Novel Basic Protein, PfN23, Functions as Key Macromolecule during Nacre Formation*

Background: The microstructure of nacre is controlled by the proteins in them. Results: When PfN23 was knocked down, the shell formation in adults and larvae was suppressed. Conclusion: The basic protein PfN23 is important for the control of crystal growth in nacre. Significance: This might provide a valuable complementary to the classic view that acidic proteins control nacre formation. The fine microstructure of nacre (mother of pearl) illustrates the beauty of nature. Proteins found in nacre were believed to be “natural hands” that control nacre formation. In the classical view of nacre formation, nucleation of the main minerals, calcium carbonate, is induced on and by the acidic proteins in nacre. However, the basic proteins were not expected to be components of nacre. Here, we reported that a novel basic protein, PfN23, was a key accelerator in the control over crystal growth in nacre. The expression profile, in situ immunostaining, and in vitro immunodetection assays showed that PfN23 was localized within calcium carbonate crystals in the nacre. Knocking down the expression of PfN23 in adults via double-stranded RNA injection led to a disordered nacre surface in adults. Blocking the translation of PfN23 in embryos using morpholino oligomers led to the arrest of larval development. The in vitro crystallization assay showed that PfN23 increases the rate of calcium carbonate deposition and induced the formation of aragonite crystals with characteristics close to nacre. In addition, we constructed the peptides and truncations of different regions of this protein and found that the positively charged C-terminal region was a key region for the function of PfN23 Taken together, the basic protein PfN23 may be a key accelerator in the control of crystal growth in nacre. This provides a valuable balance to the classic view that acidic proteins control calcium carbonate deposition in nacre.

[1]  K. Simkiss.  Biomineralization and detoxification , 1977, Calcified Tissue Research.

[2]  H. Nagasawa,et al.  Characterization of Prismalin-14, a novel matrix protein from the prismatic layer of the Japanese pearl oyster (Pinctada fucata). , 2004, The Biochemical journal.

[3]  T. Samata,et al.  A new matrix protein family related to the nacreous layer formation of Pinctada fucata , 1999, FEBS letters.

[4]  T Morita,et al.  A carbonic anhydrase from the nacreous layer in oyster pearls. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[5]  M. Fritz,et al.  Perlwapin, an abalone nacre protein with three four-disulfide core (whey acidic protein) domains, inhibits the growth of calcium carbonate crystals. , 2006, Biophysical journal.

[6]  M. Fritz,et al.  Perlinhibin, a cysteine-, histidine-, and arginine-rich miniprotein from abalone (Haliotis laevigata) nacre, inhibits in vitro calcium carbonate crystallization. , 2007, Biophysical journal.

[7]  Benedetto Marelli,et al.  Silk fibroin derived polypeptide-induced biomineralization of collagen. , 2012, Biomaterials.

[8]  Xiaodong Li,et al.  Unveiling the formation mechanism of pseudo-single-crystal aragonite platelets in nacre. , 2009, Physical review letters.

[9]  H. Lowenstam,et al.  Vaterite: a mineralization product of the hard tissues of a marine organism (Ascidiacea). , 1975, Science.

[10]  Benjamin Marie,et al.  Molluscan shell proteins: primary structure, origin, and evolution. , 2008, Current topics in developmental biology.

[11]  K. Wada,et al.  Development and morphology of the pearl oyster larvae, Pinctada fucata , 1995 .

[12]  Masato Yano,et al.  Shematrin: a family of glycine-rich structural proteins in the shell of the pearl oyster Pinctada fucata. , 2006, Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology.

[13]  I. Khalaila,et al.  A gastrolith protein serving a dual role in the formation of an amorphous mineral containing extracellular matrix , 2008, Proceedings of the National Academy of Sciences.

[14]  Xin Yi Chan,et al.  Patterning a spiralian embryo: a segregated RNA for a Tis11 ortholog is required in the 3a and 3b cells of the Ilyanassa embryo. , 2011, Developmental biology.

[15]  Steve Weiner,et al.  Mollusk shell formation: a source of new concepts for understanding biomineralization processes. , 2006, Chemistry.

[16]  Takashi Kato,et al.  An Acidic Matrix Protein, Pif, Is a Key Macromolecule for Nacre Formation , 2009, Science.

[17]  Ilhan A. Aksay,et al.  Biomimetic Synthesis of Macroscopic-Scale Calcium Carbonate Thin Films. Evidence for a Multistep Assembly Process , 1998 .

[18]  L. Xie,et al.  A Novel Extrapallial Fluid Protein Controls the Morphology of Nacre Lamellae in the Pearl Oyster, Pinctada fucata* , 2007, Journal of Biological Chemistry.

[19]  H. Nagasawa,et al.  A novel calcium-binding peptide from the cuticle of the crayfish, Procambarus clarkii. , 2004, Biochemical and biophysical research communications.

[20]  A. De Paepe,et al.  Matrix proteins with high affinity for calcium ions are associated with mineralization within the elastic fibers of pseudoxanthoma elasticum dermis. , 1996, The American journal of pathology.

[21]  M. Martindale,et al.  β-catenin and early development in the gastropod, Crepidula fornicata. , 2010, Integrative and comparative biology.

[22]  B. Wilkinson.  Biomineralization, paleoceanography, and the evolution of calcareous marine organisms , 1979 .

[23]  Q. Feng.  Principles of calcium-based biomineralization. , 2011, Progress in molecular and subcellular biology.

[24]  Gu Jing,et al.  Cloning and Characterization of Prisilkin-39, a Novel Matrix Protein Serving a Dual Role in the Prismatic Layer Formation from the Oyster Pinctada fucata* , 2009, Journal of Biological Chemistry.

[25]  Steve Weiner,et al.  Macromolecules in mollusc shells and their functions in biomineralization , 1984 .

[26]  X. H. Wu,et al.  Control of crystal phase switching and orientation by soluble mollusc-shell proteins , 1996, Nature.

[27]  Xin Yi Chan,et al.  Nanos Is Required in Somatic Blast Cell Lineages in the Posterior of a Mollusk Embryo , 2008, Current Biology.

[28]  H. Lowenstam,et al.  Minerals formed by organisms. , 1981, Science.

[29]  I. Khalaila,et al.  A Protein Involved in the Assembly of an Extracellular Calcium Storage Matrix* , 2010, The Journal of Biological Chemistry.

[30]  S. Weiner,et al.  Biomineralization: A pavement of pearl , 1997, Nature.

[31]  S. Weiner,et al.  Control of Aragonite or Calcite Polymorphism by Mollusk Shell Macromolecules , 1996, Science.

[32]  S. Weiner,et al.  Crystallization Pathways in Biomineralization , 2011 .

[33]  T. Fujikawa,et al.  Structures of mollusc shell framework proteins , 1997, Nature.

[34]  P. Hansma,et al.  Molecular Cloning and Characterization of Lustrin A, a Matrix Protein from Shell and Pearl Nacre of Haliotis rufescens * , 1997, The Journal of Biological Chemistry.

[35]  S. Weiner,et al.  Mollusc larval shell formation: amorphous calcium carbonate is a precursor phase for aragonite. , 2002, The Journal of experimental zoology.

[36]  Craig B. Thompson,et al.  Hierarchical Control of Lymphocyte Survival , 1996, Science.

[37]  S. Weiner,et al.  Mollusk Shell Acidic Proteins: In Search of Individual Functions , 2003, Chembiochem : a European journal of chemical biology.

[38]  A. P. Wheeler,et al.  Molluscan shell matrix phosphoproteins: Correlation of degree of phosphorylation to shell mineral microstructure and to in vitro regulation of mineralization , 1991 .

[39]  Zhiyong Tang,et al.  Nanostructured artificial nacre , 2003, Nature materials.

[40]  S. Mann,et al.  Crystallization at Inorganic-organic Interfaces: Biominerals and Biomimetic Synthesis , 1993, Science.

[41]  Frédéric Marin,et al.  Molluscan shell proteins , 2004 .

[42]  Rongqing Zhang,et al.  Identification of Genes Directly Involved in Shell Formation and Their Functions in Pearl Oyster, Pinctada fucata , 2011, PloS one.

[43]  S. Weiner,et al.  Structure of the nacreous organic matrix of a bivalve mollusk shell examined in the hydrated state using cryo-TEM. , 2001, Journal of structural biology.

[44]  M. Fritz,et al.  Purification and characterization of perlucin and perlustrin, two new proteins from the shell of the mollusc Haliotis laevigata. , 2000, Biochemical and biophysical research communications.

[45]  Koichi Morimoto,et al.  A novel nacre protein N19 in the pearl oyster Pinctada fucata. , 2007, Biochemical and biophysical research communications.

[46]  P. Simon,et al.  Fluorapatite-Gelatine-Nanocomposites: Self-Organized Morphogenesis, Real Structure and Relations to Natural Hard Materials , 2006 .

[47]  D. Hood,et al.  Pure aragonite synthesis , 1962 .