The calcified eggshell matrix proteome of a songbird, the zebra finch (Taeniopygia guttata)

Karlheinz Mann

The proteins of avian eggshell organic matrices are thought to control the mineralization of the eggshell in the shell gland (uterus). Proteomic analysis of such matrices identified many candidates for such a role. However, all matrices analyzed to date come from species of one avian family, the Phasianidae. To analyze the conservation of such proteins throughout the entire class Aves and to possibly identify a common protein toolkit enabling eggshell mineralization, it is important to analyze eggshell matrices from other avian families. Because mass spectrometry-based in-depth proteomic analysis still depends on sequence databases as comprehensive and accurate as possible, the obvious choice for a first such comparative study was the eggshell matrix of zebra finch, the genome sequence of which is the only songbird genome published to date. The zebra finch eggshell matrix comprised 475 accepted protein identifications. Most of these proteins (84 %) were previously identified in species of the Phasianidae family (chicken, turkey, quail). This also included most of the so-called eggshell-specific proteins, the ovocleidins and ovocalyxins. Ovocleidin-116 was the second most abundant protein in the zebra finch eggshell matrix. Major proteins also included ovocalyxin-32 and -36. The sequence of ovocleidin-17 was not contained in the sequence database, but a presumptive homolog was tentatively identified by N-terminal sequence analysis of a prominent 17 kDa band. The major proteins also included three proteins similar to ovalbumin, the most abundant of which was identified as ovalbumin with the aid of two characteristic phosphorylation sites. Several other proteins identified in Phasianidae eggshell matrices were not identified. When the zebra finch sequence database contained a sequence similar to a missing phasianid protein it may be assumed that the protein is missing from the matrix. This applied to ovocalyxin-21/gastrokine-1, a major protein of the chicken eggshell matrix, to EDIL3 and to lactadherin. In other cases failure to identify a particular protein may be due to the absence of this protein from the sequence database, highlighting the importance of better, more comprehensive sequence databases. The results indicate that ovocleidin-116, ovocleidin-17, ovocalyxin-36 and ovocalyxin-32 may be universal avian eggshell-mineralizing proteins. All the more important it is to elucidate the role of these proteins at the molecular level. This cannot be achieved by proteomic studies but will need application of other methods, such as atomic force microscopy or gene knockouts. However, it will also be important to analyze more eggshell matrices of different avian families to unequivocally identify other mineralization toolkit proteins apart from ovocleidins and ovocalyxins. Progress in this respect will depend critically on the availability of more, and more comprehensive, sequence databases. The development of faster and cheaper nucleotide sequencing methods has considerably accelerated genome and transcriptome sequencing, but this seems to concur with frequent publication of incomplete and fragmented sequence databases.