The gastrulating chick blastoderm contains 16-kDa and 14-kDa galactose-binding lectins possibly associated with an apolipoportein

Extracts from chick blastoderms were subjected to affinity chromatography on lactoside-Sepharose. Lactose-eluted fractions were examined by gradient SDS-PAGE with silver staining, as well as by immunoblot analysis using antibodies to the chicken galactose-binding lectins of 14 kDa and 16 kDa and to an apolipoprotein of chicken very low density lipoprotein (Apo-VLDL-II). Fractions containing the highest lectin activity contained four main bands. One, unidentified, comigrated with albumin; two bands were identified by immunoblotting as the 14-kDa and 16-kDa lectins. The fourth band comigrated with Apo-VLDL-II and in immunoblot analysis reacted with antibodies to this apolipoprotein. In our electrophoretic system this protein migrates close to bovine trypsin inhibitor and has an apparent molecular weight of 6500 ± 500. The present studies establish the identity of this previously described 6.5 kDa protein (Zalik et al. J. Cell. Sci. 88, 483, 1987) as Apo-VLDL-II. While the 16-kDa lectin was present consistently in all the affinity-purified preparations, the relative frequencies of the 14-kDa lectin and Apo-VLDL-II varied. In sections of primitive streak blastoderms, lectin immunofluorescence was present in the lowest, most ventral area of the primitive groove and in the cells emerging laterally from the groove to form the endoderm. Cells of the extraembryonic endoderm also displayed high lectin immunoreactivity. The localization of the lectins is similar to the one described previously for Apo-VLDL-II. Double immunofluorescence staining indicates that Apo-VLDL-II and the lectin(s) colocalize. The copurification and colocalization of Apo-VLDL-II and the lectins in the chick blastoderm suggest that this apolipoprotein may associate with the galactose-binding lectins or may display lectin activity.     

Expression of the 14 kDa and 16 kDa galactoside-binding lectins during differentiation of the chick yolk sac

The gastrulating chick embryo expresses two galactoside-binding lectins of 14 kDa and 16 kDa. These lectins are present in the area pellucida and area opaca, and in the latter are concentrated in the endoderm. Since the area opaca is the progenitor of the yolk sac, we studied the galactose-binding lectins during the development of this extraembryonic organ. In the yolk sac, lectin expression surges between 2 and 4 days, and thereafter remains constant throughout development. Using monoclonal antibodies (mAbs) specific to the 16 kDa yolk sac lectin, and a panel of polyclonal antibodies to the 14 kDa and 16 kDa lectins we studied lectin expression. The mAbs inhibit the hermagglutinating activity of extracts from chick yolk sac, embryonic pectoral muscle, and adult liver, but have no effect on the hemagglutinating activity of extracts from the adult intestine. Immunolocalization studies with the mAbs and polyclonal antibodies indicate that in the less differentiated endodermal cells of the area vitellina the 16 kDa lectin is present in discrete lectin-rich inclusions. In contrast, within the maturing endodermal epithelium of area vasculosa the 16 kDa lectin is present around the intracellular yolk platelets, and is associated with the cytoplasmic matrix. The 16 kDa lectin is also found at the apical cell surface of the yolk sac epithelium, in some regions closely associated with the plasma membrane. The 14 kDa lectin is distributed intracellularly surrounding the yolk platelets of the maturing yolk sac endoderm. The surge in expression of the 16 kDa lectin at the time of expansion of the area opaca suggests that it may be involved in the spreading of this area. Our findings also indicate that as the yolk sac endoderm differentiates into an epithelium intracellular lectin expression changes from predominantly organelle associated to cytoplasm associated. The association of both lectins with yolk suggest that the lectins may also be involved in the processing of intracellular and extracellular yolk proteins. These results, in con junction with previous findings indicating the presence of these lectins in the extracellular matrix (Didier et al., Histochemistry 100:485, 1993; Zalik et al., Intl J Dev Biol 38:55–68, 1994) indicate that these lectins play multiple roles in embryonic development.     

Purification and Characterization of Lectin from Lathyrus sativus

Lectin has been isolated and purified from Lathyrus sativus using ammonium sulphate precipitation followed by affinity chromatography. The molecular weight as determined by HPLC was found to be 42kD. The lectin is a tetramer, consisting of two types of subunits of which the heavier subunit consists of 2 polypeptides of mol wt of about 21 kD and 16 kD while the smaller subunits consists of two polypeptides of about 5kD as revealed by SDS-PAGE. The most potent sugar inhibitor of the Lathyrus lectin was found to be α-methyl D-mannoside. The N-terminal amino acid sequence was similar to that of pea lectin sequence.     

Molecular assembly, secretion, and matrix deposition of type VI collagen

Monoclonal antibodies reactive with the tissue form of type VI collagen were used to isolate the type VI collagen polypeptides from cultured fibroblasts and muscle cells. Two [35S]methionine-labeled polypeptides of 260 and 140 kD were found intracellularly, in the medium, and in the extracellular matrix of metabolically labeled cells. These polypeptides were disulfide cross-linked into very large complexes. The 260- and 140-kD polypeptides were intimately associated and could not be separated from each other by reduction without denaturation. In the absence of ascorbic acid, both polypeptides accumulated inside the cell, and their amounts in the medium and in the matrix were decreased. These results suggest that both the 260- and the 140-kD polypeptides are integral parts of the type VI collagen molecule. Examination of type VI collagen isolated from the intracellular pool by electron microscopy after rotary shadowing revealed structures corresponding to different stages of assembly of type VI collagen. Based on these images, a sequence for the intracellular assembly of type VI collagen could be discerned. Type VI collagen monomers are approximately 125 nm long and are composed of two globules separated by a thin strand. The monomers assemble into dimers and tetramers by lateral association. Only tetramers were present in culture media, whereas both tetramers and multimers were found in extracellular matrix extracts. The multimers appeared to have assembled from tetramers by end-to-end association into filaments that had prominent knobs and a periodicity of approximately 110 nm. These results show that, unlike other collagens, type VI collagen is assembled into tetramers before it is secreted from the cells, and they also suggest an extracellular aggregation mechanism that appears to be unique to this collagen.     

Budding-specific lectin induced in epithelial cells is an extracellular matrix component for stem cell aggregation in tunicates.

We have examined immunocytochemically the expression, localization and in vivo function of a calcium-dependent and galactose-binding 14 x 10(3) Mr lectin purified from the budding tunicate, Polyandrocarpa misakiensis. Lectin granules first appeared in the inner epithelium of a double-walled bud vesicle. Soon after the bud entered the developmental phase, the granules were secreted into the mesenchymal space, where the lectin-positive extracellular matrix (ECM) developed. The lectin was also produced and secreted by granular leucocytes during budding. Hemoblasts, pluripotent stem cells in the blood, were often found in association with the ECM and they aggregated with epithelial cells to form organ rudiments. The lectin showed a high binding affinity for hemoblast precursors. The blockage of epithelial transformation of stem cells by galactose in in vivo bioassy was ineffective in the presence of the lectin. Polyclonal anti-lectin antibody prevented the hemoblasts spreading on the ECM and moving toward the epithelium, but it did not block the cell-cell adhesion of hemoblasts. By three days of bud development, lectin granules and ECM have almost disappeared from the developing bud together with a cessation of hemoblast aggregation. These results show that Polyandrocarpa lectin is a component of the ECM induced specifically in budding and suggest strongly that it plays a role in bud morphogenesis by directing the migration of pluripotent stem cells to the epithelium.     

Developmental fate of the yolk protein lipovitellin in embryos and larvae of winter flounder, Pleuronectes americanus.

The developmental fate of the vitellogenin-derived yolk protein, lipovitellin (Lv), was investigated in winter flounder embryos and yolk-sac larvae. Since Lv is present as only one major polypeptide in ovulated winter flounder eggs, unlike the multiple yolk polypeptides found in the mature eggs of most teleosts, this system is presented as a simpler model of yolk protein structure and utilization during teleostean development. Winter flounder Lv is cleaved during embryogenesis from a 94 kD polypeptide at fertilization to 67 kD and 26 kD polypeptides at hatching. The rate of this proteolytic processing is slow during early embryonic development, but enters a more rapid phase between days 8 and 12 post-fertilization in embryos reared at 4-5 degrees C, and approaches 50% completion at day 10. Lv processing is essentially complete 3 days before hatching; nevertheless, major degradation of the Lv peptide by the developing winter flounder does not occur until after hatching. The Stokes radius of Lv changes only moderately following processing, from 4.50 nm in unfertilized eggs to 4.19 nm in late embryos and newly hatched larvae, whereas the processed Lv retains its heat stability relative to other yolk polypeptides. Nearly 50% of its lipid content, however, is released from the Lv particle during embryogenesis, concomitant with cleavage of the Lv 94 kD polypeptide. Lv processing may thus render a portion of the yolk protein-associated lipid more accessible to the developing embryo, whereas other yolk components are retained for later use by the winter flounder larva. Alternately, removal of lipid may lead to proteolytic vulnerability of the Lv polypeptide. In either case, only a portion of the lipid moiety of the Lv particle appears to play a significant nutritive role for the embryo, whereas its protein component is reserved for larval use. J. Exp. Zool. 284:686-695, 1999.     

Patterns of expression of a 15K beta-D-galactoside-specific lectin during early development of the avian embryo

We have determined, by immunohistochemical and biochemical techniques, the distribution of an endogenous beta-D-galactoside-binding lectin between the early primitive streak stage and the 5th day of embryonic development of the chick.The lectin, which was purified from the pectoral muscle of 16-day-old chick embryos, migrates on SDS-PAGE as a single polypeptide of relative molecular mass 15 x 10(3). Antibodies to this pure lectin interact with the 15K (K = 10(3) M(r)) polypeptide as well as with a 6.5K polypeptide; this second component appears to be antigenically related to the 15K lectin, as antibodies affinity purified on the 15K band recognize both polypeptides. In early stages of development, lectin immunoreactivity was present in most cells of the epiblast and hypoblast in the region of the primitive streak, while towards the edge of the area pellucida the epiblast was stained less intensely. During gastrulation, strong immunoreactivity was present also in migrating cells and in the mesoblast, while at the margin of the area pellucida the epiblast was negative. Up to the 10-somite stage, lectin immunoreactivity was present in the somites, neural tube and presumptive cardiac region; the non-neural ectoderm and the extracellular matrix were not labeled; the predominant immunoreactive component at this stage of development was the 6.5K polypeptide. Later in development, the lectin immunoreactivity gradually disappeared from the dermamyotome and nervous system to reappear conspicuously as soon as a differentiated myotome could be detected. Immunoreactivity was very high in the myotome, skeletal and cardiac muscles and transient in smooth muscles. The only region of the nervous system that continued to express the lectin throughout development was the trigeminal (semilunar) ganglion; in all regions of the nervous system, the lectin immunoreactivity disappeared early in development to be re-expressed only much later. The lining epithelium of the digestive tract and other endodermal derivatives expressed the lectin transiently. In the extraembryonic membranes, immunoreactivity to the lectin was observed in the yolk sac and in both layers of the amnion. The striking regulation of the expression of this endogenous lectin suggests that its functions are linked to cell proliferation and/or to the selective expression of a developmentally-timed cell phenotype.     

Modulation of growth of Aplysia neurons by an endogenous lectin.

We have purified and characterized a galactose-binding lectin from the gonads of the mollusk Aplysia californica that modulates neurite outgrowth from cultured Aplysia neurons. Agglutination of sheep red blood cells (RBC) by this lectin, termed Aplysia gonad lectin (AGL), is inhibited strongly by galactose and to a lesser extent by fucose. On SDS-PAGE, AGL appears as a single species with a molecular weight of 34 kD under reducing conditions, and 65 kD under nonreducing conditions. This suggests that AGL is a disulfide-linked dimer in its native state. Amino terminal sequence analysis of purified AGL indicates a similarity to another galactose-binding lectin, phytohemagglutinin-E (E-PHA), found in red kidney beans. By using polyclonal antibodies prepared against AGL, we have found that the lectin is present in the gonads and eggs but not in other tissues of adult Aplysia californica. We have examined biological actions of AGL on Aplysia neurons growing in primary cell culture. AGL affects several properties of these neurons. The addition of 100 nM AGL to cultured neurons enhances neurite outgrowth from the cell soma, resulting in a greater number of primary processes. In addition, AGL acts as a neurotrophic agent, increasing neurite viability in vitro. This trophic effect is not seen with concanavalin A (con A), another lectin known to affect several properties of cultured Aplysia neurons. The results are consistent with the suggestion that AGL may play a role in neuronal differentiation and/or maintenance of viability.     

The binding of very low density and low density lipoproteins to the plasma membrane of the hen's oocyte : A morphological study

The binding of lipoproteins to the oocyte plasma membrane of the domestic fowl (Gallus domesticus) was examined by electron microscopy in preparations of the ovarian follicle in the main phase of yolk formation. Numerous particles, 26 nm in diameter, were present on the untreated membrane. They were dissociated from the membrane by incubation at 4 °C in buffer at pH 6.2 and with heparin at pH 7.4. Added calcium was not required for binding, though the number of bound particles was reduced by treatment with EDTA. Very low density (VLD) lipoproteins from laying hen's plasma were found to bind to the denuded membrane and to correspond in size to the native particles. The results suggest that the binding characteristics are similar in quality to those determined for the binding of low density (LD) lipoproteins to mammalian cells. The oocytes, however, bound 100-fold more particles per unit length of membrane. VLD and LD lipoproteins from immature hens also adhered to the denuded membrane, although their apoprotein composition was very different from that of laying hen VLD lipoproteins. LD lipoproteins from immature hens and VLD lipoproteins from laying hens both contained apo-B, which formed about 80 and 35%, respectively, of the total apolipoproteins. Apo-VLDL-II is the other major apoprotein identified in laying-hen VLD lipoproteins. Apo-VLDL-II was not positively identified as a component of immature-hen LD lipoproteins and could only have been present as a minor component. Despite their great difference in apo-VLDL-II content, immature-hen LD lipoproteins and laying-hen VLD lipoproteins showed similar dissociation constants for binding to the oocyte plasma membrane. This evidence strongly suggests that the cell surface receptors recognize the B apoprotein of avian VLD lipoproteins.     

Separation of bovine heart galactose lectin from endogenous glycoproteins co-purified with the lectin during affinity chromatography

During affinity chromatographic purification of bovine heart 14 kDa galactose-binding lectin (galectin 1) on lactose-Sepharose, several high molecular weight non-lectin glycoproteins were co-purified with the lectin. Glycoprotein binding to the affinity matrix was neither hydrophobic nor ionic, but galactose-dependent since lactose abolished binding. Purification of galectin from the co-purified glycoproteins by affinity electrophoresis in presence of the specific sugar lactose increased agglutination activity about 65-fold, indicating that a complex containing galectin molecules bound sugar specifically to endogenous glycoproteins with sugar binding sites still available had been retained on lactose-Sepharose.     

Storage, ultrastructural targeting and function of toposomes and hyalin in sea urchin embryogenesis.

This study compares by immunogold labeling the ultrastructural localization of a hexameric 22S glycoprotein, called toposome, with that of hyalin in unfertilized eggs and cells of hatched sea urchin blastulae. Nearly all hyalin is present in the electron translucent compartment of the cortical granules and in the translucent non-cortical pigment granules. In the blastula both of these intracellular stores have vanished and hyalin now forms a broad band below the apical lamina. By contrast, in the egg toposomes are present on the surface, as well as stored in yolk granules and in the electron dense lamellar compartment of the cortical granules. In the hatched blastula, toposomes that have been modified by limited proteolysis in the yolk granules, are associated with the plasma membranes of all newly formed cells, while the toposomes originating from the cortical granules have been incorporated as unmodified 160 kDa polypeptides into an extracellular double layer enveloping the embryo on the outside of the hyaline layer. From evidence discussed in detail, we conclude that the extracellular toposomes rivet the apical lamina to the surface and underlying cytoskeleton of the microvilli, while the modified toposomes from the yolk granules are responsible for position specific intercellular adhesion as they are released to the surface of newly formed cells. We propose that all the material stored in yolk granules is utilized for the assembly of new membranes.     

Modulation of growth of Aplysia neurons by an endogenous lectin

We have purified and characterized a galactose-binding lectin from the gonads of the mollusk Aplysia californica that modulates neurite outgrowth from cultured Aplysia neurons. Agglutination of sheep red blood cells (RBC) by this lectin, termed Aplysia gonad lectin (AGL), is inhibited strongly by galactose and to a lesser extent by fucose. On SDS-PAGE, AGL appears as a single species with a molecular weight of 34 kD under reducing conditions, and 65 kD under nonreducing conditions. This suggests that AGL is a disulfide-linked dimer in its native state. Amino terminal sequence analysis of purified AGL indicates a similarity to another galactose-binding lectin, phytohemagglutinin-E (E-PHA), found in red kidney beans. By using polyclonal antibodies prepared against AGL, we have found that the lectin is present in the gonads and eggs but not in other tissues of adult Aplysia californica. We have examined biological actions of AGL on Aplysia neurons growing in primary cell culture. AGL affects several properties of these neurons. The addition of 100 nM AGL to cultured neurons enhances neurite outgrowth from the cell soma, resulting in a greater number of primary processes. In addition, AGL acts as a neurotrophic agent, increasing neurite viability in vitro. This trophic effect is not seen with concanavalin A (con A), another lectin known to affect several properties of cultured Aplysia neurons. The results are consistent with the suggestion that AGL may play a role in neuronal differentiation and/or maintenance of viability. © 1992 John Wiley & Sons, Inc.     

Distribution of yolk polypeptides in the follicle cells during the differentiation of the follicular epithelium in Sarcophaga bullata egg follicles

In S. bullata, the ovaries contribute to the synthesis of yolk polypeptides. A specific antiserum for yolk polypeptides was used to visualize the presence of yolk polypeptides in the follicle cells during their differentiation. After vitellogenesis has started, all follicle cells contain yolk polypeptides. The squamous follicle cells covering the nurse cells and the border cells lose yolk polypeptides before mid-vitellogenesis, whereas the follicle cells over the oocyte contain yolk polypeptides until after late vitellogenesis. All follicle cells are immunonegative afterwards. In vitro translation of poly(A)+ RNA demonstrated that the presence of yolk polypeptide mRNA correlates well with follicle cell immunopositivity for yolk polypeptides. This suggests that the follicle cells synthesize the ovarian yolk polypeptides. Differences in cellular and nuclear morphology, total and poly(A)+ RNA synthesis and the rate of yolk polypeptide synthesis were shown to be correlated with the presence or absence of yolk polypeptides in the differentiating follicular epithelium. The possible relationship between these different aspects of follicle cell differentiation, follicle cell polyploidy and the extracellular current pattern around follicles are discussed.     

Expression of genes for two C-type lectins during budding of the ascidian Polyandrocarpa misakiensis

Two closely related cDNA fragments, named pTC14-1 and pTC14-2, encoding C-type lectins were cloned from the budding ascidian Polyandrocarpa misakiensis by means of the polymerase chain reaction. The amino acid sequence deduced from pTC14-1 was identical to that of a 14-kDa calcium-dependent galactose-binding lectin, TC-14, that had been purified from this species. Between the two clones, nucleotide sequence similarity was 90%, whilst that of the deduced amino acid sequences was 82%. The cDNA inserts of these clones hybridized weakly with each other. Antisense RNA probes prepared from these clones gave intense hybridization signals on Northern blots of the W strain, but very weak signals on those of the other strains. Therefore, both clones were suggested to originate from the W strain, but from two separate genes, since the base substitution was scattered throughout the entire translated region. The amount of TC14-1 mRNA increased during bud development, and peaked at 36 h after separation of the bud from the parental body wall. At this stage, extracellular matrix containing TC-14 lectin developed in the mesenchymal space around the morphogenetic region of the bud. There was much less TC14-2, than TC14-1 mRNA at every stage of bud development. TC14-1 and TC14-2 mRNAs were detected on Northern blots of RNAs from adults and growing buds, suggesting that these genes can be used as the earliest markers of budding in this species.     

Nucleoside Diphosphate Kinase Is a Possible Component of the Ethylene Signal Transduction Pathway

In etiolated seedlings of Pisum sativum and leaves of Arabidopsis thaliana, in vivo ethylene treatment resulted in an increase in in vitro phosphorylation of 17 kD (P. sativum) or 16 and 17 kD (A. thaliana) polypeptides. These polypeptides were identified as nucleoside diphosphate kinase (NDPK) based on both biochemical properties and interaction with antibodies against NDPK from P. sativum. Using the receptor-directed antagonist of ethylene action 2,5-norbornadiene and the ethylene-insensitive mutants of A. thaliana etr1-1 and eti5, ethylene specificity and receptor dependence of NDPK phosphorylation have been demonstrated. In pea epicotyls, ethylene treatment also led to increase in nucleoside transferase activity unlike in A. thaliana leaves. The increases in nucleoside transferase activity and NDPK phosphorylation were very rapid and transient. The results suggest a role for NDPK as a possible component of the ethylene signal transduction chain.     

Selachian tooth development: II. Immunolocalization of amelogenin polypeptides in epithelium during secretory amelogenesis in Squalus acanthias

We have determined the distribution of amelogenin polypeptides in an order of elasmobranchs using indirect immunofluorescence with rabbit polyclonal antibodies prepared to purified murine amelogenins. We find that amelogenins are definitely present within the inner enamel epithelium prior to the production of the extracellular matrix component termed "enameloid" (row II developing tooth organs). During subsequent stages of selachian tooth development (row III tooth organs), immunofluorescence staining data indicated localization of amelogenin antigens within epithelium as well as the enameloid extracellular matrix. The results from these immunohistochemical studies suggest that the 16-20 kdalton amelogenins, which are characteristic of murine inner enamel epithelial cells undergoing terminal biochemical differentiation into secretory ameloblasts, may also be regarded as molecular markers for amelogenesis in developing teeth in the spiny dogfish, Squalus acanthias.     

Immunochemical identification of the growth hormone and prolactin in the hypophyseal polypeptide spectrum of chickens

Forty-eight fractions of polypeptides including 39 fractions with a molecular weight of 14-95 kD were identified in chick adenohypophysis by sodium dodecyl sulphate electrophoresis in 10-20% gradient polyacrylamide gel slabs. The immunochemical identification of the polypeptides was performed with the aid of the electroblotting of proteins and antisera to human STH, to bovine prolactin, and to the tissue-specific antigen A-1 of chick adenohypophysis. Antisera to human STH and to antigen A-1 reacted with the same major polypeptide fraction, m.w. 26 kD, characteristic of the caudal lobe of the adenohypophysis. Immunoreactive prolactin was present in chick adenohypophysis in the form of a polypeptide fraction with a molecular weight of 25 kD and in the form of two minor fractions of polypeptides with molecular weights of 27 and 28 kD. The data obtained indicate the identity of the adenohypophyseal tissue-specific antigen A-1 to chick STH.     

Mutations in genes downstream of the rpoN gene (encoding σ54) of Klebsiella pneumoniae affect expression from σ54-dependent promoters

Two open reading frames (ORFs), designated ORF95 and ORF162, downstream of the Klebsiella pneumoniae sigma 54 structural gene (rpoN) have been sequenced and shown to encode polypeptides of 12 kD and 16 kD, respectively. ORFs homologous to ORF95 are present downstream of four out of five rpoN genes sequenced to date from a range of Gram-negative bacteria, and ORF162 is also conserved, at least in Pseudomonas putida. Chromosomal mutations have been created in each gene using a kan cassette and both have the same phenotype, i.e. they cause an increase in the level of expression from sigma 54-dependent promoters. We propose that the products of both genes function to modulate the activity of E sigma 54, although a physiological role for these proteins has not yet been identified.     

Differences in expression of a variant actin between low and high metastatic B16 melanoma

The polypeptides of mouse B16 melanoma lines of defined metastatic potential have been analyzed by two-dimensional electrophoresis. Parent B16 melanoma and two independently isolated B16-F1 lines, which are low metastatic, exhibited a new polypeptide, Ax (pI 5.2; Mr = 43,000), comprising approximately 30% of the total actin, in addition to normal beta- and gamma-actin. The Ax is present in the Triton-insoluble fraction (cytoskeleton and nuclear matrix) as well as in the Triton-soluble fraction at a constant ratio of about 0.5 to beta- plus gamma-actin. The Ax polypeptide has been identified as a variant form of actin by immunostaining with anti-actin antibody and by a comparison of its tryptic patterns with those produced by beta- and gamma-actin polypeptides; the Ax is also identified as a component of microfilaments. On the other hand, the Ax polypeptide disappears or its expression is very low in high metastatic lines, two independently isolated B16-F10s and B16-BL6. By in vitro translation, we have identified the mRNA species that code for Ax in B16-F1, but not in B16-F10.