Purification and characterization of calcium-binding protein from chick chorioallantoic membrane

A procedure is described for the purification of the calcium-binding protein (CaBP) from the chorioallantoic membrane of the chick embryo. With this scheme, a 180- to 200-fold purification was achieved with a 40% yield. Characterization of the CaBP revealed that its properties differ from those of previously studied calcium-binding proteins. The CaBP has a molecular weight of 95,000 to 100,000 and appears to be composed of four subunits of identical molecular weight (22,000 to 25,000). The CaBP is a basic protein as indicated by its high electrophoretic mobility under acidic conditions and its relatively high isoelectric point of 8.06. The calcium-binding activity of the CaBP is sulfhydryl dependent and highly specific for calcium ions (10 high affinity sites, ka = 2.35 X 10(7) m-1; 100 to 120 low affinity sites, ka = 2.00 X 10(5) M-1). Amino acid analysis indicated that the CaBP contains 2 to 10 residues of a modified amino acid, gamma-carboxyglutamate (gamma-CGlu). The presence of gamma-CGlu residues suggested that vitamin K may be involved in the expression of the CaBP in the chorioallantoic membrane.     

Vitamin K-dependent gamma-glutamyl carboxylase activity in the chick embryonic chorioallantoic membrane.

During embryonic development of the chick, the onset of calcium transport by the chorioallantoic membrane (CAM) is concomitant with the appearance of a calcium-binding protein (CaBP). The development-specific expression of the CaBP in the CAM is inhibited by vitamin K antagonism in ovo with the anticoagulant, warfarin. However, the CaBP remains immunologically detectable in the CAM of warfarin-treated embryos, suggesting the presence of a precursor form of the CaBP. Previously, we have demonstrated that CaBP expression in CAM organ cultures is inducible by vitamin K. Furthermore, the CaBP contains several residues of the modified amino acid, gamma-carboxyglutamic acid (gamma-CGlu), which has been shown to be formed by vitamin K-dependent carboxylation of glutamic acid in several plasma clotting proteins. This study reports the presence of a post-translational, vitamin K-dependent gamma-glutamyl carboxylase activity in the CAM. Our results show that explants of CAM incorporate H14CO3 in an age-specific and vitamin K-dependent manner. Incorporation of H14CO3 by the CAM is further potentiated by warfarin treatment of the embryos, presumably owing to an elevation of the amount of endogenous uncarboxylated protein precursor(s). Among the subcellular (nuclear, mitochondrial, microsomal, and soluble) fractions of the CAM, only microsomes exhibit specific incorporation of of H14CO3 into gamma-CGlu. The CAM microsomal carboxylation activity is post-translational, vitamin K-dependent, specific for prenylated homologs of vitamin K, sensitive to warfarin, and appears to be unrelated to the activities of biotin-dependent carboxylases or phosphoenolpyruvate carboxykinase. Optimal carboxylation activity occurs after incubation of the microsomes with H14CO3 for 60 min at 37 degrees C in the presence of over 100 microgram of vitamin K1/ml.     

Vitamin A and bone formation. Different responses to retinol and retinoic acid of chick bone cells in organ culture

The response of bone cells in organ culture to retinol and retinoic acid was studied. Both stimulated incorporation of [3H]thymidine into DNA by 16-day embryonic chick calvaria, but the time-course of the responses differed; the peak responses to retinol and retinoic acid occurred at about 18 h and 48 h, respectively. Although retinol inhibited chick bone collagen synthesis retinoic acid had no effect, but it did stimulate non-collagenous protein synthesis, whereas the effect on the latter of retinol was, if anything, inhibitory. When present with retinol, retinoic acid was able to attenuate the inhibitory effect of the former on chick bone collagen synthesis, but preincubation with retinoic acid had no such effect. In neonatal murine calvarial cultures, retinoic acid inhibited collagen synthesis selectively in the same manner as did retinol. The ability of chick osteoblasts to respond differently to retinol and retinoic acid suggests that both forms of the vitamin may have a role in bone formation and that their intracellular models of action may differ although the attenuation response indicates there may be some interaction between the two.     

Characterization of a novel transformation-sensitive heat-shock protein (HSP47) that binds to collagen

The synthesis of a major collagen-binding glycoprotein of molecular weight 47,000 was previously shown to be regulated by malignant transformation as well as by heat shock in chick embryo fibroblasts. The 47-kDa protein purified from chick embryos was characterized biochemically, and was found to exist as a monomer in native form. Its composition was enriched in basic amino acids and glycine, with fewer acidic residues and virtually no cysteine. N-terminal amino acid sequencing covering 36 residues revealed a single, novel sequence with an internal tandem repeat of Asp-Lys-Ala-Thr-Thr-Leu-Ala and Asp-Arg-Ser-Thr-Thr-Leu-Ala.     

Phosphoproteins of chicken bone matrix. Proof of synthesis in bone tissue

Twelve-day-old embryonic chick mandibles were cultured in vitro for 6 days. Measurements of the weights of the explants, their mineral and protein components, and the EDTA-extractable proteins established that bone tissue synthesizes O-phosphoserine- and O-phosphothreonine-containing phosphoproteins which are similar to those present in embryonic and postnatal chicken bone matrix. The synthesis of the phosphoproteins was further confirmed by the demonstration that radioactively labeled O-phosphoserine and O-phosphothreonine were identified in bone and in the EDTA-extractable phosphoproteins after pulse-labeling chick mandibles in vitro with radioactively labeled serine and threonine, respectively.     

Characteristics of the vitamin K-dependent carboxylating system in human placenta.

Gamma-carboxyglutamic acid, formed during the post-translational vitamin K-dependent carboxylation of glutamic acid residues in polypeptides has been identified not only in coagulation factors II (prothrombin),, VII, IX and X [1--4], but also in several other plasma proteins [3,5,6] and in protein of bone [7,8] and kidney [9]. In rat liver, carboxylation is mediated through an enzyme system located in the microsomal membrane [10]. The enzyme system requires CO2, O2 and the reduced (hydroquinone) form of the vitamin, as well as a suitable substrate [10,11]. Rat liver microsomes also convert vitamin K1 (phylloquinone) to its stable 2,3-epoxide [12]. Several studies suggest a link between carboxylation and the formation of the epoxide [12--14]. In one of these [14], a survey of rat tissues for vitamin K1 epoxidation revealed that, in addition to liver, this activity was also possessed by kidney, bone, spleen and placenta. In preliminary experiments, vitamin K-dependent carboxylating systems have been found in rat and chick kidney [9], in chick bone [15] and in rat spleen and placenta (unpublished observations). In this communication, we describe some of the basic characteristics of the vitamin K-dependent carboxylating system as found in human placental microsomes.     

Sequence of carbonic anhydrase II cDNA from chick retina

Sequences of three cDNA clones for carbonic anhydrase II (CA-II) from chick retina are presented. The longest cDNA clone encodes all but the first three amino acids of CA-II, and the encoded sequence generally agrees with published fragments of CA-II sequence from chick red blood cells. It is 70% identical to human CA-II; the active-site residues are conserved, but the chick protein has six extra cysteines. There is a long 3'-untranslated region which contains a second open reading frame, but this is not conserved. There appears to be a single CA-II gene in the chick. Some anomalies in cDNA synthesis and in Bal31 deletion are noted.     

Expression of cellular retinoic acid binding protein II (chick-CRABP II) in the chick embryo

We previously demonstrated the presence of cellular retinoic acid binding protein II, chick-CRABP II, in chick embryos. In the present study, we investigated the distribution of chick-CRABP II in 14-day chick embryos by means of immunoblot analysis. Chick-CRABP II was expressed in skin, muscle, bone with tendon of the embryos, but not expressed in the nervous system. In adult chick tissues, chick-CRABP II was not detected on immunoblotting; Chick-CRABP II in adults amounts to less than 10 ng/mg soluble protein. These observations suggest that chick- CRABP II is an embryonic protein involved in the development of specific tissues, such as bone, muscle and skin.     

Incorporation of proline analogs into procollagen. Assay for replacement of imino acids by cis-4-hydroxy-L-proline and cis-4-fluoro-L-proline

Previously, several proline analogs have shown to be incorporated into protein and, in particular, into procollagen polypeptides. Here a new technique was used to determine the extent to which two proline analogs, cis-4-hydroxy-l-proline and cis-4-fluoro-l-proline, replaced proline and hydroxyproline in newly synthesized pro-α and pro-γ chains of procollagen. Matrix-free chick embryo tendon cells, when incubated with 1.53 mm, cis-4-hydroxy-l-proline, synthesized collagenous polypeptides in which from 13 to 19% of the total imino acid residues were replaced with the analog. Incubation of cells with 1.50 mm, cis-4-fluoro-l-proline resulted in the synthesis of polypeptides in which 27% of the imino acid residues were replaced by the analog. With lower concentrations, proportionally less of the analog was incorporated into protein. The observations here extend previous indications that proline analogs in relatively low concentrations may have a specific effect on the synthesis of collagen.     

Vitamin A and bone formation. Effect of an excess of retinol on bone collagen synthesis in vitro.

The influence of an excess of retinol on bone formation was studied by using cultures of embryonic-chick calvaria. Retinol decreased collagen synthesis in a dose-dependent manner, non-collagenous protein synthesis being relatively unaffected. Collagen synthesis was significantly inhibited after 24 h of culture with retinol and was progressively decreased, compared with control cultures containing no retinol, as the period of culture was increased. The effect of retinol on collagen synthesis could be reversed by incubation of calvaria for further periods in retinol-free medium. Incorporation of [3H]thymidine and [3H]uridine into DNA and RNA respectively was not altered by culturing calvaria with retinol for 22 h. These latter findings, and the selectivity for collagen synthesis, all suggested that the effect observed was not a cell-toxicity phenomenon. The effect of retinol on collagen synthesis by chick calvarial osteoblasts was probably direct and not mediated by osteoclasts, since a negligible number of the latter cells is present in chick calvaria. In cultures of neonatal murine calvaria, which contain many osteoclasts, retinol similarly inhibited synthesis of collagen, but not of non-collagenous protein; the concentrations of retinol necessary to produce the response were similar to those required to stimulate bone resorption in vitro.     

Histochemical aspects of the differentiation of adventitious cartilage on the membrane bones of the embryo chick

Summary The histochemistry of the adventitious cartilage of the chick has been studied and compared with both primary cartilage and the bone on which the adventitious cartilage develops. The distribution of DNA, RNA, collagen, acid mucopolysaccharide, mucoprotein, glycogen, lipid, alkaline phosphatase and inorganic phosphate has been studied. Adventitious cartilage was found to have the histochemistry of primary hypertrophic cartilage and to calcify. The appearance of lipid and alkaline phosphatase activity coincided with the onset of calcification.The proliferating osteogenic and chondrogenic cells of the chick embryo have been classified and compared histochemically. Collagen synthesis was found to be high in the osteogenic cells and acid mucopolysaccharide and mucoprotein synthesis high in the chondrogenic cells.It has been postulated that the morphogenetic switch from osteogenesis to adventitious chondrogenesis most probably involves a change in the rate of collagen and acid mucopolysaccharide synthesis by the germinal cells of the membrane bones.     

Synthesis and intracellular localization of chick acid alpha-glucosidase in chick erythrocyte-human fibroblast heterokaryons. A model system for the study of lysosomal enzyme synthesis

The synthesis and localization of chick acid alpha-glucosidase has been studied in chick erythrocyte-human fibroblast heterokaryons. Monospecific antibodies raised against purified chick liver acid alpha-glucosidase were used. It was found that the acid alpha-glucosidase in the heterokaryons is of chick origin, and is localized in the same lysosomes as the human lysosomal enzymes. It is concluded that chick erythrocyte-human fibroblast heterokaryons provide a useful model system for the study of lysosomal enzyme synthesis and routing.     

Prostaglandin production in myotube cultures. Influence on protein turnover.

The production of prostaglandins (PG) E2 and F2 alpha and their possible role in regulation of protein turnover in cultured skeletal-muscle cells were examined. Primary chick myoblasts and myotubes, and L8 myotubes, produced PGE2 and PGF2 alpha from endogenous arachidonic acid. PG production by all three cell types was increased manyfold by the addition of exogenous arachidonic acid. Arachidonate-stimulated PG production was inhibited by the addition of indomethacin (0.1 mM). When L8 and chick myotubes were treated with PGE2, PGF2 alpha, arachidonic acid (0.01 mM) or indomethacin (0.1 mM), no significant alterations in rates of protein synthesis or degradation were observed. Rates of protein synthesis and degradation in these cells were responsive to the addition of 10% fetal-bovine serum under identical experimental conditions. Thus, in contrast with incubated adult skeletal muscle, it appears that the production of prostaglandin metabolites from arachidonic acid is unrelated to regulation of protein turnover in cultured muscle cells.     

Collagen cross-linking. Effect of D-penicillamine on cross-linking in vitro.

D-Pencillamine is believed to inhibit collagen cross-link biosynthesis by forming thiazolidine rings with lysyl-derived aldehydes that are intermediates in bifunctional cross-link synthesis. Recently, we showed that aldehyde biosynthesis catalyzed by lysyl oxidase occurs after the onset of fibril formation and that nascent aldehydes form Schiff-base cross-links rapidly in fibrils. This suggested that the accessibility of D-penicillamine to most aldehydes formed during cross-link synthesis might be limited. To study this, reconstituted chick bone collagen fibrils were incubated in vitro with highly purified lysyl oxidase and D-penicillamine. As reported in previous studies in vivo, allysine content increased and polyfunctional cross-link synthesis decreased with D-penicillamine. However, the concentration of bifunctional cross-links increased rather than decreased due to a 2-fold increase in N6:6'-dehydro-5,5'-dihydroxylysinonorleucine. Hydroxyallysine, an intermediate in formation of this Schiff base, decreased. A time study indicated that allysine levels increased primarily after the bulk of Schiff base synthesis. These results indicate that D-penicillamine does not inhibit bifunctional cross-link synthesis as previously suggested. Its principal effect is to block synthesis of polyfunctional cross-link products from Schiff base cross-link precursors and to cause accumulation of these precursors. This effect may be due to interference with the close molecular packing required for polyfunctional cross-link synthesis. These results also suggest a mechanism for the relative insensitivity of tissues such as bone with high hydroxylysine content to D-penicillamine. In this study, D-penicillamine caused selective accumulation of allysyl and not hydroxyallysyl residues. In bone as opposed to soft tissues, hydroxyallysyl residues are intermediates in synthesis of almost all cross-links.     

Human skeletal growth factor stimulates collagen synthesis and inhibits proliferation in a clonal osteoblast cell line (MC3T3-E1)

Human skeletal growth factor (hSGF), an 11-kD polypeptide purified from human bone, has been proposed to be a local regulator of bone formation. To investigate the underlying cellular mechanisms in an in vitro model system, we examined the effects of hSGF on proliferation and collagen synthesis in cells of the clonal osteoblast cell line MC3T3-E1. This line was isolated from newborn mouse calvarial cells and retains many characteristics of mature osteoblasts (Sudo, H., et al., (1984) J. Cell Biol. 96:191). A 14-hr treatment with hSGF increased noncollagenous protein synthesis to 215% of unstimulated controls and increased collagen synthesis to 630% of controls as determined by [3H]proline incorporation and high-pressure liquid chromatographic separation of [3H]proline and [3H]hydroxyproline in acid hydrolysates of trichloroacetic acid-insoluble protein. HSGF did not increase cell number over a 48-hr period and caused a reversible inhibition of DNA synthesis. Half-maximal hSGF concentration for stimulation of [3H]proline incorporation and inhibition of [3H]thymidine incorporation was 100 ng/ml. HSGF also inhibited DNA synthesis in cells stimulated by serum. In contrast, hSGF stimulated both collagen synthesis and DNA synthesis in primary cultures of chick embryo bone cells, which may be developmentally less mature than MC3T3-E1 cells. The results suggest that hSGF directly stimulated mature osteoblast matrix synthetic activity and that hSGF has differential effects on proliferation of osteoblast progenitor cells and mature osteoblasts.     

Synthesis and intracellular localization of chick acid α-glucosidase in chick erythrocyte-human fibroblast heterokaryons : A model system for the study of lysosomal enzyme synthesis

The synthesis and localization of chick acid α-glucosidase has been studied in chick erythrocyte-human fibroblast heterokaryons. Monospecific antibodies raised against purified chick liver acid α-glucosidase were used. It was found that the acid α-glucosidase in the heterokaryons is of chick origin, and is localized in the same lysosomes as the human lysosomal enzymes. It is concluded that chick erythrocyte-human fibroblast heterokaryons provide a useful model system for the study of lysosomal enzyme synthesis and routing.     

The structure of anchorin CII, a collagen binding protein isolated from chondrocyte membrane

cDNA clones for anchorin CII (Mr = 34,000), a collagen-binding protein, were isolated from a lambda gt 11 cDNA library prepared from chick cartilage mRNA. Several overlapping clones were characterized which gave rise to an open reading frame coding for 329 residues and a 3'-untranslated segment of 500 base pairs. The clones were identified as coding for anchorin by hybrid select translation analysis and by comparing the deduced amino acid sequence with the sequence of 10 tryptic peptides of the protein. A hydrophobic domain of 25 residues interrupted with 3 polar residues was identified with the carboxyl-terminal portion. There was no evidence for an aminoterminal signal peptide. Northern analysis revealed that the 5' probe hybridizes to a single 1.7-kilobases (kb) mRNA species, whereas the 3' probe hybridizes to two mRNA species of 1.7 kb and 5 kb, which are present in many cells including chondrocytes, crop cells, and fibroblasts. The level of anchorin mRNA in chick embryo fibroblasts was increased by infection with Rous sarcoma virus.     

Complete amino acid sequence of 14 kDa beta-galactoside-binding lectin of chick embryo

The complete amino acid sequence of a soluble beta-galactoside-binding lectin (subunit MW 14,500) of chick embryo was determined. The protein consists of 134 amino acids beginning with serine and ending with glutamic acid, and its N-terminal was blocked with acetate. The agreement of the present result with that obtained from nucleotide sequence analysis (Y. Ohyama et al. (1986) Biochem. Biophys. Res. Commun. 134, 51-56) indicates the lack of a cleavable leader sequence. Internal homologies were observed in several regions along the polypeptide chain. The highest homology (55% identity) was found between residues 42-58 and residues 112-128. This suggests that chick 14 kDa lectin may have evolved via several gene duplications.