MATERIA MELANICA: A PIGMENTED PERCEPTION

Patrick A. Riley
Cell Pathology Research Laboratories, Division off Molecular Pathology
Institute of Medical Research, UCL, Cleveland Street, London W1P 6DB

On July the 11th 1991 there was a total eclipse of the sun visible from Mexico at midday. It is clear from the enormous corona that this star is an immense source of energy. The primacy of this energy source in sustaining life on this planet was recognised in the Bronze Age - prehistoric solar religion. In particular, the early Egyptian civilization who embued the sun with divinity: the God Ra riding daily across the sky in his chariot. Although it is 96 million miles away the amount of energy falling on the earth's surface greatly exceeds any other source of energy. It is presently the ultimate source of all energy in the biosphere.

Origin of Life

It seems likely that life actually originated in conditions which permitted the utilisation of geochemical sources of energy. The generation of the first self-replicating systems (either proteins or nucleic acids) arose from the "primaeval soup" which, according to Orgel, had the consistency of a well-known packet soup made up to the manufacturers' specification.

Life as Negentropic State

We may define life as an "improbable state of order perpetuated in time". Life is thus an exception to the general time-dependent trend of increasing entropy which is characteristic of our present universe. To sustain this creation of order (anabolic activity) energy is required and this is derived from the catabolism or breakdown of order in other molecules. It is likely that molecules with large energy yields similar ATP. existed initially in plentiful supply in the primitive geochemically-generated environment.

Metabolic Coupling: Compartmentation

It is clear that an advantage would accrue to systems that could strongly couple catabolism to anabolic activity and this would result from containment of the reactants. This compartmentation (for example, by membranes, which would form spontaneously from amphypathic molecules such as phospholipids) permits the action of natural selection and therefore the advent of evolution in the sense described by Darwin. It should be noted that selection cannot operate in an open system - an example of this perhaps provided by economic systems.

Generation of an Electrochemical Gradient

Production of a semipermeable enclosed system enables the generation of an electrochemical gradient as a source of metabolic energy and this potential energy can be utilized by permitting the gradient To equilibrate by passing through a system which is coupled to an appropriate synthetic reaction. The mechanism of generating an electrochemical gradient involves electron transport across the containing membrane from an electron donor to an electron acceptor on the other side. Primitive systems may have used mechanisms based on external donors of electrons such as ammonia, sulphur dioxide, reduced iron and nitrite.

The basic system of generating an electrochemical gradient (of which an example still exists in Escherichia coli) requires linked exterior and interior catalytic reactions. In this case formate is oxidised to carbon dioxide and two electrons are transferred through an intermediate shuttle molecule to another catalytic site where fumarate is reduced to succinate. This latter reaction requires the donation of two protons and thus the electron transfer is the equivalent of a proton pump moving, in effect. one proton from the interior to the exterior of the organism. The electrochemical gradient achieved by this reaction is then able to drive important synthetic reactions such as ATP synthesis.

Autotropism: Evolution of Photochemical systems

The major evolutionary breakthrough was the development of autotropism in organisms by the evolution of photochemical reaction centres. This took place about 3x109 years ago in ancestors of the (present day) green sulphur bacteria. These prescient organisms took advantage of the photochemical principle enunciated by Einstein that a single quantum of light is sufficient to drive a chemical reaction. In the most primitive system (known as photosystem I) a magnesium-based porphyrin) absorbs one photon of light and excites an electron from the metal which is rapidly transferred to adjacent molecules in the complex producing a charge separation. The electron is passed to a shuttle mechanism, in this case ferredoxin, which passes it to an NADP reductase. The missing electron in the photoreceptor pigment is replaced by an electron from a suitable environmental donor molecule, in this case probably hydrogen sulphide since this has greater reducing potential (-230 mV compared to + 180 mV for water).

The next important step in the development of early living systems was the evolution of photosystem II in precursors of purple bacteria. This system uses light energy to excite electrons which are passed through a cytochrome complex which is able to use part of the energy to pump protons. The lower energy electon re-enters the cytochrome complex and is re-excited by light and passes round the system again. This is, perhaps, the first example of a purposeful electrical circuit.

Finally, a combination of both these photosystems in relatives of the cyanobacteria which occurred about 2.7x109 years ago gave rise to a system absorbing light energy which was able to use water (which was in plentiful supply) as the electron donor and which generated oxygen as the product. In this system, the electron excited by the first quantum of light is used to drive a proton pump but is then passed to another photosystem where absorption of a second quantum raises the redox potential of the electron which is used tcl reduce NADP. Various types of shunts and recycling are possible, for example the second photosystem can alternatively drive the prclton pump mechanism.

Transition from Reducing to Oxidizing atmosphere

Clearly the great significance of the evolution of photosystems was the generation of oxygen. There appears to have been a delay in the rise of atmospheric oxygen. The large banded deposits of iron oxide dated about 2x109 years ago suggest that this lag in the rise of oxygen concentration was due to the oxidation of a large pool of reduced iron in the oceans. The rise in oxygen in the atmosphere is dated about 1.5 x 109 years ago and the advent of atmospheric oxygen changed the chemistry of living systems. As Szent-Gyorgvi has put it, "life processes changed from the a to the state in which oxygen is the universal electron acceptor in the biosphere".

Evolution of microorganisms with oxygen-dependent metabolism

The abundance of organic molecules and oxygen permitted the evolution of micrclorganisms with electron transport systems adapted to transport electrons from NADH to oxygen. Such electron transport systems accept from NADH electrons which pass to oxygen through a series of proton-pumping complexes so that there is a gain in the electrochemical gradient. These systems were the forerunners of present-day mitochondrial electron transport.

Evolution of Eucarvotes (Evolution of Organelles/Endosymbiosisi

The next step in evolution seems to have involved phagocytosis of these organisms to form various types of endosymbiont. Probably the first step in this series involved autophagocytosis by a non-photosynthetic organism to give rise to the forerunners of nucleus and endop]asmic reticulum. Such a process would have the advantage of segregating the genetic material and increasing the effective surface area of the organism. It is possible that similar autoingestion processes gave rise to the Golgi apparatus and to peroxnomes.

Phagocytosis of oxygen-utilizing micro-organisms by primitive cells are thought to account for mitochondria, and plants may have evolved later by phagocytosis of photosynthetic bacteria to give chloroplasts.

All these organelles are self-repljcating and contain genetic andior epigenetic information required for their replication. Interestingly, genes coding for most of the components of mitochondria and chloroplasts have been transferred from these organelles to the nucleus. The reason for this is not clear but it is possible that this reduces the potential for oxidative damage to DNA at the sites of oxygen metabolism Transfer into the nucleus may therefore preserve the integrity of otherwise vulnerable regions of the DNA of organelles. The consequence of the gene transfer is that these endosymbiotic organelles require either all or most of their components such as lipids and proteins to be synthesized elsewhere in the cell and be transferred to them. Delivery depends on phospholipid exchange proteins for phospholipids and on various complex sorting signals for proteins.

Protein traffic: Sorting Signals

The general scheme for protein traffic in the cell may be represented as a binary pathway; certain proteins remaining in the cytosol with some being transferred secondarily to the nucleus, mitochondria, chloroplasts or peroxisomes; and other proteins being translocated into the endoplasmic reticulum space (which is topologically exterior to the cell) and then variously transported to the Colgi apparatus and sorted into lysosomes, secretory vesicles and plasma membrane-destined vesicles. The signalling systems are extremely complex and the full process, involving a number of accessory proteins that assist translocation and sorting such as chaperonins, may be divided to three categories: (1) amino acid sequences in the form of signal peptides or signal "patches" in the folded protein; (2) modification of amino acids to generate secondary signals, such as phosporylation of serine, methylation of glutamic acid, acetylation of lysine, the acylation of tyrosine, or the formation of acylated thiol esters at the carboxy terminus; (3) protein glycosylation, the major series being through the N-glycosylation of serine. Single sugar additions, such as in the N-acetylglucosamine "anchor" which attaches proteins to the cytosolic portion of the ER, or phosphatidyl inositol attachment to the carboxy terminus which seems to anchor some proteins to the inner surtace of the endoplasmic reticulum, are relatively rare. The major process involves the dolichol- mediated oligosaccharide transfer which occurs on the interior of the endoplasmic reticulum.

Subsequent processing occurs by limited proteolysis, and modification of the oligosaccharides. These modifications include phosphorylation of mannose and reorganization of the components of the oligosaccharides and take place mainly in the Golgi apparatus. Transfer between the various compartments occurs by vesicular transport which carries proteins through the cis, medial and trans compartments of the Golgi apparatus where various signal modifications take place. The proteins are finally sorted into categories in the trans-Golgi network and give rise to the sets of vesicles currently recognized, i.e. lysosomes, secretory vesicles and plasma membrane vesicles. The mechanims of budding of these vesicles appears to differ. Lysosomes appear to be formed from clathrin-coated vesicles containing mannose phosphate receptors. The sorting signals and budding mechanisms of other vesicular traffic are not clear.

The Nature of the Melanosome

With these processes of generation of intracellular organelles in mind we may begin to formulate a view about the nature of the organelle which is of specific interest to us as Pigment Cell Biologists - namely the melanosome. Although the long controversy in the past regarding the possible mitochondrial origin of melanosomes was effectively settled by the classic demonstration in Oxford by Seiji, Fitzpatrick and Birbeck (using the then novel technique of differential gradient centrifugation) that the melanosome could legitimately be regarded as a separate organelle, much of the present knowledge of melanosomes is the result of work carried out in Japan. As well as paying tribute to the work of Seiji we must acknowledge the important contributions of Jimbow and in particular the series of elegant studies by Mishima and his collaborators.

There has recently been a proposal by Gisella Moellman that the melanosome is a type of peroxisome. This suggestion was made on the basis of the catalase activity detected in melanosomes, and the amino acid sequence of tyrosinase which appears to have three amino acids of the carboxy terminus which resemble the peroxisomal signal sequence. This is an interesting idea and is consistent with the historical suggestion of Medawar that pigment spread in vertebrates occurs by transfection of adjacent pigment cells by self-replicating particles.

However, there are a number of features which, in my view, are inconsistent with the peroxisomal hypothesis. For example, melanosomes have a single membrane structure and appear to grow by vesicular fusion, like endolysosomes. No evidence has been reported of replicating melanosomes. They do not contain nucleic acid. Therefore, if they were to be epigenetically transmitted like peroxisomes, melanosomes would need to be present in all (or most) cells, and certainly in oocytes. I am not aware of any evidence of such epigenetic transfer of melanosomes in most species (although there may be some examples e.g. amphibia).

Secondly, the tyrosinases that have been analysed are known to be glycosylated and therefore cannot be destined for cytosolic distribution and subsequent incorporation into a self-replicating organelle such as a peroxisome. Moreover, activity attributed to tyrosinase has been detected by many investigators in the trans-membrane domain.

Thirdly, co-segregation of tyrosinase activity appears to be (predominantly) with lysosomal enzymes. The enzymes that have been detected in isolated and purified melanosomes include: acid phosphatase, aryl sulphatase, beta-galactosidase, b -N-acetylglucosaminidase, beta-glucuronidase, cathepsin D and alpha-mannosidase. This points strongly to co-segregation of tyrosinase with lysosomal enzymes, although some other enzymes have also been detected such as tryptophan-2,3-dioxygenase, tyrosine aminotransferase, ATPase, and gamma-glutamyl transferase which are not classical lysosomal enzymes.

Fourthly, there is evidence from some recent work by Alison Winder that fibroblasts transfected with the tyrosinase gene generate pigment products in lysosome-like vesicles.

If we add to this the suggestion that the carboxyl terminus of the tyrosinase is in the transmembrane portion of the enzyme (and therefore could not act as a signal for transfer to peroxisomes) and the recent demonstration by Sandra Naish-Byfield that the catalatic activity of tyrosinase is, at least in part, due to alternative copper- centred catalysis, I believe the case for suggesting that melanosomes are peroxisome-like organelles is greatly weakened.

If the melanosome is not a peroxisome in what category does it belong? From an evolutionary point of view it is probably a secretory vesicle since for example, in arthropods tyrosinase is externalised for its role in cuticular hardening. Also, in a sense, the triggered release of products such as in the spray glands of millipedes and bombadier beetles and certain cephalopods suggests a secretory role for the organelle. However. In higher plants and animals the vesicle is generally retained in the cell - although not invariably.

Evolution of Tyrosinase (Trends in Phylogeny)

In the presence of so distinguished and knowledgable an audience, it may be an error to venture into this dark forest of speculation. However, as this is a privileged occasion, I will touch on the subject. It would appear that the ancestral gene occurred very early in evolution, since tyrosinase appears to be a feature both of plants and animals. In the animal kingdom some divergence seems to have taken place leading to haemocyanins in the descendants of the protostomes.

Since evolution is conservative what could have given rise to tyrosinase? One possibility, I venture to suggest, might be from a precursor gene for cytochrome a since cytochrome a and a3 have a copper-binding site and also haem attachment sites. It is possible that such an origin could account for some of the features of the aminoacid sequence that are of current interest.

Evolutionary Significance of Orthoquinones

The major significance of tyrosinase appears to be as a mechanism for generating reactive orthoquinones. Orthoquinones have a different electronic structure, and different reactivity, to paraquinones. The latter are much used by living systems in electron transport as reversible electron acceptors and donors. In general, orthoquinones more readily undergo covalent binding to the ring by nucleophiles. Their evolutionary function may be connected with this property and orthoquinones appear to be important in micro-organisms, including fungi and bacteria as a type of generalised antibiotic. Orthoquinones also appear to be significant in protecting plants, and particularly fruits, from opportunistic predators such as insects or grubs, and we are all familiar with the blackening of fruits (e.g. bananas) when they are damaged. Orthoquinones are used by insects in their immune system and also in defensive sprays. In all probability they also form a significant repellent component of cephalopod ink. Another protective function appears to be the use of orthoquinones in strengthening protein coats by tanning of spores and seed pods and, of course, in the sclerotization of the insect cuticle.

Naturally, the chemical reactivity of orthoquinones also means that they are potentially precursors of polymeric pigments which we term melanin. It seems not improbable that melanogenesis evolved as a detoxification pathway for orthoquinones generated for the purposes to which I have just alluded. Polymerisation may have been utilized to maintain an acceptable steady-state concentration of orthoquinone either at the periphery of the organism or in melanosomes.

Whilst the precise mechanism of melanogenesis has not yet been clarified, the process finally results in the generation of a bathochromic indolic polymer of irregular structure with important physicochemical properties, including a wide spectral absorbtion, semiconductor properties, stable radical properties, phonon-photon coupling, easy formation of charge-transfer complexes, and strong cationic binding. The biological applications and significance of these properties is properly the topic of another dissertation.

Conclusion

I am concious that what I have presented is a sketchy and speculative evolutionary view of melanogenesis; it has, of course, posed more questions than it has answered.

Nevertheless, I believe that our salvation as a species lies in our rationality and we are bound together by having Science as our philosophy (I mean 'Science' in the Popperian sense). In this discourse I have employed the principle of parsimony, first expounded by William of Ockham but, of course, we must remain cognizant of the complexity of our universe. You will have detected that I am guided by the maxim of Alfred North Whitehead 'seek simplicity and distrust it'.

Acknowledgements

This text is an abridged version of the Inaugural Lecture delivered to the 3rd ESPCR Scientific Meeting in Amsterdam, September 1991.

I am grateful to Dr Wiete Westerhof, Chairman of the Organizing Committee for the invitation to give this lecture. I am indebted to many individuals who have helped, directly or indirectly, to embue me with knowledge and to mould my views. In particular I thank Dr Jan Borovansky, Professor Peter Campbell, Dr Sandra Naish-Byfield and Dr Anthony Smith. Any blame, however, attaches to me alone.