Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd‐I (positions 1–905) comprising the N‐terminal globule and rod‐like domain and Nd‐II corresponding mainly to the C‐terminal globule (position 906–1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N‐terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N‐terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31–56 kDa) connected by either a rod‐like or a thin segment. Recombinant nidogen formed stable complexes (Kd less than or equal to 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd‐I binding to collagen IV and fragment Nd‐II binding to laminin fragment P1. In addition, recombinant nidogen but not Nd‐I was able to bridge between laminin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes.

The C-terminal domain NC1 of mouse collagen XVIII (38 kDa) and the shorter mouse and human endostatins (22 kDa) were prepared in recombinant form from transfected mammalian cells. The NC1 domain aggregated non-covalently into a globular trimer which was partially cleaved by endogenous proteolysis into several monomers (25-32 kDa) related to endostatin. Endostatins were obtained in a highly soluble, monomeric form and showed a single N-terminal sequence which, together with other data, indicated a compact folding. Endostatins and NC1 showed a comparable binding activity for the microfibrillar fibulin-1 and fibulin-2, and for heparin. Domain NC1, however, was a distinctly stronger ligand than endostatin for sulfatides and the basement membrane proteins laminin-1 and perlecan. Immunological assays demonstrated endostatin epitopes on several tissue components (22-38 kDa) and in serum (120-300 ng/ml), the latter representing the smaller variants. The data indicated that the NC1 domain consists of an N-terminal association region (~50 residues), a central protease-sensitive hinge region (~70 residues) and a C-terminal stable endostatin domain (~180 residues). They also demonstrated that proteolytic release of endostatin can occur through several pathways, which may lead to a switch from a matrix-associated to a more soluble endocrine form.

The calcareous egg is produced by all birds and most reptiles. Current understanding of eggshell formation and mineralization is mainly based on intensive studies of one species - the domesticated chicken Gallus gallus. The majority of constituents of the chicken eggshell have been identified. In this article we review eggshell microstructure and ultrastructure, and the results of recent genomic, transcriptomic and proteomic analyses of the chicken eggshell matrix to draw attention to areas of current uncertainty such as the potential role of amorphous calcium carbonate and the specific nature of the molecules that initiate (nucleate) mammillary cone formation and terminate palisade layer calcification. Comparative avian genomics and proteomics have only recently become possible with the publication of the Taeniopygia guttata (zebra finch) genome. Further rapid progress is highly anticipated with the soon-to-be-released genomes of turkey (Meleagris gallopavo) and duck (Anas platyrhynchos). These resources will allow rapid advances in comparative studies of the organic constituents of avian eggshell and their functional implications.

The major difference between inorganic minerals and biominerals is the presence of an organic matrix consisting of proteins, glycoproteins, proteoglycans, and polysaccharides, which is synthesized by specialized cells under genetic control before or during mineralization. The organic matrix is thought to play a major role in the assembly of the biomineral and determination of its mechanical properties. The recent elucidation of the chicken genome provided an opportunity to explore the matrix proteome of a biomineral using up-to-date MS-based technology. We identified 520 proteins in this matrix including the ten matrix proteins already known before. The identified proteins were divided into three abundance groups using the exponentially modified protein abundance index described recently which was roughly calibrated with the few known data on protein yield derived from Edman sequence analysis. A small group of 32 highly abundant proteins contained the presently known eggshell-specific proteins and all of the other known eggshell matrix constituents identified before with much less sensitive conventional methods. The present study, which is the first comprehensive proteomic study of a vertebrate biomineral, is intended as a starting point for the detailed molecular characterization of eggshell matrix proteins, their interactions in the matrix network and functional studies.