Chapter 9
Invertebrate Calcium Concretions
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Novel Biomineralization Systems Harold Silverman, Jody M. Myers, and Thomas H. Dietz Department of Zoology and Physiology, Louisiana State University, Baton Rouge, LA 70803 Molecules influencing natural biomineralization processes have been instructive models for choosing and designing chemical agents for industrial mineralization processes and, perhaps more importantly, in industrial strategies for the inhibition of mineralization. Vertebrate bones, teeth and invertebrate shells have been the most extensively studied. This report documents a naturally occurring and widespread biomineralization phenomenon, the invertebrate calcium concretions. These concretions may lead to the discovery of additional novel peptides, peptide complexes or other polymers that may have important implications to the initiation and inhibition of mineralization processes. We review the invertebrate concretions presently known and the extent of our chemical knowledge of these concretions, with an emphasis on the concretion forming system in bivalves, where a novel phosphate-binding protein-complex has been identified. Many promising molecules for industrial use, either as inhibitors or initiators of mineralization processes, mimic molecules initially discovered in, and isolated from, biological mineralization systems (e.g.,]^. Biomineralization systems that have been extensively studied include vertebrate bone and teeth, and invertebrate shells and tests. These systems produce biominerals largely in an extracellular environment. The mineral in all of these cases is associated with a small amount of organic material (as low as about 0.1% by weight) (e.g., 3^4). The organic material is composed of a variety of peptides, gjycopeptides, and some nonpolar molecules. While limited in terms of quantity, these components are thought to act in concert to regulate the mineralization process in vivo. There are specific examples of polyanionic proteins that are thought to be both inhibitory and/ or stimulatory to mineralization (5-10). Some of these molecules can be phosphorylated, a process common to molecular regulation (e.g., 11,12). While there are data with reference to these molecules there is at present no complete understanding how these molecules control mineralization (3,13,14). Many of 0097-6156/91/0444-0125$06.00/0 © 1991 American Chemical Society In Surface Reactive Peptides and Polymers; Sikes, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
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the molecules have been, at least partially, characterized and several have unique structures that have potential industrial applications. As an example, peptides containing polycarboxylic acids are of interest for their apparent ability to inhibit mineralization (2). More recently, some of the proteolipids have become of interest for their ability to initiate or pattern the initial mineralization process. Indeed, there is evidence that the initial mineralization events in bone and cartilage may be intravesicular being specifically associated with particular membrane components and their enzymatic activity (e.g., 15,16).
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Calcium Concretions As reviewed by Brown in 1982 (17), calcified concretions, corpuscles, granules, bodies, and a host of other named mineralized products are produced in virtually all of the invertebrate Phyla and in the vertebrates. At the time of Brown's review, perhaps the best studied concretions in terms of chemical composition were those found in cestodes (e.g., 18) and those that were end-products of cellular degradation process or tertiary lysosomes (19 for review). While many of these concretions have a similar morphology and a size ranging from 0.5 to 3 μτη (although large concretions are 100-200 μτη in diameter), the chemical composition of the concretions are different from species to species and even between tissues in the same animal. Most are calcium-based minerals with either carbonate, phosphate, or oxalate serving as the primary anion. They can be rather complex, with other divalents replacing calcium to some extent, and some have more than one predominant anion. The mode of concretion production among tissues is remarkably different; some concretions being produced extracellularly and others being produced intracellularly. All of these differing concretion systems provide largely unstudied models of biomineralization which could yield biological polymers of interest. Perhaps more importantly, the concretion systems allow for the study of intracellular mineralization. This process has not been as extensively studied by comparison with extracellular mineralization, and may provide new clues to the molecular events associated with biomineralization in general. Alternatively, novel mechanistic events, molecules, or both may be present which could at some point be exploited for their chemical properties. The chemical composition of only a few of the many concretion systems have been studied in detail with reference to their chemical compositions. Our understanding of even the more well-studied concretion systems is largely limited to their inorganic composition. Examples of well-studied systems include the concretions produced in the hepatopancreas of snails, the concretions produced in Malpighian tubules in association with molting in face fly larvae, and concretions found in the gills of freshwater unionid mussels. The tertiary lysosome system found in the digestive and/or excretory organs of some molluscs and crustaceans also has been characterized, but will not be covered here. Taking the three examples cited above, and comparing chemical differences and similarities gives some insight
In Surface Reactive Peptides and Polymers; Sikes, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
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Invertebrate Calcium Concretions
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into the diversity of chemical structure and function for these calcium mineralized structures.
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Gastropod Concretions There are at least two different types of concretions in the gastropods which have two relatively unique and independent functions. The last major review of this system was written by Fornie and Chetail (20). The following is intended as an overview of what is known about the chemical composition of these system and is not meant to be an exhaustive review. One type of concretion is produced in cells of the hepatopancreas of the animal. These concretions are thought to be produced intracellularly in association with Golgi and endoplasmic reticulum (21,22). They are composed largely of calcium pyrophosphate mineral (23,24) and can contain a variety of divalent cations substituted for calcium. In animals from metal-polluted sites the polluting divalent is preferentially accumulated into these intracellularly produced concretions or granules (25). Thus, these concretions are produced by cells with access to the external environment and appear to be used for the binding and eventual elimination of toxic divalents by secretion of the concretion into the digestive tract (22,25). The organic content of these granules is minimal according to C, H, and Ν analysis, and has not been analyzed in further detail. Carbonate mineral does not appear to be a major component of these concretions (23). In contrast, there are connective tissue cells (amoebocytes) in gastropods which produce carbonate-based concretions (20,26,27). The divalents present are mainly calcium and magnesium and the concretions do not tend to accumulate significant quantities of other divalent cations. In two species, Pomacea paludosa and Pila virens the calcium carbonate is largely in the form of amorphous crystal while some vaterite crystals also are observed (27,28). These granules apparently are produced intracellularly by connective tissue cells of gastropods (e.g., 28,29). These granules are used to supply calcium to reproductive organs for passage to the egg capsule (26,27). Many others have suggested that they also are used for damaged shell regeneration (e.g., 30,31). Their function in this role is not as clear and has been disputed by some investigators (32,33). The egg capsule is particularly rich in calcium, and capsule formation appears to be related to a solubilization of granules during egg production and laying. Thus, in the same animal there are at least two widely divergent concretion systems both in termi of biological function and chemical properties. Neither of these concretion systems has had any extensive analyses for organic constituents. According to histochemical data, the calcium storage concretions of P. paludosa contain acid mucopol ysaccharides, and amino acid analysis of concretion digests suggest a peptide component rich in aspartic and glutamic acid (28).
In Surface Reactive Peptides and Polymers; Sikes, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
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Insect Concretions Several organs in various insects have the capacity to produce concretions. Again the object here is not to provide an exhaustive review but to give an introduction to the better studied insect concretion systems. The most common location for concretion accumulations are the Malpighian tubules (34,35). These concretions have different functions in different insects depending on the ecological physiology of the organism. The composition of the concretions also appears to differ among insects even though they are associated with the same organ, the Malpighian tubules. In the face fly larvae the granules are formed from calcium extracted from the larval food. Calcium is stored in granules of calcium phosphate with small quantities of magnesium and carbonate (14%) also present (35,36). The mineral phase is amorphous calcium phosphate, perhaps orthophosphate, although the data are not conclusive (37). The organic matrix of these concretions is a minor component (
