A Novel Disulfide-Rich Protein Motif from Avian Eggshell Membranes

Abstract

Under the shell of a chicken egg are two opposed proteinaceous disulfide-rich membranes. They are fabricated in the avian oviduct using fibers formed from proteins that are extensively coupled by irreversible lysine-derived crosslinks. The intractability of these eggshell membranes (ESM) has slowed their characterization and their protein composition remains uncertain. In this work, reductive alkylation of ESM followed by proteolytic digestion led to the identification of a cysteine rich ESM protein (abbreviated CREMP) that was similar to spore coat protein SP75 from cellular slime molds. Analysis of the cysteine repeats in partial sequences of CREMP reveals runs of remarkably repetitive patterns. Module a contains a C-X4-C-X5-C-X8-C-X6 pattern (where X represents intervening non-cysteine residues). These inter-cysteine amino acid residues are also strikingly conserved. The evolutionarily-related module b has the same cysteine spacing as a, but has 11 amino acid residues at its C-terminus. Different stretches of CREMP sequences in chicken genomic DNA fragments show diverse repeat patterns: e.g. all a modules; an alternation of a-b modules; or an a-b-b arrangement. Comparable CREMP proteins are found in contigs of the zebra finch (Taeniopygia guttata) and in the oviparous green anole lizard (Anolis carolinensis). In all these cases the long runs of highly conserved modular repeats have evidently led to difficulties in the assembly of full length DNA sequences. Hence the number, and the amino acid lengths, of CREMP proteins are currently unknown. A 118 amino acid fragment (representing an a-b-a-b pattern) from a chicken oviduct EST library expressed in Escherichia coli is a well folded, highly anisotropic, protein with a large chemical shift dispersion in 2D solution NMR spectra. Structure is completely lost on reduction of the 8 disulfide bonds of this protein fragment. Finally, solid state NMR spectra suggest a surprising degree of order in intact ESM fibers.

The extracellular matrix of eggshell displays anti-inflammatory activities through NF-κB in LPS-triggered human immune cells

“…Collagens present in ESM are also interesting bioactive candidates, as collagen is today an ingredient in different wound healing products, and the structural properties of collagen fibers with effect on cell proliferation and remodeling activities have been outlined 13. The functions of CREMPS, recently discovered in large quantities in ESM and shown to be avian specific,14 has not been determined, but their presence in large amounts in the ESM indicates that these proteins also could be interesting candidates in relation to wound healing. Hen egg white glycoprotein, ovotransferrin, also present in ESM, has recently been demonstrated to possess anti-inflammatory properties 15.…”

Short-Term Effects of Ovomet®, Eggshell Membrane, in Joint Pain. A Double-Blind and Placebo Study

“…Ovomet ® contains naturally the above cited components, all of them being important constituents of joints and playing a crucial role in their health, mobility and flexibility. Moreover, Ovomet ® can be used as a source of sulfur due to the number of disulphide bonds and sulfur amino acids that have been quantified [11]. It is well known that sulfur plays a very important role in nutrition and in maintaining the cartilage matrix [12].…”

Effects of 50 Days Ovomet Supplementation on Biomechanical Parameters and Subjective Pain Perception among Old Institutionalized Patients. A Preliminary Study

“…The eggshell membrane is one of the food supplements that have had very good results in this sense [2][3][4][5]. Eggshell membrane is comprised of a large part of collagen, mainly type I, V and X [6][7][8]; glycosaminoglycans (hyaluronic acid, chondroitin sulfate and different proteins that are involved in the antiinflammatory and immune system regulation ability of the eggshell membrane (lysozyme, ovotransferrin, ovocleidin-17 and ovocleidin-116) [8,9]. In addition, the eggshell membrane can be used as a source of sulfur due to the number of disulfide bonds and sulfur amino acids that have been quantified [10].…”