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• W2037257086 abstract "The role of avian eggshell matrix proteins in shell formation is poorly understood. This calcitic biomaterial forms in a uterine fluid where the protein composition varies during the initial, calcification, and terminal phases of eggshell deposition. A specific antibody was raised to a 116-kDa protein, which is most abundant in uterine fluid during active eggshell calcification. This antiserum was used to expression screen a bacteriophage cDNA library prepared using mRNA extracted from pooled uterine tissue harvested at the midpoint of eggshell calcification. Plasmids containing inserts of differing 5′-lengths were isolated with a maximum cDNA sequence of 2.4 kilobases. Northern blotting and reverse transcriptase-polymerase chain reaction demonstrated that the 2.35-kilobase message was expressed in a uterine-specific manner. The hypothetical translational product from the open reading frame corresponded to a novel 80-kDa protein, which we have named ovocleidin-116. After removal of the predicted signal peptide, its N-terminal sequence corresponded almost exactly with that determined from direct microsequencing of the 116-kDa uterine protein (this work) and with that previously determined for the core protein of a 120-kDa eggshell dermatan sulfate proteoglycan (Corrino, D. A., Rodriguez, J. P., and Caplan, A. I. (1997) Connect. Tissue Res. 36, 175–193). Ultrastructural colloidal gold immunocytochemistry of ovocleidin-116 demonstrated its presence in the organic matrix, in small vesicles found throughout the mineralized palisade layer, and the calcium reserve assembly of the mammillary layer. Ovocleidin-116 thus is a candidate molecule for the regulation of calcite growth during eggshell calcification. The role of avian eggshell matrix proteins in shell formation is poorly understood. This calcitic biomaterial forms in a uterine fluid where the protein composition varies during the initial, calcification, and terminal phases of eggshell deposition. A specific antibody was raised to a 116-kDa protein, which is most abundant in uterine fluid during active eggshell calcification. This antiserum was used to expression screen a bacteriophage cDNA library prepared using mRNA extracted from pooled uterine tissue harvested at the midpoint of eggshell calcification. Plasmids containing inserts of differing 5′-lengths were isolated with a maximum cDNA sequence of 2.4 kilobases. Northern blotting and reverse transcriptase-polymerase chain reaction demonstrated that the 2.35-kilobase message was expressed in a uterine-specific manner. The hypothetical translational product from the open reading frame corresponded to a novel 80-kDa protein, which we have named ovocleidin-116. After removal of the predicted signal peptide, its N-terminal sequence corresponded almost exactly with that determined from direct microsequencing of the 116-kDa uterine protein (this work) and with that previously determined for the core protein of a 120-kDa eggshell dermatan sulfate proteoglycan (Corrino, D. A., Rodriguez, J. P., and Caplan, A. I. (1997) Connect. Tissue Res. 36, 175–193). Ultrastructural colloidal gold immunocytochemistry of ovocleidin-116 demonstrated its presence in the organic matrix, in small vesicles found throughout the mineralized palisade layer, and the calcium reserve assembly of the mammillary layer. Ovocleidin-116 thus is a candidate molecule for the regulation of calcite growth during eggshell calcification. polyacrylamide gel electrophoresis 3-(cyclohexylamino)propanesulfonic acid ovocleidin-116 phosphate-buffered saline polymerase chain reaction The avian eggshell forms in the uterine (shell gland) region of the oviduct in an acellular milieu that is supersaturated with respect to calcium and bicarbonate and which contains a variety of proteins whose concentrations vary during the sequential process of shell formation (1Nys Y. Hincke M.T. Arias J.L. Garcia-Ruiz J.M. Solomon S.E. Poult. Avian Biol. Rev. 1999; (in press)Google Scholar, 2Arias J.L. Fink D.J. Xiao S-Q. Heuer A.H. Caplan A.I. Int. Rev. Cytol. 1993; 145: 217-250Crossref PubMed Scopus (169) Google Scholar, 3Hamilton R.M.G. Food Microstructure. 1986; 5: 99-110Google Scholar, 4Parsons A.H. Poult. Sci. 1982; 61: 2013-2021Crossref Google Scholar). The eggshell matrix proteins that have been identified can be subdivided into at least three groups. Osteopontin, also found in bone matrix, is an eggshell component, as are a number of egg white proteins (ovalbumin, ovotransferrin, and lysozyme) (5Pines M. Knopov V. Bar A. Matrix Biol. 1996; 14: 765-771Crossref Scopus (104) Google Scholar, 6Hincke M.T. Connect. Tissue Res. 1995; 31: 227-233Crossref PubMed Scopus (103) Google Scholar, 7Gautron J. Hincke M.T. Panheleux M. Garcia-Ruiz J.M. Nys Y. Goldberg M. Robinson C. Proceedings of the Sixth International Conference on the Chemistry and Biology of Mineralized Tissues. American Academy of Orthopaedic Surgeons, Rosemont, IL1999Google Scholar). The third group are proteins that N-terminal amino acid sequencing and immunochemistry indicate are eggshell-specific. One of these, ovocleidin-17, exhibits similarity in its N-terminal sequence to a variety of snake venom lectins and to the C-type lectin-like domain of certain proteoglycans (8Hincke M.T. Tsang C.P.W. Courtney M. Hill V. Narbaitz R. Calcif. Tissue Int. 1995; 56: 578-583Crossref PubMed Scopus (122) Google Scholar). 1M. T. Hincke, manuscript in preparation. 1M. T. Hincke, manuscript in preparation.The proteins are initially secreted by epithelial and glandular cells into the uterine fluid, whose protein composition varies remarkably during the initial, active calcification and terminal phases of eggshell mineralization (9Gautron J. Hincke M.T. Nys Y. Connect. Tissue Res. 1997; 36: 195-210Crossref PubMed Scopus (113) Google Scholar). Sequential deposition of these matrix proteins into the forming eggshell results in their different localization patterns as the inner (mammillary) and outer (palisade) layers of the mineralized shell are assembled (10Hincke M.T. Bernard A.-M. Lee E.R. Tsang C.P.W. Narbaitz R. Br. Poult. Sci. 1992; 33: 505-516Crossref PubMed Scopus (42) Google Scholar). Partially purified eggshell matrix and uterine fluid proteins can dramatically delay the precipitation of calcium carbonate from a metastable solution of calcium chloride and sodium bicarbonate (9Gautron J. Hincke M.T. Nys Y. Connect. Tissue Res. 1997; 36: 195-210Crossref PubMed Scopus (113) Google Scholar, 11Gautron J. Bain M.M. Solomon S.E. Nys Y. Br. Poult. Sci. 1996; 37: 853-866Crossref PubMed Scopus (57) Google Scholar). We have demonstrated that such partially purified mixtures contain eggshell osteopontin and that dephosphorylation dramatically reduces this inhibitory activity. 2M. T. Hincke and M. St. Maurice, submitted for publication. 2M. T. Hincke and M. St. Maurice, submitted for publication. In addition, the carbohydrate moieties of dermatan sulfate and keratan sulfate proteoglycans have been demonstrated immunochemically in eggshell matrix (12Arias J.L. Carrino D.A. Fernandez M.S. Rodriguez J.P. Dennis J.E. Caplan A.I. Arch. Biochem. Biophys. 1992; 298: 293-302Crossref PubMed Scopus (89) Google Scholar, 13Fernandez M.S. Araya M. Arias J.L. Matrix Biol. 1997; 16: 13-20Crossref PubMed Scopus (99) Google Scholar). In vitro, fractions containing partially purified eggshell dermatan sulfate proteoglycan alter the morphology and decrease the size of growing calcite crystals (14Wu T.-M. Fink D.J. Arias J.L. Rodriguez J.P. Heuer A.H. Caplan A.I. Slavkin H.C. Price P. Chemistry and Biology of Mineralized Tissues. Elsevier Science Publishing Co., Inc., New York1992: 133-141Google Scholar, 15Wu T.-M. Rodriguez J.P. Fink D.J. Carrino D.A. Blackwell J. Caplan A.I. Heuer A.H. Matrix Biol. 1994; 14: 507-513Crossref Scopus (52) Google Scholar, 16Carrino D.A. Dennis J.E. Wu T.M. Arias J.L. Fernandez M.S. Rodriguez J.P. Fink D.J. Heuer A.H. Caplan A.I. Connect. Tissue Res. 1996; 35: 325-329Crossref PubMed Scopus (55) Google Scholar). However, despite these studies, the direct roles for specific matrix components in eggshell formation remain undefined. There has been a lack of precise molecular detail regarding the uterine-specific eggshell matrix proteins, information that would allow comparisons with similar proteins in other calcifying tissues. In the present study, we report the cloning, characterization, and ultrastructural localization of a 116-kDa protein that is a major constituent of the uterine fluid during the calcification stage of shell formation. Furthermore, sequence comparisons reveal that it corresponds to the core protein of a unique, partially characterized dermatan sulfate proteoglycan that possesses little homology to proteoglycans previously isolated from bone or cartilage. Egg-laying hens (ISA BROWN) were housed individually in cages located in a windowless, air conditioned poultry house. They were subjected to a cycle of 14 h of light:10 h of darkness and were fed ad libitum on a layers' diet as recommended by the Institut National de la Recherche Agronomique (France). Cages were equipped with a computerized system to record the precise time of daily egg laying (oviposition). Ovulation was considered to occur 0.5 h after oviposition (11Gautron J. Bain M.M. Solomon S.E. Nys Y. Br. Poult. Sci. 1996; 37: 853-866Crossref PubMed Scopus (57) Google Scholar). Eggs were expelled with an intravenous injection of 50 μg/hen of prostaglandin (prostaglandin F2α) at 6–9 h (initial stage), 18–19 h (active calcification phase), and 22–23 h (terminal phase of shell calcification) after the preceding oviposition (11Gautron J. Bain M.M. Solomon S.E. Nys Y. Br. Poult. Sci. 1996; 37: 853-866Crossref PubMed Scopus (57) Google Scholar). Uterine fluid was collected immediately after the egg expulsion by gravimetry into a plastic test tube placed at the entrance of the everted vagina. Aliquots of uterine fluid were immediately diluted 1:1 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)3 buffer (0.0625m Tris-HCl, pH 6.8, 2% SDS, 5% β-mercaptoethanol, 25% glycerol, 0.01% bromphenol blue). A portion of the fluid was kept to measure protein concentration, and the remainder was rapidly frozen in liquid nitrogen and stored at −20 °C. Protease inhibitors were used throughout all extraction procedures (17Termine J.D. Wientroub S. Fischer L.W. Dickson G.R. Methods of Calcified Tissue Preparation. Elsevier Science Publishers B.V., Amsterdam1984: 547-563Google Scholar). The first extraction protocol was based upon methods developed to extract matrix proteins from bone and cartilage (17Termine J.D. Wientroub S. Fischer L.W. Dickson G.R. Methods of Calcified Tissue Preparation. Elsevier Science Publishers B.V., Amsterdam1984: 547-563Google Scholar) and has been previously described (9Gautron J. Hincke M.T. Nys Y. Connect. Tissue Res. 1997; 36: 195-210Crossref PubMed Scopus (113) Google Scholar, 11Gautron J. Bain M.M. Solomon S.E. Nys Y. Br. Poult. Sci. 1996; 37: 853-866Crossref PubMed Scopus (57) Google Scholar). Briefly, eggshell powder was extracted with 4 m guanidine hydrochloride, 0.05m Tris-HCl, pH 7.4, for 4 days. The resulting supernatant was dialyzed, desalted, and then concentrated by ultrafiltration (Amicon Cell, cutoff 5 kDa). This soluble material is referred to as the extramineral eggshell extract. The pellet was demineralized with 0.5 m EDTA, 0.05 m Tris, pH 7.4, extracted with 4 m guanidine hydrochloride, 0.05 m Tris, pH 7.4, and then centrifuged. The resulting supernatant was desalted and concentrated as described above and is referred to as the intramineral eggshell extract. The second extraction protocol was an adaption of one previously published (13Fernandez M.S. Araya M. Arias J.L. Matrix Biol. 1997; 16: 13-20Crossref PubMed Scopus (99) Google Scholar). A membrane extract was prepared by treating ground shell membranes with 4 m guanidine hydrochloride. The supernatant was desalted by dialysis and concentrated in an Amicon cell. This sample is referred to as the membrane extract. The ground shell was dialyzed against 50% acetic acid to demineralize. After further dialysis against demineralized water, the soluble material was concentrated as before and is referred to as the matrix soluble 1 fraction. The insoluble shell material was extracted as above for the membranes and is referred to as the matrix insoluble 1 fraction. Matrix proteins from tibiotarsal bones and from the upper proliferating zone of the cartilage growth plate of broiler chickens (4 weeks old) were extracted by established procedures (17Termine J.D. Wientroub S. Fischer L.W. Dickson G.R. Methods of Calcified Tissue Preparation. Elsevier Science Publishers B.V., Amsterdam1984: 547-563Google Scholar) and are referred to as cartilage- or bone-EDTA extracts. Laying hens were sacrificed 2–4 h or 16–17 h after oviposition. Various tissues (0.5–2 g of magnum, white and red isthmus, uterus, liver, kidney, duodenum, and muscle) were homogenized in 10 ml of Tris buffer (50 mm Tris-HCl, 77 mm NaCl, pH 7.4, containing protease inhibitors). Homogenates were centrifuged (10,000 ×g, 30 min) before freezing in liquid nitrogen for storage at −20 °C. Blood was collected from the same birds just before sacrifice and centrifuged at 2500 × g to obtain a sample of plasma. The protein concentration of all samples and extracts was determined by a Pierce micromethod (18Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211906) Google Scholar) using ovalbumin as the protein standard. Tissue samples were diluted 1:1 in SDS-PAGE buffer and boiled for 5 min before separation on gels. SDS-PAGE was performed on a 4–20% gradient gel (19Laemnli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205511) Google Scholar, 20Hames B.D. Rickwood D. Gel Electrophoresis of Proteins: A Practical Approach. IRL Press, Oxford1981Google Scholar). Proteins were stained with Coomassie Blue or electroblotted (0.8 mA/Cm2) for 1 h onto polyvinylidene difluoride membranes (Hyperbond, Beckman) in 10 mm CAPS buffer, pH 11, 10% methanol for N-terminal amino acid microsequencing or to nitrocellulose membranes (Bio-Rad) in 25 mm Tris, 192 mm glycine, 10% methanol for Western blot analysis. Two preparative SDS-PAGE gradient gels (3 mm width) were prepared. About 8 mg of uterine fluid protein collected at the active phase of shell calcification were distributed into the sample lanes of both gels and electrophoresed. Gels were stained with Coomassie Blue, destained, and then thoroughly rinsed with demineralized water. The 116-kDa band was excised from both gels and ground to a fine powder with a Spex freezer mill. The powder was suspended in sterile 154 mm NaCl solution, mixed 1:1 with Freund's complete adjuvant, and injected into two rabbits. The animals were boosted after 3 weeks with the same antigen preparation in Freund's incomplete adjuvant. Animals were boosted 6 times to obtain a satisfactory titer. This antiserum is termed α-116. Proteins were electrotransferred to nitrocellulose membrane, which were washed in PBS-Tween (0.01m phosphate buffer, 0.0027 m potassium chloride, 0, 137 m sodium chloride, pH 7.4, Tween 20 0.1%), and then blocked for 1 h in 5% nonfat dry milk in PBS-Tween. The membranes were washed in PBS-Tween (2 × 5 min) and then incubated for 1.5 h with diluted antiserum (1:5000–1:10,000 in PBS-Tween, 1% bovine serum albumin). After 3 washes in PBS-Tween, the membranes were incubated for 1 h with 1:10,000 anti-rabbit Ig, peroxidase-linked species-specific whole antibody (NA 934, Amersham Pharmacia Biotech) in PBS-Tween, 1% bovine serum albumin. Membranes were washed again (4 times) in PBS-Tween and then twice in PBS. The enhanced chemiluminescence (ECL) method was used according to the manufacturer's instructions to reveal immunoreactive bands. Normal eggs from White Leghorn hens were cracked open and extensively washed with distilled water. Shell membranes were removed following brief treatment with 1 N HCl at room temperature as described previously (10Hincke M.T. Bernard A.-M. Lee E.R. Tsang C.P.W. Narbaitz R. Br. Poult. Sci. 1992; 33: 505-516Crossref PubMed Scopus (42) Google Scholar). Ground shell (140 g) was decalcified in a solution of 0.65m EDTA, pH 7.5, 10 mm 2-mercaptoethanol, 0.05% NaN3. The resulting suspension was centrifuged and dialyzedversus 10 mm Tris-HCl, pH 7.5, 1 mm2-mercaptoethanol, 0.05% NaN3 and loaded onto a previously equilibrated column of Trisacryl-DEAE. After washing the column extensively, all retained proteins were eluted with 250 ml of 1m NaCl in the equilibration buffer. This solution was applied to a column of hydroxyapatite (8 ml of resin) that had been previously equilibrated with 5 mm potassium phosphate, pH 7.5, 250 mm NaCl, 1 mm 2-mercaptoethanol, 0.05% NaN3. A linear gradient to 0.4 mpotassium phosphate was developed, which eluted 3 peaks (A 280). These were analyzed by SDS-PAGE and pooled for dialysis versus water and lyophilization. The second peak (fraction 27–35) contained ovocleidin-17 and osteopontin in addition to several other bands that were found by microsequencing to be derived from OC-116 (see Fig. 5). Sequencing was performed as described previously (8Hincke M.T. Tsang C.P.W. Courtney M. Hill V. Narbaitz R. Calcif. Tissue Int. 1995; 56: 578-583Crossref PubMed Scopus (122) Google Scholar). Proteins from uterine fluid sample (25 μg of protein) harvested from the calcification stage of shell formation were separated by SDS-PAGE on a 7% gel and then transferred to polyvinylidene difluoride (Bio-Rad Laboratories). Protein bands were visualized by Coomassie Blue staining. The 116-kDa band (which ran as 130 kDa on the 7% gel) was excised and washed extensively with sterile water. N-terminal microsequencing was performed at the Service de Sequence de Peptide de l'Est du Quebec, Laval, Quebec and revealed the following sequence: (T/V)PV(S/G)LPAR(A/I)(R/V)GN(D/C)PGQHQILLK. Pooled RNA was prepared by the guanidinium isothiocyanate extraction method (21Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62898) Google Scholar) from shell gland mucosal tissue harvested at the middle phase of eggshell calcification from 4 White Leghorn hens. Messenger RNA was purified by affinity chromatography on oligo(dT)-Sepharose, and utilized to prepare a vector-ligated, directional bacteriophage cDNA library using the Stratagene ZAP-cDNA synthesis kit and Gigapack II packaging system exactly according to the manufacturer's protocols. The phage library was screened by standard methods using antiserum raised to the 116-kDa eggshell matrix protein (1/1000) and anti-rabbit IgG-alkaline phosphatase conjugate (1/3000) as a secondary antibody with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. Protein synthesis was induced with nitrocellulose circles (Amersham Pharmacia Biotech Hybond C) pretreated with 10 mmisopropyl-1-thio-β-d-galactopyranoside. Phages with positive inserts were purified to homogeneity by three rounds of screening and rescued into Bluescript plasmid using helper phage, according to the kit instructions. Plasmid DNA was isolated by alkaline lysis miniprep, and the cDNA inserts were sequenced by automated protocols at the sequencing service, Station de Pathologie Aviaire et de Parasitologie, INRA-Center de Tours, Nouzilly, France. RNA was extracted from oviduct tissues harvested at different stages of eggshell calcification (2 birds/stage) (RNA InstaPure, Eurogentech). Northern blotting was performed by established methods (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) following migration of 15 μg of total RNA on a 1% agarose gel containing formaldehyde and transfer of RNA to Hybond N membrane. A BstEII restriction fragment (OC-116 nucleotides 234–959) was radiolabeled by random priming. Following overnight hybridization with this labeled probe at 42 °C, the membrane was washed (42 °C: 2× SSC, 0.1% SDS, 15 min; 0.5× SSC, 0.1% SDS, twice for 15 min; 55 °C: 0.5× SSC, 0.1% SDS). Labeled bands were visualized with x-ray film and quantified with a Storm PhosphorImager. Southern blotting of chicken genomic DNA (prepared from White Leghorn liver (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar)) digested with EcoRI and BamHI was also performed using the OC-116 BstEII restriction fragment as a probe. Reverse transcription was performed using random priming and Superscript II RNase H-reverse transcriptase (Life Technologies, Inc.). PCR primers to selectively amplify nucleotides 536–1227 had the following sequences: upper, 5′-TGGAGATGGCGATGGTG-3′; and lower, 5′-TGGCTGGAAGGCTATGC-3′. Samples of hen uterus and eggshell were fixed in aldehyde immediately after tissue harvest. These were washed, dehydrated in ethanol, and embedded in LR White acrylic resin as described previously (23McKee M.D. Nanci A. Microsc. Res. Tech. 1995; 31: 44-62Crossref PubMed Scopus (94) Google Scholar). Survey sections of embedded tissue were viewed by light microscopy, and selected regions were trimmed for thin sectioning and transmission electron microscopy. Grid-mounted tissue sections were processed for immunocytochemistry by incubation with primary antibodies, and immunolabeling patterns were visualized after conventional staining with uranyl acetate and lead citrate using protein A-colloidal gold complex (23McKee M.D. Nanci A. Microsc. Res. Tech. 1995; 31: 44-62Crossref PubMed Scopus (94) Google Scholar). Incubated grids were examined in a JEOL JEM 1200FX operated at 60 kV. Other samples of eggshell were fractured, critical-point dried, and examined at 3 kV in a JEOL JSM 6400V field emission scanning electron microscope. The protein composition of hen uterine fluid varies dramatically between the initial, active calcification and terminal stages of shell formation (Fig. 1). Particularly evident during the active calcification stage are bands at 180, 116, 66, 45, 36, 32, 21, and 14 kDa. In the present study, polyclonal antibodies were raised in rabbit toward the prominent 116-kDa protein. Western blotting with this antiserum showed immunoreactive bands of 180, 116, 66, and 45 kDa in uterine fluid only at this stage of shell calcification (Fig.2). Only eggshell matrix extracted by decalcification from the calcified shell (intramineral eggshell extract and matrix soluble 1 fraction) showed similar immunoreactive bands (Fig. 2). Western blot analysis revealed that the 180- and 116-kDa bands were only seen in extracts of uterine tissue and not in other regions of the oviduct or any other tissue (Fig.3). However, these bands could hardly be detected in uterine tissue harvested at the initial stage of shell formation (data not shown). No immunoreactivity was detected in intra- or extramineral extracts of cartilage or bone or in hen egg white (data not shown). These results suggested that the antiserum raised to the 116-kDa band recognized a protein that was specifically synthesized and secreted by the uterine cells during the active stage of eggshell calcification, which subsequently became incorporated into the calcifying matrix.Figure 2Western blotting to detect the 116-kDa protein in uterine fluid and eggshell extracts. Samples were subjected to SDS-PAGE and electrotransferred to nitrocellulose membrane (“Materials and Methods”). Primary antiserum raised to the 116-kDa protein was utilized at 1:5000 dilution. All samples were 20 μg of protein except uterine fluid (calcification, 17 μg; and terminal, 19 μg) and membranes (1.3 μg). Uterine fluid samples were harvested at different stages of eggshell formation: 1, initial; 2, active calcification; 3, terminal. Eggshell extract samples were as follows: 4, intramineral; 5, extramineral; 6, matrix soluble 1; 7, matrix soluble 2; 8, membranes. The calculated masses (kDa) of the immunoreactive bands in lane 2 are indicated on theleft (arrows).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Western blotting to detect the 116-kDa protein in oviduct and other hen tissues. Extracts from different tissues (all 20 μg of protein) were subjected to SDS-PAGE and electrotransferred to nitrocellulose membrane (“Materials and Methods”). Primary antiserum raised to the 116-kDa protein was utilized at a 1:5000 dilution. Lanes were loaded as follows:1, magnum; 2, white isthmus; 3, red isthmus; 4, uterus; 5, plasma; 6,liver; 7, kidney; 8, duodenum; 9,muscle. Lane 10 contained molecular weight markers (Bio-Rad, prestained). The arrows indicate the immunoreactive bands seen only in the uterus (145–166 and 99–118 kDa).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To clone the coding sequences for the 116-/180-kDa immunoreactive bands, an expression screening approach was taken. A cDNA library prepared with mRNA purified from hen uterine tissue harvested at the midpoint of eggshell calcification was utilized. Expression screening this cDNA library with α-116 yielded a number of clones with inserts of variable length that differed in their 5′-sequence. These were assembled to produce a single composite cDNA sequence. In total, 24 independently purified clones were sequenced, two of which were full-length. All phage inserts that were immunoreactive with α-116 were derived from this sequence, confirming the specificity of the antiserum in this context. The total composite cDNA sequence was 2407 bases in length with a poly(A) tail. The total composite cDNA sequence is seen in Fig.4. No significant similarity was found with any nucleotide sequence in the NIH GenBank or EST data bases. 4The nucleotide sequence for ovocleidin-116 has been deposited in the GenBank data base under GenBank Accession numberAF148716. The context of the first start codon at nucleotide 24 is compatible with the Kozak consensus sequence for initiation of transcription in higher eukaryotes (24Kozak M. J. Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2788) Google Scholar). An open reading frame of 2343 base pairs was found; the corresponding hypothetical 80-kDa protein sequence is indicated in Fig.4. A signal peptide cleavage site is predicted between residues 18 and 19 (25Nielsen H. Engelbrecht J. Brunak S. von Heijne G. Protein Eng. 1997; 10: 1-6Crossref PubMed Scopus (4911) Google Scholar). The putative signal peptide (1Nys Y. Hincke M.T. Arias J.L. Garcia-Ruiz J.M. Solomon S.E. Poult. Avian Biol. Rev. 1999; (in press)Google Scholar, 2Arias J.L. Fink D.J. Xiao S-Q. Heuer A.H. Caplan A.I. Int. Rev. Cytol. 1993; 145: 217-250Crossref PubMed Scopus (169) Google Scholar, 3Hamilton R.M.G. Food Microstructure. 1986; 5: 99-110Google Scholar, 4Parsons A.H. Poult. Sci. 1982; 61: 2013-2021Crossref Google Scholar, 5Pines M. Knopov V. Bar A. Matrix Biol. 1996; 14: 765-771Crossref Scopus (104) Google Scholar, 6Hincke M.T. Connect. Tissue Res. 1995; 31: 227-233Crossref PubMed Scopus (103) Google Scholar, 7Gautron J. Hincke M.T. Panheleux M. Garcia-Ruiz J.M. Nys Y. Goldberg M. Robinson C. Proceedings of the Sixth International Conference on the Chemistry and Biology of Mineralized Tissues. American Academy of Orthopaedic Surgeons, Rosemont, IL1999Google Scholar, 8Hincke M.T. Tsang C.P.W. Courtney M. Hill V. Narbaitz R. Calcif. Tissue Int. 1995; 56: 578-583Crossref PubMed Scopus (122) Google Scholar, 9Gautron J. Hincke M.T. Nys Y. Connect. Tissue Res. 1997; 36: 195-210Crossref PubMed Scopus (113) Google Scholar, 10Hincke M.T. Bernard A.-M. Lee E.R. Tsang C.P.W. Narbaitz R. Br. Poult. Sci. 1992; 33: 505-516Crossref PubMed Scopus (42) Google Scholar, 11Gautron J. Bain M.M. Solomon S.E. Nys Y. Br. Poult. Sci. 1996; 37: 853-866Crossref PubMed Scopus (57) Google Scholar, 12Arias J.L. Carrino D.A. Fernandez M.S. Rodriguez J.P. Dennis J.E. Caplan A.I. Arch. Biochem. Biophys. 1992; 298: 293-302Crossref PubMed Scopus (89) Google Scholar, 13Fernandez M.S. Araya M. Arias J.L. Matrix Biol. 1997; 16: 13-20Crossref PubMed Scopus (99) Google Scholar, 14Wu T.-M. Fink D.J. Arias J.L. Rodriguez J.P. Heuer A.H. Caplan A.I. Slavkin H.C. Price P. Chemistry and Biology of Mineralized Tissues. Elsevier Science Publishing Co., Inc., New York1992: 133-141Google Scholar, 15Wu T.-M. Rodriguez J.P. Fink D.J. Carrino D.A. Blackwell J. Caplan A.I. Heuer A.H. Matrix Biol. 1994; 14: 507-513Crossref Scopus (52) Google Scholar, 16Carrino D.A. Dennis J.E. Wu T.M. Arias J.L. Fernandez M.S. Rodriguez J.P. Fink D.J. Heuer A.H. Caplan A.I. Connect. Tissue Res. 1996; 35: 325-329Crossref PubMed Scopus (55) Google Scholar, 17Termine J.D. Wientroub S. Fischer L.W. Dickson G.R. Methods of Calcified Tissue Preparation. Elsevier Science Publishers B.V., Amsterdam1984: 547-563Google Scholar, 18Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211906) Google Scholar) is underlined in Fig. 4. The proposed N terminus of the mature protein corresponds to the results of direct microsequencing of the 116-kDa band (see “Materials and Methods,” (T/V)PV(S/G)LPAR(A/I)(R/V)GN(D/C)PGQHQILLK) and is virtually identical to that recently reported for a 120-/200-kDa eggshell dermatan sulfate proteoglycan (PVSLPARARGNCPGQHILLKGCNTK) (26Corrino D.A. Rodriguez J.P. Caplan A.I. Connect. Tissue Res. 1997; 36: 175-193Crossref PubMed Scopus (67) Google Scholar). Additionally, we have indep" @default.
• W2037257086 created "2016-06-24" @default.
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• W2037257086 date "1999-11-01" @default.
• W2037257086 modified "2023-10-14" @default.
• W2037257086 title "Molecular Cloning and Ultrastructural Localization of the Core Protein of an Eggshell Matrix Proteoglycan, Ovocleidin-116" @default.
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