derivative found in vertebrate eyes that has only toxicity but no beneficial function [124–130]. In a sense, A2E ultimately results from the choice of monostable pigments in evolution due to their constant release of free all-*trans* retinal and the demand for 11-*cis* retinal in daylight.


**Table 4.** Biological functions and toxicities of vitamin A derivatives in vertebrates.
