The Hodgkin-associated Ki-1 antigen exists in an intracellular and a membrane-bound form

The Hodgkin-associated Ki-1 antigen occurs in two different molecular forms. The 120-kDa membrane-associated form is a phosphorylated glycoprotein, which is derived from a non-phosphorylated intracellular 84-kDa apoprotein that is co-translationally N-glycosylated with a carbohydrate portion of 6 kDa. The other form of the Ki-1 antigen is a non-glycosylated phosphoprotein of 57 kDa which only occurs intracellularly. Both forms of the antigen are phosphorylated at serine residues. Enzymatic cleavage with sialidase reduced the 120-kDa membrane antigen by about 15 kDa, while its 90-kDa precursor and the 57-kDa intracellular form of the Ki-1 antigen remained unaltered. Pulse-chase experiments revealed that the 57-kDa and 90/120-kDa molecules are synthesized independently of each other. Four to eight hours after synthesis, the degradation of the 120-kDa molecule to a 105-kDa membrane-associated intermediate begins. This is further processed and appears in the cell supernate as a 90-kDa molecule. Hodgkin's disease-derived, Epstein-Barr virus-transformed cell lines and the acute T cell leukemia line MOLT-4 contain both forms of the Ki-1 antigen, whereas only the 57-kDa intracellular antigen is expressed in U266/B1 myeloma cells, in the Burkitt lymphoma cell lines Raji and Daudi and in acute promyelocytic HL-60 leukemia cells.     

Oral/aboral ectoderm differentiation of the sea urchin embryo depends on a planar or secretory signal from the vegetal hemisphere

A monoclonal antibody that recognizes oral ectoderm and esophagus of sea urchin larvae was newly produced. Distribution of the antigen, named Hpoe, was examined by indirect immunofluorescence microscopy. Hpoe did not exist in eggs and appeared during the cleavage stage. In hatched blastulae, Hpoe was detected on the apical surface of all cells. As embryogenesis progressed, Hpoe disappeared from the primary mesenchyme, archenteron and aboral ectoderm. Hpoe reappeared in foregut at the prism stage and was restricted to the oral ectoderm and esophagus at the pluteus stage. Using this antigen as a molecular marker of oral/aboral ectoderm differentiation, the role of the vegetal hemisphere in ectoderm differentiation was examined. All animal hemispheres isolated from 16-cell stage embryos, mesenchyme blastulae, early gastrulae and mid gastrulae developed into epithelial balls and every cell expressed Hpoe. These epithelial balls failed in oral/aboral ectoderm differentiation. Twenty millimolar LiCI-treated whole embryos developed into exo-gastrulae but Hpoe restriction in ectoderm occurred in these exo-gastrulae. These results show that oral/aboral ectoderm differentiation requires an inductive interaction from the vegetal hemisphere and indicate that the inductive interaction depends on a planar or secretory signal, rather than the contact of the esophagus and ectoderm.     

Morphological changes in the oral (buccopharyngeal) membrane in urodelan embryos: development of the mouth opening

The ultrastructure of the oral (buccopharyngeal) membrane in the embryo of the urodelan, Hynobius tokyoensis, was examined by transmission (TEM) and scanning electron microscopy (SEM). The oral membrane consists of the stomodeal ectoderm and foregut endoderm, and is three to five cell layers thick at stage 24. The oral membrane gradually thickens as development proceeds. The stomodeal collar, derived from the ectoderm, is folded inward along the foregut endoderm. Tooth germs are formed partly by cells of the stomodeal collar and partly by mesenchymal cells and calcification takes place before hatching. Secretory granules, which are markers of epithelial differentiation, appear in some cells of the foregut endoderm. Within the oral membrane, the cells of the stomodeal collar become the basal cells, and the endodermal cells of the foregut become the apical cells of the future oral epithelium. Gaps are formed by the epithelial differentiation of the endodermal cells of the foregut in the oral membrane. The gaps connect with each other, with the stomodeum, and with the foregut. As a result of these events, the mouth opens at stage 43, just after hatching.     

Biochemical analysis of a human epithelial surface antigen: differential cell expression and processing

Epithelial surface antigen (ESA) is a glycoprotein with a distribution in vivo that is largely confined to human epithelial cells. Previous studies using a mouse monoclonal antibody (MH99) detecting ESA had shown that the antigen immunoprecipitated from most epithelial cancer cell lines has two chains (38,000 and 32,000 Da) when separated under reducing conditions and only one (38,000 Da) under nonreducing conditions. We now show that the 38-kDa band observed under nonreducing conditions consists of two species, one a 38-kDa single chain protein and the other a disulfide-linked dimer consisting of the 32-kDa chain bonded to a previously unrecognized 6-kDa chain. Pulse-chase studies have shown that ESA is synthesized as a 34-kDa protein which is glycosylated to a 38-kDa glycoprotein containing both high mannose and complex carbohydrate chains. With longer chase periods, a 32-kDa species also appears. Peptide mapping, together with the pulse-chase data, suggests that the 32- and 6-kDa species are formed from the 38-kDa protein, probably by limited proteolysis. Epithelial cell lines differ in their ratios of 38/32-kDa species, some cell lines having only the 38-kDa form. Incubation of radiolabeled extracts of cells having only the 38-kDa protein with unlabeled extracts of the other cell types resulted in progressive conversion of the 38-kDa species to the 32- and 6-kDa forms. Only cell lines expressing both forms of ESA are able to carry out this cleavage of the 38-kDa protein. This is a novel mechanism for generating cell-type related differences in cell surface glycoprotein expression. Finally, sequential immunoprecipitation experiments showed that the antigen detected by Ab MH99 is closely related or identical to that detected by Ab 17-1A, a previously described colon cancer antigen.     

Purification and characterization of a 32-kDa protein that localizes to the sea urchin extraembryonic matrix, the hyaline layer.

We have purified a 32 kilodalton (kDa) protein that localized with isolated, intact hyaline layers prepared from 1-h-old embryos. The protein appeared not to bind calcium and was not quantitatively released from 1-h-old embryos in the absence of Ca2+ and Mg2+. Using polyclonal antiserum prepared against the 32-kDa protein, the antigen was detected throughout embryonic development. By the hatched blastula stage of development, the 32-kDa protein was replaced by a species of slightly smaller molecular mass. Quantitative determination indicated that the 32-kDa protein accounted for approximately 6% of the total protein present in the sea urchin egg. This result is suggestive of a structural role for the 32-kDa protein that is required throughout embryonic development, although perhaps in a modified form from the hatched blastula stage on.     

Molecular cloning and characterization of chick sialoprotein associated with cones and rods, a developmentally regulated glycoprotein of interphotoreceptor matrix

MY-174 is an IgM class monoclonal antibody originally established against chick PG-M/versican. The antibody specifically stains the photoreceptor layer, where we recently reported an absence of PG-M/versican. In this study, we re-characterized the antibody and identified the molecule that reacts to MY-174 at the photoreceptor layer. Immunohistochemistry localized the antigen to the matrix surrounding photoreceptors. A variety of glycosidase digestions showed that the antigen is the 150-kDa glycoprotein that has sialylated N- and O-linked glycoconjugates having a molecular mass of more than 30-kDa. The peptide sequences obtained from purified MY-174 antigen showed we had sequenced a full-length cDNA with an open reading frame of 2787 base pairs, encoding a polypeptide of 928 amino acids, with 56 and 54% identities to human and mouse sialoprotein associated with cones and rods (SPACRs), respectively, and with the structural features observed in SPACRs. The specific sialylated O-glycoconjugates here are involved in the epitope structure for MY-174. SPACR first appeared by embryonic days 15-16, and expression increased with developmental age, paralleling the adhesion between neural retina and retinal pigment epithelium. Thus, we concluded that the MY-174 antigen at the photoreceptor layer, a developmentally regulated glycoprotein, is identical to chick SPACR and may be involved in a novel system mediating adhesion between neural retina and retinal pigment epithelium.     

Alpha-enolase is restricted to basal cells of stratified squamous epithelium.

We have developed a monoclonal antibody against a 50-kDa protein that binds preferentially to basal cells in the limbus of rat, rabbit, and human corneas (J. D. Zieske, G. Bukusoglu, and M. A. Yankauckas, Invest. Ophthalmol. Visual Sci. 33, 143-152, 1992). Here we report on the purification and identification of the antigen. The 50-kDa antigen was purified from rabbit limbal and corneal epithelium using HPLC methodology including anion exchange (DEAE) followed by reverse-phase (C18) chromatography. The purified 50-kDa protein was then digested with endoproteinase Lys-C, and a reproducible profile comprising approximately 20 peptides was observed by reverse-phase HPLC of the digest. Sequence analysis of five peptides ranging in length from 4 to 20 residues revealed that the 50-kDa protein was alpha-enolase, a glycolytic enzyme. Overall, 57 amino acids were identified with a 95% sequence homology. Localization of alpha-enolase in rat epithelium by immunofluorescence microscopy demonstrated that simple epithelium contained low or undetectable levels of the enzyme. Stratified squamous epithelium, however, showed high levels of alpha-enolase, which was localized specifically to cells of the basal layer. Epidermal, corneal limbal, oral mucosal, vaginal, and laryngeal epithelium all showed cytoplasmic binding specific to the basal cells. These data indicate that the glycolytic enzyme alpha-enolase is preferentially localized in the basal cell layer of stratified squamous epithelium and suggest that glycolytic activity is concentrated in these cells. The localization pattern suggests that a major change in metabolism occurs as cells leave the mitotically active basal cell layer and migrate toward terminal differentiation in the suprabasal cell layers.     

Immunocytochemical analysis of embryonic compartmentation with a monoclonal antibody against a cytokeratin-related antigen

Summary Mab 113F4, a monoclonal antibody recognizing an antigen in the outer synaptic layer of the chick neural retina, also recognizes an antigen appearing in all three germ layers of the gastrulating chick embryo. However, as neurulation proceeds, the antigen is down-regulated in three distinct patterns. First, the antigen is lost specifically from those trunk ectodermal cells destined to form the neural plate and, later, the neural tube. It remains absent from any neural derivative until day 13 when it appears in the outer synaptic layer of the neural retina, coincident with synaptogenesis in this region. Second, the entirety of the head ectoderm loses this antigen as the head lifts off the blastoderm. This down-regulation is followed later by a similar loss of antigen expression in the trunk ectoderm. Third, expression in the mesoderm becomes limited to the lateral plate and extraembryonic epithelia. Endodermal derivatives continue to express the antigen throughout development. Antigen 113F4 is localized within the cytoplasm and is organized in a fibrillar pattern. The intracellular localization of this antigen and its characteristic spatio-temporal tissue distribution are consistent with the antigen being a cytokeratin or cytokeratin-related antigen. The changes in tissue distribution suggest a possible role in tissue modelling in response to inductive interactions during development.     

Immunocytochemical analysis of embryonic compartmentation with a monoclonal antibody against a cytokeratin-related antigen.

Mab 113F4, a monoclonal antibody recognizing an antigen in the outer synaptic layer of the chick neural retina, also recognizes an antigen appearing in all three germ layers of the gastrulating chick embryo. However, as neurulation proceeds, the antigen is down-regulated in three distinct patterns. First, the antigen is lost specifically from those trunk ectodermal cells destined to form the neural plate and, later, the neural tube. It remains absent from any neural derivative until day 13 when it appears in the outer synaptic layer of the neural retina, coincident with synaptogenesis in this region. Second, the entirety of the head ectoderm loses this antigen as the head lifts off the blastoderm. This down-regulation is followed later by a similar loss of antigen expression in the trunk ectoderm. Third, expression in the mesoderm becomes limited to the lateral plate and extraembryonic epithelia. Endodermal derivatives continue to express the antigen throughout development. Antigen 113F4 is localized within the cytoplasm and is organized in a fibrillar pattern. The intracellular localization of this antigen and its characteristic spatio-temporal tissue distribution are consistent with the antigen being a cytokeratin or cytokeratin-related antigen. The changes in tissue distribution suggest a possible role in tissue modelling in response to inductive interactions during development.     

An RGDS peptide-binding receptor, FR-1R, localizes to the basal side of the ectoderm and to primary mesenchyme cells in sand dollar embryos

Immunoblotting using polyclonal antibodies (pAb) raised against an FR-1 receptor (FR-1R), a 57 kDa Arg-Gly-Asp-Ser (RGDS)-binding protein, of the sand dollar Clypeaster japonicus showed that the pAb monospecifically bound to the protein. FR-1R was present in purified plasma membrane, suggesting that the protein is a membrane-bound protein. The molecular structure of FR-1R did not change throughout the early embryogenesis, whereas its expression changed significantly during this period. FR-1R was present in the cortex of unfertilized eggs and was then transferred to the hyaline layer soon after the fertilization. The hyaline layer retained FR-1R immunoreactivity during early embryogenesis. FR-1R appeared on the basal side of the ectoderm at the morula stage and was retained basolaterally, at least, to the early gastrula stage. In mesenchyme blastulae, FR-1R was also present on the surface of primary mesenchyme cells (PMC). FR-1R was localized on the basal side of the ectoderm in early gastrulae, exclusively at the place where PMC formed ventrolateral aggregates, and at the apical tuft ectoderm. In vitro, PMC bound to FR-1R and its binding was inhibited in the presence of a synthetic RGDS peptide or the pAb. The pAb introduced into the blastocoele perturbed PMC migration and gastrulation. FR-1R was weakly recognized by antihuman integrin beta5 subunit pAb.     

Storage and mobilization of extracellular matrix proteins during sea urchin development

After fertilization, sea urchin embryos surround themselves with an extracellular matrix, or hyaline layer, to which cells adhere during early development. Hyalin, the major protein component of the hyaline layer has been isolated and partially characterized in several laboratories. Although other proteins are present in the hyaline layer, little is known about their origin, distribution, or functions. The present report characterizes a set of hyaline layer proteins that are secreted after fertilization from a class of vesicles that are distinct from cortical granules. The group of proteins in these vesicles were identified by a monoclonal antibody (8d11) which recognizes a carbohydrate epitope common to each of these molecules. 8d11 polypeptides range in molecular weight from 105 to 225 kDa. Oogonia and oocytes in early stages of vitellogenesis do not express the antigen. The proteins are first observed by immunofluorescence during oogenesis as a peripheral band in mid-vitellogenic oocytes. Following germinal vesicle breakdown 8d11 moves to be distributed evenly throughout the cytoplasm. The proteins are transported to the egg surface by a cytochalasin-sensitive mechanism after fertilization, and secreted predominately within the first 30 min of development. 8d11 proteins are depleted in areas of cell contact during early embryogenesis, and become concentrated on the apical surface of ectoderm cells where they are assembled into high-molecular-weight aggregates. Three of the molecules in this group may be proteins previously described as "apical lamina" proteins. These observations provide evidence of a third pathway (cortical granules and basal lamina granules being the other two) for synthesis, storage, and exocytosis of matrix proteins that are release after fertilization.     

Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus

The major envelope glycoprotein (gp350) of Epstein-Barr virus has been expressed and secreted in the yeast Saccharomyces cerevisiae as a 400-kDa glycoprotein. This is the first example of the secretion of such a large, heavily glycosylated heterologous protein in yeast. Since gp350 proved highly toxic to S. cerevisiae, initial cellular growth required repression of the expression of gp350. Using temperature- or galactose-inducible promoters, cells could be grown and the expression of gp350 then induced. After induction, the glycoprotein accumulated both intracellularly as well as in the culture medium. Only the most heavily glycosylated form was secreted, suggesting a role for N-linked glycans in directing secretion. The extent of O-linked glycosylation of the yeast-derived protein was similar to that of the mature viral gp350. N-linked glycosylation varied slightly depending upon culture conditions and host strain used and was more extensive than that associated with the mature viral gp350. Although there is no evidence that more than a single mRNA for the glycoprotein was expressed from the recombinant plasmid, variously sized glycoproteins accumulated in yeast at early stages after induction, probably reflecting intermediates in glycosylation. The yeast-derived glycoproteins reacted with animal and human polyclonal antibodies to gp350 as well as with a neutralizing murine monoclonal antibody to gp350, suggesting that this glycoprotein retains several epitopes of the native glycoprotein.     

Formation of the approximately 350-kDa Apg12-Apg5.Apg16 multimeric complex, mediated by Apg16 oligomerization, is essential for autophagy in yeast

Autophagy, responsible for the delivery of cytoplasmic components to the lysosome/vacuole for degradation, is the major degradative pathway in eukaryotic cells. This process requires a ubiquitin-like protein conjugation system, in which Apg12 is covalently bound to Apg5. In the yeast Saccharomyces cerevisiae, the Apg12-Apg5 conjugate further interacts with a small coiled-coil protein, Apg16. The Apg12-Apg5 and Apg16 are localized in the cytosol and pre-autophagosomal structures and play an essential role in autophagosome formation. Here we show that the Apg12-Apg5 conjugate and Apg16 form a approximately 350-kDa complex in the cytosol. Because Apg16 was suggested to form a homo-oligomer, we generated an in vivo system that allowed us to control the oligomerization state of Apg16. With this system, we demonstrated that formation of the approximately 350-kDa complex and autophagic activity depended on the oligomerization state of Apg16. These results suggest that the Apg12-Apg5 conjugate and Apg16 form a multimeric complex mediated by the Apg16 homo-oligomer, and formation of the approximately 350-kDa complex is required for autophagy in yeast.     

Localization of a 24-kilodalton glycoprotein in adult Schistosoma mansoni using immunofluorescence and immunoelectron microscopy

Studies on schistosome protective immune responses have focused mainly on antigens of the parasite's syncytial surface. One of the characterized schistosome antigens, a 24-kDa glycoprotein, has been considered important in mechanisms of immune evasion by the parasites. In the present study, using affinity-purified antibodies to the 24-kDa protein for immunofluorescence and immunoelectron microscopy, we demonstrated an association of the 24-kDa antigen with the discoid bodies (the major syncytial inclusion bodies; DBs) and the surface membrane complex (most likely the apical plasma membrane) of adult Schistosoma mansoni. This is consistent with previous observations that the 24-kDa antigen appeared to be localized to the syncytial membrane and DB fractions. The present results also support the suggestion that the DBs are the precursor organelles of the apical plasma membrane.