Isolation of ovocleidin-116 from chicken eggshells, correction of its amino acid sequence and identification of disulfide bonds and glycosylated Asn.

Fractionation of the soluble chicken eggshell matrix by chromatographic methods yielded 13 endogenous proteolytic fragments of the eggshell-specific proteoglycan core protein ovocleidin-116. The N-terminal amino acid sequences of these fragments in general confirmed the recently cDNA-deduced sequence of ovocleidin-116, with one exception. One fragment yielded a completely new sequence and was instrumental in detecting a frame shift error in the nucleotide sequence. The correction yielded a new sequence which was 38 amino acids shorter than before and contained a 57-amino acid long novel C-terminal sequence. The predicted sequence of ovocleidin-116 contained two consensus N-glycosylation sites, only one of which (Asn62) was found to be fully modified. A disulfide bond was identified between Cys31 and 42 implying that Cys329 and 421 form a second disulfide bond. Finally, the yield of fragments indicated that ovocleidin-116 is a major component of the chicken eggshell matrix.     

Ovocalyxin-32, a novel chicken eggshell matrix protein. isolation, amino acid sequencing, cloning, and immunocytochemical localization

The eggshell is a highly ordered structure resulting from the deposition of calcium carbonate concomitantly with an organic matrix upon the eggshell membranes. Mineralization takes place in an acellular uterine fluid, which contains the ionic and matrix precursors of the eggshell. We have identified a novel 32-kDa protein, ovocalyxin-32, which is expressed at high levels in the uterine and isthmus regions of the oviduct, and concentrated in the eggshell. Sequencing of peptides derived from the purified protein allowed expressed sequence tag sequences to be identified that were assembled to yield a full-length composite sequence whose conceptual translation product contained the complete amino acid sequence of ovocalyxin-32. Data base searches revealed that ovocalyxin-32 has limited identity (32%) to two unrelated proteins: latexin, a carboxypeptidase inhibitor expressed in the rat cerebral cortex and mast cells, and a skin protein, which is encoded by a retinoic acid receptor-responsive gene, TIG1. High level expression of ovocalyxin-32 was limited to the isthmus and uterus tissue, where immunocytochemistry at the light and electron microscope levels demonstrated that ovocalyxin-32 is secreted by surface epithelial cells. In the eggshell, ovocalyxin-32 localizes to the outer palisade layer, the vertical crystal layer, and the cuticle of the eggshell, in agreement with its demonstration by Western blotting at high levels in the uterine fluid during the termination phase of eggshell formation. Ovocalyxin-32 is therefore identified as a novel protein synthesized in the distal oviduct where hen eggshell formation occurs.     

Identification and localization of lysozyme as a component of eggshell membranes and eggshell matrix.

The avian eggshell is a composite biomaterial composed of non-calcifying eggshell membranes and the overlying calcified shell matrix. The calcified shell forms in a uterine fluid where the concentration of different protein species varies between the initial, rapid calcification and terminal phases of eggshell deposition. The role of these avian eggshell matrix proteins during shell formation is poorly understood. The properties of the individual components must be determined in order to gain insight into their function during eggshell mineralization. In this study, we have identified lysozyme as a component of the uterine fluid by microsequencing, and used western blotting, immunofluorescence and colloidal-gold immunocytochemistry to document its localization in the eggshell membranes and the shell matrix. Furthermore, Northern blotting and RT-PCR indicates that there is a gradient to the expression of lysozyme message by different regions of the oviduct, with significant albeit low levels expressed in the isthmus and uterus. Lysozyme protein is abundant in the limiting membrane that circumscribes the egg white and forms the innermost layer of the shell membranes. It is also present in the shell membranes, and in the matrix of the calcified shell. Calcite crystals grown in the presence of purified hen lysozyme exhibited altered crystal morphology. Therefore, in addition to its well-known anti-microbial properties that could add to the protective function of the eggshell during embryonic development, shell matrix lysozyme may also be a structural protein which in soluble form influences calcium carbonate deposition during calcification.     

Purification,characterization, and in vitro mineralization studies of a novel goose eggshell matrix protein,ansocalcin

Biomineralization is an important process in which hard tissues are generated through mineral deposition, often assisted by biomacromolecules. Eggshells, because of their rapid formation via mineralization, are chosen as a model for understanding the fundamentals of biomineralization. This report discusses purification and characterization of various proteins and peptides from goose eggshell matrix. A novel 15-kDa protein (ansocalcin) was extracted from the eggshell matrix, purified, and identified and its role in mineralization evaluated using in vitro crystal growth experiments. The complete amino acid sequence of ansocalcin showed high homology to ovocleidin-17, a chicken eggshell protein, and to C-type lectins from snake venom. The amino acid sequence of ansocalcin was characterized by the presence of acidic and basic amino acid multiplets. In vitro crystallization experiments showed that ansocalcin induced pits on the rhombohedral faces at lower concentrations (<50 microg/ml). At higher concentrations, the nucleation of calcite crystal aggregates was observed. Molecular weight determinations by size exclusion chromatography and sodium dodecyl sulfate -polyacrylamide gel electrophoresis showed reversible concentration-dependent aggregation of ansocalcin in solution. We propose that such aggregated structures may act as a template for the nucleation of calcite crystal aggregates. Similar aggregation of calcite crystals was also observed when crystallizations were performed in the presence of whole goose eggshell extract. These results show that ansocalcin plays a significant role in goose eggshell calcification.     

Differential label‐free quantitative proteomic analysis of avian eggshell matrix and uterine fluid proteins associated with eggshell mechanical property

Eggshell strength is a crucial economic trait for table egg production. During the process of eggshell formation, uncalcified eggs are bathed in uterine fluid that plays regulatory roles in eggshell calcification. In this study, a label‐free MS‐based protein quantification technology was used to detect differences in protein abundance between eggshell matrix from strong and weak eggs (shell matrix protein from strong eggshells and shell matrix protein from weak eggshells) and between the corresponding uterine fluids bathing strong and weak eggs (uterine fluid bathing strong eggs and uterine fluid bathing weak eggs) in a chicken population. Here, we reported the first global proteomic analysis of uterine fluid. A total of 577 and 466 proteins were identified in uterine fluid and eggshell matrix, respectively. Of 447 identified proteins in uterine fluid bathing strong eggs, up to 357 (80%) proteins were in common with proteins in uterine fluid bathing weak eggs. Similarly, up to 83% (328/396) of the proteins in shell matrix protein from strong eggshells were in common with the proteins in shell matrix protein from weak eggshells. The large amount of common proteins indicated that the difference in protein abundance should play essential roles in influencing eggshell strength. Ultimately, 15 proteins mainly relating to eggshell matrix specific proteins, calcium binding and transportation, protein folding and sorting, bone development or diseases, and thyroid hormone activity were considered to have closer association with the formation of strong eggshell.     

Cloning of ovocalyxin-36, a novel chicken eggshell protein related to lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and plunc family proteins

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 egg-shell 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 permeability-increasing 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.     

Polymorphisms in eggshell organic matrix genes are associated with eggshell quality measurements in pedigree Rhode Island Red hens

Novel and traditional eggshell quality measurements were made from up to 2000 commercial pedigree hens for a candidate gene association analysis with organic eggshell matrix genes: ovocleidin-116 , osteopontin ( SPP1 ), ovocalyxin-32 ( RARRES1 ), ovotransferrin ( LTF ), ovalbumin and ovocalyxin-36 , as well as key genes in the maintenance and function of the shell gland [ estrogen receptor ( ESR1 ) and carbonic anhydrase II ( CAII )]. Associations were found for (i) ovalbumin with breaking strength and shell thickness; (ii) ovocleidin-116 with elastic modulus, shell thickness and egg shape; (iii) RARRES1 with mammillary layer thickness; (iv) ESR1 with dynamic stiffness; (v) SPP1 with fracture toughness and (vi) CAII with egg shape. The marker effects are as large as 17% of trait standard deviations and could be used to improve eggshell quality.     

Phosphoproteins of the chicken eggshell calcified layer

The chicken eggshell matrix is a complex mixture of proteins and proteoglycans. It also contains phosphoproteins that are thought to affect mineralization of the matrix. Several of the matrix phosphoproteins, such as the major component osteopontin, have already been identified as phosphoproteins in other tissues, but the phosphorylation status of the eggshell matrix forms was unknown. The phosphopeptides, obtained after cleavage of the matrix proteins with several different cleavage methods, were enriched by anion-exchange chromatography and reversible binding to titanium oxide and identified by LC-MS(n) or pseudo-MS(n) analysis following neutral loss scanning. Altogether we identified 39 phosphorylated matrix proteins, 22 of which were not known to be phosphorylated before. Eight of the proteins were identified as eggshell matrix components for the first time. Together these proteins contained more than 150 different phosphorylation sites, 103 of which were determined with high confidence. Among the major phosphorylated proteins of the chicken eggshell matrix were osteopontin and the eggshell-specific proteins ovocleidin-17, ovocleidin-116, and ovocalyxin-32.     

Crystallization and preliminary x-ray analysis of ovocleidin-17 a major protein of the gallus gallus eggshell calcified layer

In this work, we report the crystallization of ovocleidin-17, the major protein of the avian eggshell calcified layer and the preliminary X-ray characterization of this soluble protein which is implied into the CaCO(3) formation of the eggshell in avians. Crystals belong to one of the trigonal space group P3 with cell dimensions a= b= 59.53 A and c = 83.33 A, and alpha=beta= 90 degrees and gamma=120 degrees. Crystals diffract up to 3.0 A.     

Genetic variation in eggshell crystal size and orientation is large and these traits are correlated with shell thickness and are associated with eggshell matrix protein markers

The size and orientation of calcium carbonate crystals influence the structure and strength of the eggshells of chickens. In this study, estimates of heritability were found to be high (0.6) for crystal size and moderate (0.3) for crystal orientation. There was a strong positive correlation (0.65) for crystal size and orientation with the thickness of the shell and, in particular, with the thickness of the mammillary layer. Correlations with shell breaking strength were positive but with a high standard error. This was contrary to expectations, as in man-made materials smaller crystals would be stronger. We believe the results of this study support the hypothesis that the structural organization of shell, and in particular the mammillary layer, is influenced by crystal size and orientation, especially during the initial phase of calcification. Genetic associations for crystal measurements were observed between haplotype blocks or individual markers for a number of eggshell matrix proteins. Ovalbumin and ovotransferrin (LTF) markers for example were associated with crystal size, while ovocleidin-116 and ovocalyxin-32 (RARRES1) markers were associated with crystal orientation. The location of these proteins in the eggshell is consistent with different phases of the shell-formation process. In conclusion, the variability of crystal size, and to a lesser extent orientation, appears to have a large genetic component, and the formation of calcite crystals are intimately related to the ultrastructure of the eggshell. Moreover, this study also provides evidence that proteins in the shell influence the variability of crystal traits and, in turn, the shell's thickness profile. The crystal measurements and/or the associated genetic markers may therefore prove to be useful in selection programs to improve eggshell quality.     

Structure of the 116-kDa polypeptide of the clathrin-coated vesicle/synaptic vesicle proton pump.

A 116-kDa polypeptide has recently been found to be a common component of vacuolar proton pumps isolated from a variety of sources. The 116-kDa subunit of the proton pump was purified from clathrin-coated vesicles of bovine brain, and internal sequences were obtained from proteolytic peptides. Oligonucleotide probes designed from these peptide sequences were utilized in polymerase chain reactions to isolate partial bovine cDNA clones for the protein. Sequences from these were then utilized to isolate rat brain cDNA clones containing the full-length coding region. RNA blots indicate the presence of an abundant 3.9-kilobase message for the 116-kDa subunit in brain, and primer extension analysis demonstrates that the cloned sequence is full-length. The rat cDNA sequences predict synthesis of a protein of 96,267 Da. Analysis of the deduced amino acid sequence of the 116-kDa subunit suggests that it consists of two fundamental domains: a hydrophilic amino-terminal half that is composed of greater than 30% charged residues, and a hydrophobic carboxyl-terminal half that contains at least six transmembrane regions. The structural properties of the 116-kDa proton pump polypeptide agree well with its proposed function in coupling ATP hydrolysis by the cytoplasmic subunits to proton translocation by the intramembranous components of the pump.     

Estrogen regulation of tissue-specific expression of complement C3

The administration of estradiol to immature rats results in the increased synthesis and secretion of a 180-kDa protein, composed of 115- and 65-kDa subunits, by the uterine luminal epithelial cells. A monoclonal antibody against the 180-kDa protein was utilized to isolate the corresponding cDNA (LE-1) from a rat uterine luminal epithelial cell cDNA lambda gt11 expression library. This LE-1 cDNA was sequenced and shown to be homologous to complement component C3. The sequence was approximately 81 and 90% homologous to human and mouse C3, respectively. The LE-1 cDNA sequence was homologous with the 3' portion of the C3 mRNA containing the alpha subunit (115 kDa). Uterine mRNA isolated from immature rats treated with 1 microgram of estradiol for 24 h demonstrated a 25-fold increase in the concentration of a 6.0-kilobase mRNA by Northern hybridization with either LE-1 or authentic human C3 cDNA probes. To further examine the possibility that the estradiol-regulated secretory protein was C3, an aliquot of radiolabeled media protein from control and estradiol-stimulated rat uteri was incubated with goat anti-rat C3 antibody. The immunoprecipitated radiolabeled protein from estradiol-treated animals was increased significantly (p less than 0.01) compared to media from control animals. Analysis of the immunoprecipitated proteins on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of 180 kDa from estradiol-stimulated uterine media, whereas no detectable proteins were immunoprecipitated from media obtained from control uteri. Also, when the immunoprecipitated protein was reduced (20 mM dithiothreitol) it dissociated into two subunits of 115 and 65 kDa. Immunohistochemical studies demonstrated the presence of C3 only in the epithelial cells of estrogen-stimulated rat uteri. In addition, the estradiol-stimulated mRNA was only detectable in uterine epithelial cell RNA. Analysis of liver RNA demonstrated a 6.0-kilobase mRNA, as in the uterus, when hybridized with LE-1. However, unlike the uterus, its concentration was not influenced by estrogen administration with up to three daily injections of 100 micrograms of diethylstilbestrol. Based on biophysical, DNA sequence, and antibody data we conclude that rat uterine epithelial cells produce C3 in response to estradiol whereas the expression in the liver was not modulated by estrogens.     

Nucleotide sequence of a cDNA for a member of the human 90-kDa heat-shock protein family

This paper describes the isolation and sequence of a human cDNA homologous to a class of proteins commonly referred to as 90-kDa heat-shock proteins. The complete nucleotide sequence of 2563 bp and the deduced amino acid sequence are presented. A single long open reading frame encodes a protein of 83,303 Da, the amino acid composition of which correlates well with that determined for the human 90-kDa heat-shock or 'stress' protein [Welch, W.J. and Feramisco, J.R., J. Biol. Chem. 257 (1982) 14949-14959]. Moreover, sequence analysis of this gene reveals extensive homology with the Drosophila 83-kDa and yeast 90-kDa heat-shock proteins. A comparison of the translated product of the human cDNA to the published yeast 90-kDa heat-shock protein reveals more than 60% homology at both the nucleotide and amino acid levels. Several regions of 50 aa or more show greater than 90% identity. This cDNA also hybridizes with an RNA species which increases upon heat shock of HeLa cells.     

cDNA-derived amino acid sequence of the 86-kDa subunit of the Ku antigen

The Ku antigen is a DNA-associated nuclear protein recognized by sera from patients with autoimmune diseases. It consists of two polypeptides of 86 and 70 kDa. cDNA clones encoding the 86-kDa subunit of the Ku antigen were isolated by probing lambda gt11 recombinant cDNA expression libraries with a monoclonal antibody specific for this protein. The amino acid sequence deduced from the cDNA comprises 732 amino acids and corresponds to a protein with molecular weight of 81.914. Nineteen residues at the NH2 terminus determined by protein sequencing corresponded to the sequence deduced from the cDNA. The predicted amino acid sequence contains a region with repeating leucine residues similar to the "leucine zipper" structure observed in the c-myc, v-myc, and c-fos oncogene products. The largest cDNA hybridized to 2.7- and 3.4-kilobase poly(A)+ mRNAs from HeLa cells. The cDNA clones expressed fusion proteins immunoreactive with the monoclonal antibody and sera from patients with autoimmune diseases.     

Ovine osteopontin: I. Cloning and expression of messenger ribonucleic acid in the uterus during the periimplantation period.

Trophoblast-derived interferon tau (IFNtau) acts on the endometrium to increase secretion of several proteins during the pregnancy recognition period in ruminants. One of these is a 70-kDa acidic protein that has not been identified. Our hypothesis was that the 70-kDa acidic protein is osteopontin (OPN). OPN is an acidic glycoprotein that fragments upon freezing and thawing or treatment with proteases including thrombin. OPN contains a Gly-Arg-Gly-Asp-Ser (GRGDS) sequence that binds to cell surface integrins to promote cell-cell attachment and cell spreading. Using antisera to recombinant human OPN, both 70-kDa and 45-kDa proteins were identified in uterine flushings from pregnant ewes by Western blotting. A clone containing the entire ovine OPN cDNA coding sequence was isolated by screening a Day 15 pregnant ovine endometrial cDNA library with a partial ovine OPN cDNA. In pregnant ewes, steady-state levels of OPN endometrial mRNA increased (P < 0. 01) after Day 17. In both cyclic and pregnant ewes, in situ hybridization analysis showed that OPN mRNA was localized on unidentified immune cells within the stratum compactum of the endometrium. In pregnant ewes, OPN mRNA was also expressed by the glandular epithelium. Results suggest that progesterone and/or IFNtau induce expression and secretion of OPN by uterine glands during the periimplantation period and that OPN may induce adhesion between luminal epithelium and trophectoderm to facilitate superficial implantation.     

Identification and characterisation of a Leishmania donovani antigen belonging to the 70-kDa heat-shock protein family

A Leishmania donovani promastigote cDNA library was screened with serum obtained from a patient infected with visceral leishmaniasis. Sequence analysis of a clone obtained from this library revealed that the 600-bp insert corresponded to the carboxy-terminal region of an antigen related to the 70-kDa heat-shock protein family. The full-length sequence of the corresponding gene (1959 nucleotides) was determined after isolation of genomic clones. Genes encoding the antigen are present on a single chromosome as a series of approximately twelve 3.7-kb direct tandem repeats. The antigen can be identified as a 70-kDa heat-shock cognate protein by virtue of its molecular mass, sequence and constitutive expression during heat shock. It is expressed at all stages of the parasite life-cycle. Antibodies against the lambda gt11 fusion protein were detected in more than 50% of serum samples obtained from patients with visceral leishmaniasis, but were not detected in sera from patients with cutaneous leishmaniasis or Chagas' disease.     

Primary structure of alpha 2-macroglobulin receptor-associated protein. Human homologue of a Heymann nephritis antigen.

The alpha 2-macroglobulin (alpha 2M) receptor complex as purified by affinity chromatography contains three polypeptides: a 515-kDa heavy chain, an 85-kDa light chain, and a 39-kDa associated protein. Previous studies have established that the 515/85-kDa components are derived from a 600-kDa precursor whose complete sequence has been determined by cDNA cloning (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gassepohl, H., and Stanley, K. (1988) EMBO J. 7,4119-4127). We have now determined the primary structure of the human 39-kDa polypeptide, termed alpha 2M receptor-associated protein, by cDNA cloning. The deduced amino acid sequence contains a putative signal sequence that precedes the 323-residue mature protein. Comparative sequence analysis revealed that alpha 2M receptor-associated protein has 73% identity with a rat protein reported to be a pathogenic domain of Heymann nephritis antigen gp 330 and 77% identity to a mouse heparin-binding protein termed HBP-44. The high overall identity suggests that these molecules are interspecies homologues and indicates that the pathogenic domain, previously thought to be a portion of gp 330, is in fact a distinct protein. Further, the 120-residue carboxyl-terminal region of alpha 2M receptor-associated protein has 26% identity with a region of apolipoprotein E containing the low density lipoprotein receptor binding domain. Pulse-chase experiments revealed that the newly formed alpha 2M receptor-associated protein remains cell-associated, while surface labeling experiments followed by immunoprecipitation suggest that this protein is present on the cell surface forming a complex with the alpha 2M receptor heavy and light chains.