Cloning of Ovocalyxin-36, a Novel Chicken Eggshell Protein Related to Lipopolysaccharide-binding Proteins, Bactericidal Permeability-increasing Proteins, and Plunc Family Proteins

Abstract

The avian eggshell is a composite biomaterial composed of noncalcifying eggshell membranes and the overlying calcified shell matrix. The shell is deposited in a uterine fluid where the concentration of different protein species varies at different stages of its formation. The role of avian eggshell proteins during shell formation remains poorly understood, and we have sought to identify and characterize the individual components in order to gain insight into their function during elaboration of the eggshell. In this study, we have used direct sequencing, immunochemistry, expression screening, and EST data base mining to clone and characterize a 1995-bp full-length cDNA sequence corresponding to a novel chicken eggshell protein that we have named Ovocalyxin-36 (OCX-36). Ovocalyxin-36 protein was only detected in the regions of the oviduct where eggshell formation takes place; uterine OCX-36 message was strongly up-regulated during eggshell calcification. OCX-36 localized to the calcified eggshell predominantly in the inner part of the shell, and to the shell membranes. BlastN data base searching indicates that there is no mammalian version of OCX-36; however, the protein sequence is 20 -25% homologous to proteins associated with the innate immune response as follows: lipopolysaccharide-binding proteins, bactericidal permeabilityincreasing proteins, and Plunc family proteins. Moreover, the genomic organization of these proteins and OCX-36 appears to be highly conserved. These observations suggest that OCX-36 is a novel and specific chicken eggshell protein related to the superfamily of lipopolysaccharide-binding proteins/bactericidal permeability-increasing proteins and Plunc proteins. OCX-36 may therefore participate in natural defense mechanisms that keep the egg free of pathogens.

Purification and Characterization of Avian β-Defensin 11, an Antimicrobial Peptide of the Hen Egg

“…AvBD1, -2, and -3 mRNAs are not expressed in the immature oviduct of younger hens or in the mature oviduct of nonlaying hens, but their oviduct expression increases when the hens are forming eggs (25,34). These results suggest modulation of AvBD expression as a function of egg-laying activity, possibly as a consequence of the dilation of the oviduct by the presence of the forming egg, as shown for osteopontin and ovocalyxin 36 (7,16). Furthermore, several studies have shown a modulation of ␤-defensin expression correlating with stimulation by pathogens.…”

“…2). In the MS-MS spectrum of the (ϩ7) precursor ion at m/z 1,325, the N-terminal sequence LPRDTSR of mature AvBD11 was determined by six fragment b ions, b 2 to b 7 , and the C-terminal sequence KEI was confirmed by three fragment y ions (y 1 to y 3 ) (Fig. 3).…”

A century of poultry genetics

“…This gene was extensively used as a model to study gene structure and expression in a vertebrate, as shown by the identification of specific sequences at the exon-intron boundaries (Breathnach et al, 1978). Molecular characterisation of egg proteins is still ongoing, for example, the identification of eggshell proteins (Gautron et al, 2007).…”

Protein constituents of the eggshell: eggshell-specific matrix proteins

“…A cDNA library from pooled RNA extracted from chicken uteruses that were harvested during the mid-phase of shell calcification was successfully screened to clone novel eggshell matrix proteins [28,29]. Expression screening of this library, using polyclonal antisera raised to partially purified eggshell matrix proteins, allowed clones with the corresponding cDNA sequences to be identified and sequenced.…”

“…The conceptual amino acid sequence was compared to partial amino acid sequencing data for proteins present in uterine fluid and eggshell extracts. This method allowed the identification of two novel eggshell matrix proteins, ovocleidin-116 [28] and ovocalyxin-36 [29]. Another associated approach was to compare the available expression sequence tag (EST) sequences to partial protein or nucleotide sequences from egg components.…”