Mouse Lefty2 and zebrafish antivin are feedback inhibitors of nodal signaling during vertebrate gastrulation.

Mammalian lefty and zebrafish antivin form a subgroup of the TGF beta superfamily. We report that mouse mutants for lefty2 have an expanded primitive streak and form excess mesoderm, a phenotype opposite to that of mutants for the TGF beta gene nodal. Analogously, overexpression of Antivin or Lefty2 in zebrafish embryos blocks head and trunk mesoderm formation, a phenotype identical to that of mutants caused by loss of Nodal signaling. The lefty2 mutant phenotype is partially suppressed by heterozygosity for nodal. Similarly, the effects of Antivin and Lefty2 can be suppressed by overexpression of the nodal-related genes cyclops and squint or the extracellular domain of ActRIIB. Expression of antivin is dependent on Nodal signaling, revealing a feedback loop wherein Nodal signals induce their antagonists Lefty2 and Antivin to restrict Nodal signaling during gastrulation.     

Biosynthesis of prostatic acid phosphatase in a normal human cell-line

The biosynthesis of the prostatic form of human acid phosphatase was studied in normal embryonic lung cells, WI-38, by metabolic labeling with tritiated leucine and [32P]phosphate, followed by specific immunoprecipitation, gel electrophoresis, and fluorography. Of the total tartrate-inhibitable acid phosphatase activity in WI-38 cells, 30% is due to the prostatic form. The primary translation product that leads eventually to the mature prostatic enzyme is a precursor polypeptide of 112 kDa. The precursor polypeptide is processed to mature polypeptides of 59, 55, and 49 kDa via an intermediate 91-kDa precursor. WI-38 cells also secrete a 113-kDa peptide into the medium. The precursor and mature polypeptides are glycosylated and phosphorylated. Upon treatment with endo-beta-hexosaminidase H, the apparent molecular weighs of the polypeptides are reduced by approximately 4 kDa and phosphate is lost.     

Transforming growth factor beta 1: importance of glycosylation and acidic proteases for processing and secretion

The role of glycosylation of the transforming growth factor-beta 1 (TGF-beta 1) precursor was investigated by treating a transfected Chinese hamster ovary (CHO) cell line expressing high levels of recombinant TGF-beta 1 (TGF-beta 3-2000 cells) with a series of glycosylational inhibitors. Tunicamycin, a nucleoside antibiotic which prevents the formation of the dolichol intermediate necessary for oligosaccharide addition of the nascent polypeptide chain, appeared to block secretory exit and led to an increase in the cellular associated, nonglycosylated pro-TGF-beta 1 form. 1-Deoxymannojirimycin and swainsonine, inhibitors of the mannosidases I and II, respectively, blocked complete glycoprotein processing of the TGF-beta 1 precursor as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by sensitivity to glycosidases. However, the abnormal TGF-beta 1 polypeptides containing the altered carbohydrate side chains were secreted readily by the CHO cells. In contrast, inhibitors of the glucosidases at the first step in glycoprotein remodeling, 1-deoxynojirimycin and castanospermine, markedly inhibited secretion of the TGF-beta 1 polypeptides from transfected CHO cells. In all cases, these inhibitors did not appear to affect proteolytic processing of the TGF-beta 1 polypeptides. Furthermore, inhibitor treatment did not affect mannose-6-phosphorylation of the TGF-beta 1 polypeptides. These results suggest that glycosylation and early stage remodeling of oligosaccharide side chains are necessary for secretion of TGF-beta 1. Treatment of the transfected CHO cells with weak bases (NH4Cl and chloroquine), or a monovalent ionophore (monensin), prevented proteolytic processing of the TGF-beta 1 precursor indicating that cleavage occurs by proteases in an acidic cellular compartment.     

Biosynthesis and secretion of an osteopontin-related 20-kDa polypeptide in the Madin-Darby canine kidney cell line

We describe a 20-kDa phosphorylated polypeptide, which is secreted constitutively at the apical surface of the kidney-derived Madin-Darby canine kidney cell line. Using polyclonal antibodies raised against this protein, we show that it is generated from a 60-kDa O-glycosylated, sulfated, and phosphorylated precursor protein by an intracellular proteolytic maturation step, which is pH-sensitive. Amino acid sequence analysis of the 20-kDa secreted polypeptide demonstrated that it displays 70% identity with the carboxyl-terminal amino acids of human osteopontin. The amino-terminal amino acid of the 20-kDa polypeptide corresponds to amino acid 213 of human osteopontin. Thrombin has been shown to cleave rat osteopontin in vivo and in vitro at amino acid 153, yielding two fragments of 28 and 26 kDa. A similar cleavage product can be detected by thrombin treatment of the 60-kDa precursor, suggesting that the precursor is identical or closely related to osteopontin. In the rat nephron, the protein has been localized along the luminal surfaces of the proximal and distal tubule and the collecting duct cells. These results show that in the kidney-derived cell line Madin-Darby canine kidney osteopontin or a closely related protein is proteolytically processed to a 20-kDa polypeptide, raising the possibility that diverse functions of osteopontin in various tissues might be attributed to specific processing to distinct polypeptides.     

Biosynthesis and post-translational processing of the precursor to brain-derived neurotrophic factor

We examined the biosynthesis and post-translational processing of the brain-derived neurotrophic factor precursor (pro-BDNF) in cells infected with a pro-BDNF-encoding vaccinia virus. Metabolic labeling, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis reveal that pro-BDNF is generated as a 32-kDa precursor that is N-glycosylated and glycosulfated on a site, within the pro-domain. Some pro-BDNF is released extracellularly and is biologically active as demonstrated by its ability to mediate TrkB phosphorylation. The precursor undergoes N-terminal cleavage within the trans-Golgi network and/or immature secretory vesicles to generate mature BDNF (14 kDa). Small amounts of a 28-kDa protein that is immunoprecipitated with BDNF antibodies is also evident. This protein is generated in the endoplasmic reticulum through N-terminal cleavage of pro-BDNF at the Arg-Gly-Leu-Thr(57)- downward arrow-Ser-Leu site. Cleavage is abolished when Arg(54) is changed to Ala (R54A) by in vitro mutagenesis. Blocking generation of 28-kDa BDNF has no effect on the level of mature BDNF and blocking generation of mature BDNF with alpha(1)-PDX, an inhibitor of furin-like enzymes, does not lead to accumulation of the 28-kDa form. These data suggest that 28-kDa pro-BDNF is not an obligatory intermediate in the formation of the 14-kDa form in the constitutive secretory pathway.     

Transforming growth factor beta 1 induces apoptosis through cleavage of BAD in a Smad3-dependent mechanism in FaO hepatoma cells

Transforming growth factor beta (TGF-beta) induces apoptosis in a variety of cells. We have previously shown that TGF-beta 1 rapidly induces apoptosis in the FaO rat hepatoma cell line. We have now studied the effect of TGF-beta 1 on the expression of different members of the Bcl-2 family in these cells. We observed no detectable changes in the steady-state levels of Bcl-2, Bcl-X(L), and Bax. However, TGF-beta 1 induced caspase-dependent cleavage of BAD at its N terminus to generate a 15-kDa truncated protein. Overexpression of the 15-kDa truncated BAD protein enhanced TGF-beta 1-induced apoptosis, whereas a mutant BAD resistant to caspase 3 cleavage blocked TGF-beta 1-induced apoptosis. Overexpression of Smad3 dramatically enhanced TGF-beta 1-induced cleavage of BAD and apoptosis, whereas antisense Smad3 blocked TGF-beta 1-induced apoptosis and BAD cleavage. These results suggest that TGF-beta 1 induces apoptosis through the cleavage of BAD in a Smad3-dependent mechanism.     

Antagonistic effects of Smad2 versus Smad7 are sensitive to their expression level during tooth development

Members of the transforming growth factor-beta (TGF-beta) superfamily regulate cell proliferation, differentiation, and apoptosis, controlling the development and maintenance of most tissues. TGF-beta signal is transmitted through the phosphorylation of Smad proteins by TGF-beta receptor serine/threonine kinase. During early tooth development, TGF-beta inhibits proliferation of enamel organ epithelial cells but the underlying molecular mechanisms are largely unknown. Here we tested the hypothesis that antagonistic effects between Smad2 and Smad7 regulate TGF-beta signaling during tooth development. Attenuation of Smad2 gene expression resulted in significant advancement of embryonic tooth development with increased proliferation of enamel organ epithelial cells, while attenuation of Smad7 resulted in significant inhibition of embryonic tooth development with increased apoptotic activity within enamel organ epithelium. These findings suggest that different Smads may have differential activities in regulating TGF-beta-mediated cell proliferation and death. Furthermore, functional haploinsufficiency of Smad2, but not Smad3, altered TGF-beta-mediated tooth development. The results indicate that Smads are critical factors in orchestrating TGF-beta-mediated gene regulation during embryonic tooth development. The effectiveness of TGF-beta signaling is highly sensitive to the level of Smad gene expression.     

Biosynthesis and intracellular processing of carbonic anhydrase in Chlamydomonas reinhardtii

Carbonic anhydrase (CA) of Chlamydomonas reinhardtii is a glycoprotein of 35 kDa which is localized outside the plasma membrane. The activity of CA was increased when the CO2 concentration during photoautotrophic growth was decreased to air level. After decreasing the CO2 concentration from 4% to 0.04%, several polypeptides including CA were induced continuously or transiently. To investigate the biosynthesis and intracellular processing of CA, the cells of wall-less mutant CW-15, which secretes CA into the culture medium, were pulse-labeled with radioactive arginine, chased, and radioactive proteins were immunoprecipitated with anti-CA serum. A 42-kDa polypeptide with isoelectric point (pI) of 7.1-7.3 was first synthesized. Within 5 min the molecular mass of this polypeptide was decreased to 35 kDa and it was then secreted into the culture medium within 30 min. This indicates that the former is the precursor form and the latter the mature form of CA. The primary translation product from poly(A)-rich RNA in a cell-free reticulocyte lysate system from a rabbit was a 38-kDa polypeptide. This was cotranslationally converted into the 42-kDa precursor in vitro in the presence of dog pancreatic microsomal membranes. As the 42-kDa precursor had a high affinity to concanavalin A, it was assumed to have a high-mannose-type oligosaccharide. The mature enzyme had a pI of 6.1-6.2 and was composed of more than two isoforms, which had a complex-type oligosaccharide with low affinity to concanavalin A. Chemical deglycosylation of the mature enzyme by trifluoromethanesulfonic acid indicated that the molecular mass of the polypeptide moiety was 32 kDa and the difference between this and the primary translation product suggests that cleavage of the polypeptide occurs during its biosynthesis.     

GM2 gangliosidoses in Spain: Analysis of the HEXA and HEXB genes in 34 Tay-Sachs and 14 Sandhoff patients

Acid α-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes glycogen to glucose. Deficiency of GAA causes Pompe disease. Mammalian GAA is synthesized as a precursor of ~ 110,000 Da that is N-glycosylated and targeted to the lysosome via the M6P receptors. In the lysosome, human GAA is sequentially processed by proteases to polypeptides of 76-, 19.4-, and 3.9-kDa that remain associated. Further cleavage between R²⁰⁰ and A²⁰⁴ inefficiently converts the 76-kDa polypeptide to the mature 70-kDa form with an additional 10.4-kDa polypeptide. GAA maturation increases its affinity for glycogen by 7-10 fold. In contrast to human GAA, processing of bovine and hamster GAA to the 70-kDa form is more rapid. A comparison of sequences surrounding the cleavage site revealed human GAA contains histidine at 201 while other species contain hydrophobic amino acids at position 201 in the otherwise conserved sequence. Recombinant human GAA (rhGAA) containing the H201L substitution was expressed in 293 T cells by transfection. Pulse chase experiments in 293 T cells expressing rhGAA with or without the H201L substitution revealed rapid processing of rhGAA^H201L but not rhGAA^WT to the 70-kDa form. Similarly, when GAA precursor was endocytosed by human Pompe fibroblasts rhGAA^H201L but not rhGAA^WT was rapidly converted to the 70-kDa mature GAA. These studies indicate that the amino acid at position 201 influences the rate of conversion of 76-kDa GAA to 70-kDa GAA. The GAA sequence rather than the lysosomal protease environment explains the predominance of the 76-kDa form in human tissues.     

Flounder and fugu have a single lefty gene that covers the functions of lefty1 and lefty2 of zebrafish during L-R patterning

The lefty gene encodes a member of the TGF-beta superfamily that regulates L-R axis formation during embryogenesis via antagonistic activity against Nodal, another TGF-beta superfamily member. Both mouse and zebrafish have two lefty genes, lefty1 and lefty2. Interestingly, the expression domains of mouse and zebrafish lefty are different from one another. At present, the orthology and functional diversity of the mouse and zebrafish lefty genes are not clear. Here, we report that flounder and two fugu species, Takifugu and Tetraodon, have a single lefty gene in their genomes. In addition, we provide evidence that the mouse lefty genes were duplicated on a single chromosome but the zebrafish lefty genes arose from a whole-genome duplication that occurred early in the divergence of ray-finned fishes. These independent origins likely explain the difference in the expression domains of the mouse and zebrafish lefty gene pairs. Furthermore, we found that the duplication corresponding to the zebrafish lefty2 gene was lost from the fugu genome, suggesting that loss of lefty2 in the fugu/flounder lineage occurred after its divergence from the zebrafish lineage. During L-R patterning, the single lefty gene of flounder covers two expression domains, the left side of the dorsal diencephalon and the left LPM, which are regulated separately by lefty1 and lefty2 in zebrafish. We infer that the lefty genes of the ray-finned fishes and mammals underwent independent gene duplication events that resulted in independent regulation of lefty expression.     

A novel mouse Smad4 mutation reduces protein stability and wild-type protein levels

Smad4 is a key signal transducer of the transforming growth factor-beta (TGF-beta) superfamily of growth factors that are critical regulators of embryonic patterning and adult tissue homeostasis. The biological activity of the TGF-beta signaling is tightly controlled at multiple levels, including the abundance of SMAD4 proteins. We previously recovered a novel allele of Smad4 in a gene-based screen in N-ethyl-N-nitrosourea (ENU)-mutagenized mouse embryonic stem cells. The mutation resulted in an unstable truncated protein that is degraded through proteasomal pathways. In the heterozygous state, this allele acts in a dominant negative fashion to reduce the wild-type protein level as well as signaling output. Biochemical characterization indicated that the truncated protein is able to form a complex with the wild-type protein, thus targeting it for proteasomal degradation as well. Phenotypic analyses of the heterozygous animals provided insight into the threshold requirement of Smad4-dependent signaling in vivo.     

TGF-beta superfamily signaling is essential for tooth and hair morphogenesis and differentiation

Members of the transforming growth factor beta (TGF-beta) superfamily of signaling molecules are involved in the regulation of many developmental processes that involve the interaction between mesenchymal and epithelial tissues. Smad7 is a potent inhibitor of many members of the TGF-beta family, notably TGF-beta and activin. In this study, we show that embryonic overexpression of Smad7 in stratified epithelia using a keratin 5 promoter, results in severe morphogenetic defects in skin and teeth and leads to embryonic and perinatal lethality. To further analyze the functions of Smad7 in epithelial tissues of adult mice, we used an expression system that allowed a controlled overexpression of Smad7 in terms of both space and time. Skin defects in adult mice overexpressing Smad7 were characterized by hyper-proliferation and missing expression of early markers of keratinocyte differentiation. Upon Smad7-mediated blockade of TGF-beta superfamily signaling, ameloblasts failed to produce an enamel layer in incisor teeth. In addition, TGF-beta blockade in adult mice altered the pattern of thymic T cell differentiation and the number of thymic T cells was significantly reduced. This study shows that TGF-beta superfamily signaling is essential for development of hair, tooth and T-cells as well as differentiation and proliferation control in adult tissues.     

A potential mucus precursor in Tetrahymena wild type and mutant cells.

By using an antibody to a specific mucus polypeptide (34 kDa) to study whole cell extracts of both a secretory mutant (SB281) and wild type (wt) Tetrahymena, we demonstrate that a 57-kDa polypeptide is a probable precursor to the 34-kDa secretory polypeptide. We postulate that the precursor accumulates in the mutant cells because it cannot be cleaved. This mutant contains no recognizable mature secretory granules (mucocysts). By immunoelectron microscopy, the 34-kDa polypeptide was localized in wt cells specifically to the mature mucocysts and to their released products. Localization in mutant cells occurred in two different types of cytoplasmic vesicles: small electron dense vesicles (0.3-0.5 microns in diameter) and large electron lucent vacuoles (1.2-3.5 microns in diameter). Immunoblot analyses of homogenates of mutant and wt cells with the anti-34-kDa serum revealed a dominant band in the mutant at Mr 57 kDa whereas the wt showed a dominant band only at Mr 34 kDa. Furthermore, the 57-kDa polypeptide is immunoprecipitated with anti-34-kDa serum from the mutant cell. Further evidence for a precursor relation of the 57-kDa polypeptide in mutant cells to the 34-kDa mucus polypeptide of wt cells was obtained by the use of drugs (monensin, chloroquine, NH4Cl) that block secretory product processing in wt cells. Extracts of drug-treated wt cells showed the presence of a 57-kDa cross reacting band even after 18 h of incubation in growth medium whereas untreated control cells contained the 34-kDa mature protein almost exclusively. These results indicate that processing of the precursor to the 34-kDa polypeptide occurs in an acidic compartment(s) possibly in either the trans Golgi network, or condensing vacuoles or both.     

The processing of a cathepsin L precursor in vitro

Subcultured rat fibroblasts secreted a cathepsin L precursor when maintained for 24 h in serum-free medium containing 20 mM ammonium ions. The precursor was identified by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using polyclonal antibodies to cathepsin L. The molecular mass of the precursor was found to be approximately 39 kDa, which confirms the result originally reported by Y. Nishimura et al. (1988, Arch. Biochem. Biophys. 263, 107-116). Treatment of the precursor containing medium with cathepsin D at pH values ranging from 3.5 to 5.5 caused a limited cleavage of the precursor molecule. The resultant polypeptides are an unstable intermediate form with Mr 35,000 and a stable single chain form of cathepsin L showing a Mr about 32,500. The cathepsin D-mediated conversion was strongly accelerated by Hg2+ ions. A further proteolytic cleavage of the 32.5-kDa polypeptide has not been observed. The enzymatic activity toward Z-Phe-Arg-NHMec at pH 5.5 increased during the conversion, indicating that active cathepsin L was formed from an inactive precursor molecule.     

Biosynthesis and processing of lysosomal acid phosphatase in cultured human cells

The biosynthesis of lysosomal acid phosphatase was studied in a normal human embryonic lung cell line, WI-38. Cells were labeled with radioactive leucine under a variety of conditions, the enzyme was immunoprecipitated using a monospecific antiserum raised against human liver lysosomal acid phosphatase, and the products were separated by electrophoresis and were visualized by fluorography. Lysosomal acid phosphatase constitutes 60% of the total tartrate-inhibitable acid phosphatase in WI-38. It is initially synthesized as a high-molecular-weight precursor polypeptide of 69 kDa. The precursor polypeptide is rapidly glycosylated and processed to a mature enzyme of 53-45 kDa via intermediates of 65 and 60 kDa in WI-38 cells. The 69-kDa precursor polypeptide is also converted to larger precursor polypeptides of 74 and 80 kDa. The multiplicity of precursor polypeptides is due at least in part to differences in the glycosylation and phosphorylation of the polypeptides. Sensitivity of phosphorylated oligosaccharide chains from precursor, mature and small polypeptides to endo-beta-hexosaminidase H-catalyzed cleavage suggests the presence of high-mannose phosphorylated oligosaccharide chains similar to those present on many other lysosomal enzymes. The effects of tunicamycin and ammonium chloride were also studied. In contrast to the effect of ammonium chloride on arylsulfatase A secretion, the lysosomal acid phosphatase in WI-38 cells was not secreted in the presence of NH4Cl. This is consistent with the existence of an alternate route for the transfer of lysosomal acid phosphatase into lysosomes. This alternate route may be the reason that I-cell fibroblasts contain a normal level of lysosomal acid phosphatase.     

Involvement of calcium in interactions between gingival epithelial cells and Porphyromonas gingivalis

Abstract Three major polypeptides of 34, 48 and 50 kDa which appear to copurify with 1,3-β-glucan synthase activity were isolated by glycerol gradient centrifugation of Chaps-solubilized proteins from the fungus Saprolegnia monoica . The antiserum produced against the 34-kDa polypeptide revealed by protein immunoblotting that this polypeptide copurified with 1,3-β-glucan synthase during enzyme purification. This antiserum adsorbs the enzymatic activity as well as the 48- and 50-kDa polypeptides. These results indicate that the 34-kDa peptide is a component of the multisubunit protein complex involved in 1,3-β-glucan synthase activity.