Structure-Function Relationship of Avian Eggshell Matrix Proteins: A Comparative Study of Two Major Eggshell Matrix Proteins, Ansocalcin and OC-17 Rajamani Lakshminarayanan, ² Jeremiah S. Joseph, ‡ R. Manjunatha Kini, ‡ and Suresh Valiyaveettil* ,² Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543 Received September 15, 2004; Revised Manuscript Received November 10, 2004 The role of individual matrix proteins in avian eggshell calcification is poorly understood despite numerous attempts to characterize and localize their presence in the eggshell matrix. Ansocalcin, the major matrix protein from goose eggshell, was found to induce the formation of calcite crystal aggregates under in vitro. Owing to its high similarity with the chicken eggshell matrix protein ovocleidin 17 (OC-17), a comparative investigation has been carried out to understand the structure-function relationship. RP-HPLC shows that ansocalcin is the major component in extracts of goose eggshells before and after bleach treatment. However, OC-17 was observed in minute quantities in the extract of bleach-treated chicken eggshells. In vitro crystal growth experiments showed that OC-17 and ansocalcin interact differently with the calcite crystals formed. Circular dichroism, intrinsic tryptophan fluorescence, and dynamic light scattering studies showed that, under the conditions used in our experiments, OC-17 does not aggregate in solution or induce the nucleation of calcite aggregates in the concentration range used. These observations indicate that OC-17 and ansocalcin play different roles in the eggshell calcification. To our knowledge, this is the first report on the comparison of properties of homologous eggshell proteins that belong to the same phylogeny. Introduction Biomineralization offers organisms the capability to produce inorganic materials with robust shape and morphol- ogy for their survival in dynamic environmental conditions. Calcified tissues account for more than 50% of the hard tissues generated. 1 An array of acidic biomacromolecules, such as proteins and proteoglycans, aid the biomineralization process, which is rapid in some cases (e.g., avian eggshells) or a slow process in other cases, occurring over a few years (e.g., bones, teeth, seashells, etc.). 2 Numerous attempts have been made to understand the molecular mechanisms of biomineralization through isolation and characterization of the soluble organic matrixes (SOM) from various mineralized tissues. 3 In a few cases, the major protein component has been purified and sequenced. 4 Avian eggshell calcification represents a unique model for biomineralization owing to its rapid formation (17-22 h). 2g The eggshell fabrication takes place in an extracellular milieu with the help of the macromolecules present/secreted by the oviduct cells as the egg moves along the oviduct. 5 The resultant calcified shell exhibits a high degree of textural gradient. A number of proteins and proteoglycans have been identified in the chicken eggshell matrix. 6 Of the three groups of proteins identified in the chicken eggshell matrix, only a few proteins have been tested in vitro to investigate their mineralization capabilities. 7 Ansocalcin, OC-17, struthiocalcin-1 (SCA-1), and struthio- calcin-2 (SCA-2) are the eggshell-specific proteins extracted from avian eggshells and sequenced completely. SCA-1 shows high identity (65%) and the same number of amino acid residues as ansocalcin, whereas SCA-2 possesses same number of amino acid residues as OC-17 and two phopo- serines located in the same region as OC-17. These proteins belong to the C-type lectin superfamily, yet lack consensus QPD motif that is required for carbohydrate binding. 8 Recently, the occurrence of two different families of C-type lectins has been demonstrated in other materials. 8c Thus, ansocalcin and OC-17 belong to different group of C-type lectin-like proteins. The traditional bioinformatic approach based on sequence similarity is the first step in inferring the structure-function relationships. It is known that homologous proteins share a common three-dimensional folding and secondary structure, although they may or may not have the same function. 9 Given the high similarity between ansocalcin and OC-17 (Figure 1), it would be interesting to compare their activity toward the nucleation of calcium carbonate crystals. Therefore, a comparative study on the structure-property relationship of the two eggshell proteins has been carried out and described in detail. Immunochemically, OC-17 has been observed predomi- nantly in the mamillary bodies and distributed through out the palisade layer of the eggshell. 10 It is understood that bleach treatment removes the shell membrane and the proteins attached externally to the eggshell. Hence, our first aim was to identify the presence of ansocalcin and OC-17 * To whom correspondence should be addressed. Tel.: (65)68744327. Fax: (65)67791691. E-mail: chmsv@nus.edu.sg. ² Department of Chemistry, National University of Singapore. ‡ Department of Biological Sciences, National University of Singapore. 741 Biomacromolecules 2005, 6, 741-751 10.1021/bm049423+ CCC: $30.25 © 2005 American Chemical Society Published on Web 01/13/2005