A cDNA expression library constructed from mantle tissue mRNA of the Mediterranean fan mussel Pinna nobilis was screened with antibodies raised against the acetic acid-soluble shell matrix of the same species. This resulted in the isolation of a 2138-base pair cDNA, containing 13 tandem repeats of 93 base pairs. The deduced protein has a molecular mass of 66.7 kDa and a isoelectric point of 4.8. This protein, which is enriched in serine and proline residues, was overexpressed, purified, and used for producing polyclonal antibodies. Immunological in situ and in vitro tests showed that the protein is localized in the nacreous aragonitic layer of P. nobilis, but not in the calcitic prisms. Because this protein of the nacre of P. nobilis exhibits some mucin-like characteristics, we propose the name mucoperlin. This is the first paper reporting the cloning of a molluscan mucin and the first molecular evidence for the involvement of a mucin in molluscan calcification. This finding corroborates our previous hypothesis that some of the proteinaceous constituents of the molluscan shell matrix would derive from mucins, common to many metazoan lineages of the late Precambrian (Marin, F., Smith, M., Isa, Y., Muyzer, G. and Westbroek, P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1554 -1559). The adaptation of an ancestral mucin to a new function, the regulation of the mineralization process, may be one of the molecular events, among others, that would explain the simultaneous emergence of organized calcification in many metazoan lineages during the Cambrian explosion.The skeletons produced by molluscs to protect and support their soft bodies are organo-mineral amalgamates (1, 2) exhibiting a high degree of order at the nanoscale (3). As a rule, the mineralized structure has mechanical properties far superior to each of its constituent components. A well known example is mother-of-pearl or nacre. Its micron-sized compact brick wall texture is more than 1000 times tougher than the chemically precipitated counterpart, aragonite (4).A complex biochemical machinery is required for the production of these highly ordered biominerals. Specialized microenvironments must be created to accommodate the growing minerals (1, 5). The constituent ions as well as macromolecules capable of directing and fine-tuning the crystallization must be supplied, while waste products of crystallization have to be removed and deleterious precipitations avoided. The actual biomineralization process follows as a remarkable case of selforganization, with the densely packed organic-inorganic composite emerging as the final product. Furthermore, the organisms must deploy upstream the calcification, signal transmitters (6, 7), and transcription factors (8, 9) able to switch their biomineralizing machineries on and off.Despite its complexity, it would appear as though the skeleton-forming machinery evolved with remarkable facility. The skeletons of many animal phyla were installed in less than 30 million years, since the beginning of the Cambrian, 544 million ...
