Chinese hamster lung cell polysomes direct the synthesis of a single molecular weight species of procollagen alpha chains.

Polysomes prepared from cultured Chinese hamster lung cells direct the synthesis of procollagen alpha chains in an heterologous cell-free system containing the postribosomal supernatant fraction prepared from wheat germ. Total protein synthesis requires both subcellular components and an exogenous energy source, and is inhibited by the antibiotics puromycin and aurin tricarboxylic acid. The ratio of collagenase-digestible to nondigestible material produced depends upon the wheat germ and not the polysome level in the reaction. Under optimal conditions, a significant fraction of the total product migrates on denaturing sodium dodecyl sulfate-polyacrylamide gels as a single molecular weight collagenase-digestible species corresponding in size to the procollagen alpha chain (Mr approximately equal to 170,000). Approximately one-third of this high molecular weight material represents products whose synthesis results from cell-free mRNA initiation, and no distinct product larger than the 170,000-dalton material is observed. These studies confirm the initial observation that collagen represents one of the major gene products of Chinese hamster lung cells and demonstrate the usefulness of this cell line for the study of mammalian collagen biosynthesis.     

Signals causing change in morphological phenotype,growth mode,and gene expression of vascular endothelial cells

Comparison of three different lines of bovine aortal endothelial cells provides a clear demonstration of reversible morphologic phenotype coincidental with change in expression and growth mode. These phenotypic forms can be externally controlled so that cells may exist either in an epithelioid contact-inhibitable state or as a fibroblastoid non-contact-inhibitable state. Clonal cell line N (normal) shows a strong tendency to maintain the epithelioid phenotype. Clonal cell line Sp (sprout) can readily and reversibly adopt the epithelioid or fibroblastoid phenotype. A factor in normal serum is responsible for maintaining the cells in the epithelioid phenotype. This factor could be a growth factor since several polypeptide growth factors are shown to drive cells from the fibroblastoid phenotype to the epithelioid phenotype within 11 hours. This growth factor-induced change is not mediated through induced DNA synthesis. Clonal cell line V (variant) normally maintains the fibroblastoid phenotype but can be directed to the epithelioid phenotype provided cells are on an appropriate collagenous matrix. Associated with these changes in morphological phenotype are depression of the expression of the pro α₂ chain of collagen type I which may be characteristic of the contact-inhibited state and of an 80,000 mol wt polypeptide synthesized only by cells in the fibroblastoid phenotype. An endothelial cell collagen ECl (mol wt 177,000) was synthesized by all cell lines regardless of phenotype whereas a suspected breakdown product EC3 (mol wt 100,000) was found only in the epithelioid phenotype. Other differences and similarities between cell lines include expression of a 135,000 mol wt glycoprotein GP (V and N), the procollagen of collagen type III (N) of fibronectin (N, V, Sp), and of the pro α₁ chain of collagen type I (Sp, V). The characteristic expression of each line and its response to signals controlling morphologic phenotype impinges on the question of whether there exist several distinct types of vascular endothelial cells with different functional potentials controlled by extracellular signals.     

Isolation and cell-free translation of a human immunoglobulin light chain messenger RNA.

Human myeloma cell line RPMI 8226 synthesizes and secretes a lambda immunoglobulin light chain. Total cellular RNA, obtained from cells grown in culture, was used for the isolation of poly(A)-containing mRNA by oligo(dT)-cellulose chromatography. The poly(A)-containing mRNA was translated in an Ehrlich ascites extract. Immunoprecipitation of the cell-free products showed that a polypeptide chain antigenically related to human lambda chain was synthesized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the cell-free product was larger than the secreted light chain. On tryptic digestion the cell-free product and the secreted light chain exhibited essentially identical peptide patterns except for an additional peptide derived from the cell-free product. We conclude that the lambda mRNA from this human myeloma cell line codes for a precursor to the secreted lambda chain as described for immunoglobulin mRNAs from murine plasmacytomas.     

Evidence that the collagen in the culture medium of Chinese hamster lung cells contains components related at the primary structural level to the alpha1(V) collagen chain

The collagenous protein synthesized by cultured Chinese hamster lung (CHL) cells and present in the culture medium has been isolated after limited pepsin digestion and differential salt precipitation. Molecular size analysis of this material indicates that the CHL cell medium collagen contains chains which exhibit an apparent molecular mass of approximately 85,000 Da. When chromatographed on CM-cellulose under denaturing conditions, the reduced and alkylated CHL cell medium collagen chains elute slightly after the human alpha1(I) chain but well before the pepsin-derived alpha1(V) chain, which is the constituent chain present in the CHL cell cellular matrix collagen. Analysis of the peptides derived by CNBr cleavage of the CHL medium collagen chains by chromatography on CM-cellulose reveals, however, that these chains contain peptides which correspond both in size and in chemical properties to those derived from the alpha1(V) collagen chain, but clearly lack two peptides (alpha1(V)-CB4 and alpha1(V)-CB5) which are normally present in pepsin-derived alpha1(V) chains. Furthermore, analysis of the CHL cell culture medium collagenous material obtained without pepsin digestion indicates the presence of collagenous chains that exhibit after reduction a molecular mass of approximately 160,000 Da, which is smaller than the proposed size of the pro alpha1(V) collagen chain. These results demonstrate that the collagenous protein present in the culture medium of CHL cells is directly related at the primary structural level to the alpha1(V) collagen chain, and it is postulated that this material represents the large fragment derived from a collagenase cleavage of the [pro alpha1(V)]3 molecules present in the cell layer. Furthermore, these results and previous reports indicate that the only identifiable genetic type of procollagen chain synthesized by this cloned cell line in culture corresponds to the pro alpha1(V) chain.     

Biosynthesis of intestinal microvillar proteins. Processing of aminopeptidase N by microsomal membranes.

The biosynthesis of small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in a cell-free translation system derived from rabbit reticulocytes. When dog pancreatic microsomal fractions were present during translation, most of the aminopeptidase N synthesized was found in a membrane-bound rather than a soluble form, indicating that synthesis of the enzyme takes place on ribosomes attached to the rough endoplasmic reticulum. The microsomal fractions process the Mr-115 000 polypeptide, which is the primary translation product of aminopeptidase N, to a polypeptide of Mr 140 000. This was found to be sensitive to the action of endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96), showing that aminopeptidase N undergoes transmembrane glycosylation during synthesis. The position of the signal sequence in aminopeptidase N was determined by a synchronized translation experiment. It was found that microsomal fractions should be added before about 25% of the polypeptide was synthesized to ensure processing to the high-mannose glycosylated form. This suggests that the signal sequence is situated in the N-terminal part of the aminopeptidase N. The size of the cell-free translation product in the absence of microsomal fractions was found to be similar to that on one of the forms of the enzyme obtained from tunicamycin-treated organ-cultured intestinal explants.     

A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins

Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins.     

Cell-free synthesis of mouse corticotropin. Evidence for two high molecular weight gene products.

Polysomes or mRNA prepared from cultured AtT-20/D16v mouse pituitary adenocarcinoma cells direct the efficient incorporation of amino acid into newly synthesized material in the presence of wheat germ translational factors. A significant franction of the total cell-free product is specifically immunoprecipitable with corticotropin antibody purified by immune affinity chromatography. Analysis of the cell-free synthesized immunoreactive products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that two high molecular weight corticotropin species (Mr congruent to 32,500 and 28,000) are synthesized in an approximate 2:1 ratio. Neither product contains carbohydrate based upon concanavalin A chromatography or exposure to polysaccharidases. The smaller molecular weight product does not appear to arise from proteolytic processing since both species are synthesized in approximately the same ratio in cell-free reaction mixtures directed by either polysomes or mRNA. These results suggest that AtT-20/D16v cells contain two distinct mRNA poluations specifying the synthesis of two different high molecular weight forms of mouse corticotropin.     

A serum factor promotes collagenase synthesis by an osteoblastic cell line.

Regulation of the synthesis of collagenase was investigated in the osteoblastic cell line, UMR 106-01. The cells were stained by the avidin-biotin-complex technique for the presence of the enzyme. By this method, it was possible to identify cells producing collagenase. Synthesis, but not secretion, was found to be constitutive in these cells with the enzyme located intracellularly in cytoplasmic vesicles and the Golgi apparatus. The amount of collagenase contained within UMR cells and the number of cells synthesizing the enzyme were proportional to the concentration of fetal bovine serum in the incubating medium. When serum was withdrawn from the osteosarcoma cells, the content of collagenase decreased with time and the enzyme became undetectable by 48 h of serum depletion. The decrease in collagenase content could be completely reversed by resupplying serum to the cells. The collagenase promoting activity of serum could not be eliminated by adsorption on activated charcoal but was retained by a dialysis membrane with a 12,000 mol wt cutoff. A range of bone-seeking hormones or agents known to affect collagenase secretion was added to the medium in an attempt to mimic the effect of serum on collagenase accumulation. None of these agonists, including parathyroid hormone, could reproduce the effect of serum on these cells, although parathyroid hormone could act as a collagenase secretagogue in the presence or absence of serum. It is concluded that fetal bovine serum contains a yet unidentified factor or factors greater than 12,000 mol wt responsible for the continued synthesis of collagenase by UMR 106-01 cells.     

Homocystinuria: biogenesis of cystathionine beta-synthase subunits in cultured fibroblasts and in an in vitro translation system programmed with fibroblast messenger RNA.

Rabbit antiserum raised against pure human hepatic cystathionine beta-synthase was used to precipitate synthase from extracts of radiolabeled cultured fibroblasts derived from 17 homocystinuric patients and two controls. Size analysis of the immunoprecipitates by SDS/polyacrylamide gel electrophoresis revealed that 15 of the 17 synthase-deficient lines synthesized synthase subunits indistinguishable in size from the control (Mr = 63,000). One mutant fibroblast line, previously shown to lack catalytic activity and antigenically cross-reacting material, contained no immunoprecipitable product. Analyses of immunoprecipitated polypeptides synthesized in vitro by cell-free translation of mRNAs prepared from selected mutants confirmed and extended the results from cell extracts. This experimental approach also allowed us to determine the biochemical and genetic defect in a patient with barely detectable synthase subunits in cell extracts. His cultured fibroblasts and those of his father contained two mRNA species, separable by size, coding for equal amounts of two immunoprecipitable polypeptides: one of normal size (Mr = 63,000); the other approximately 7,000 daltons smaller (Mr = 56,000). His mother's fibroblasts made only the Mr = 63,000 species. We conclude that this patient is a compound heterozygote, and that one of his mutant alleles results in the synthesis of a synthase polypeptide missing about 60 amino acid residues.     

Characterization of the protein species synthesized in vivo and in vitro by an aphid endosymbiont

The endosymbionts isolated from rifampicin-injected insects were less active in synthesizing proteins in vitro than were the uninjected ones. It was confirmed using [³H]leucine that the pea aphid, when injected with cycloheximide, actively synthesized only one protein species, symbionin. The synthesis of symbionin is highly sensitive to chloramphenicol. When separated two-dimensionally, symbionin was represented by a single spot. It is an acidic protein with mol. wt of about 63,000. Two days after an injection of cycloheximide the aphid began to synthesize a protein in addition to symbionin. The protein's mol. wt was less than that of symbionin and its synthesis was also sensitive to chloramphenicol. On a two-dimensional gel, proteins synthesized in the isolated endosymbiont were separated into more than 100 species. Under incomplete anaerobic conditions, the isolated symbiont synthesized several unique proteins.     

The immunology of procollagen. I. Development of antibodies to determinants unique to the proalpha l chain

Rabbits immunized with proα1 chains derived from embryonic chick cranial bone procollagen developed antibodies directed specifically to the NH₂-terminal non-triple-helical sequence unique to the precursor chain. Antibodies were detected by a sensitive radioimmunoassay which employed radiolabeled chick proα1 chains, rabbit antisera absorbed with an excess of α1 chain, and a sheep anti-rabbit γG globulin serum. The specificity of such rabbit antisera was demonstrated by inhibition assays with unlabeled proα1 chains and with CNBr and collagenase fragments obtained from the additional sequence in the precursor chain. The availability of antibodies to this relatively immunogenic region of procollagen provides a means of assaying for the collagen precursor in biosynthetic experiments and may permit the intracellular localization of the macromolecule by antibody-labeling techniques.     

Translation of Taenia crassiceps mRNA in cell-free heterologous systems.

1. mRNA isolated from larval Taenia crassiceps directs efficiently the synthesis of proteins in cell-free heterologous systems. 2. Part of the newly synthesized proteins in a reticulocyte system are precipitable by a rabbit antiserum against T. crassiceps proteins. 3. Analysis of the antiserum-protein dissociated complex by sodium dodecyl sulfate polyacrylamide gel electrophoresis indicates that most of the proteins synthesized are of low molecular weight (13,000-22,000) although a protein of mol. wt. 260,000 is also produced. 4. Whether the newly synthesized proteins which are precipitable by specific antisera correspond to parasite antigens or to proteins with closely antigenic similarities remains to be established.     

Isolation and translation of calvaria procollagen messenger ribonucleic acids.

Procollagen messenger ribonucleic acid (mRNA) was isolated from the calvaria of 15-day-old chick embryos by chromatographing total RNA over oligo(dT)-cellulose two times, and then fractionating the twice-bound RNA on 85% Me2SO/0-20% sucrose gradients. When analyzed on 99% formamide gels, the 27-30S fraction had three sharp fluorescent bands, one corresponding to 27S ribosomal RNA (rRNA), the others having mobilities lower than 27S corresponding to molecular weights of 1 700 000 and 1 800 000. In wheat-germ, cell-free extracts, the 27-30S fraction directed the synthesis of two prominent collagenase sensitive polypeptides with mobilities corresponding to the calvaria pro-alpha chain markers. Twelve percent of this [3H]proline-labeled, wheat-germ product could be hydroxylated with prolyl hydroxylase.     

Isolation and cell-free translation of immunoglobulin messenger RNA.

The mRNA's for both the heavy chain (H³¹⁵) and the light chain (L³¹⁵) of the mineral oil-induced plasmacytoma-315 myeloma protein have been isolated and partially purified from both total cellular RNA and RNA derived from membrane-bound polysomes. The yields of both L³¹⁵ mRNA and, in particular, of H³¹⁵ mRNA were increased when total cellular RNA was used as starting material. Total poly(A)-containing mRNA and partially purified mRNA obtained by preparative sucrose gradient sedimentation stimulated protein synthesis in cell-free extracts derived from Ehrlich ascites tumor cells or wheat germ. Cell-free products antigenically and structurally related to both the authentic L³¹⁵ and H³¹⁵ secreted by intact cells were synthesized in the Ehrlich ascites cell-free system in response to the appropriate mRNA's. Only the L³¹⁵ mRNA was efficiently translated in the cell-free system derived from wheat germ.     

Cell-free synthesis and assembly of prolyl 4-hydroxylase: the role of the beta-subunit (PDI) in preventing misfolding and aggregation of the alpha-subunit.

Prolyl 4-hydroxylase (P4-H) catalyses a vital post-translational modification in the biosynthesis of collagen. The enzyme consists of two distinct polypeptides forming an alpha 2 beta 2 tetramer (alpha = 64 kDa, beta = 60 kDa), the beta-subunit being identical to the multifunctional enzyme protein disulfide isomerase (PDI). By studying the cell-free synthesis of the rat alpha-subunit of P4-H we have shown that the alpha-subunit can be translocated, glycosylated and the signal peptide cleaved by dog pancreatic microsomal membranes to yield both singly and doubly glycosylated forms. When translations were carried out under conditions which prevent disulfide bond formation, the product synthesized formed aggregates which were associated with the immunoglobulin heavy chain binding protein (BiP). Translations carried out under conditions that promote disulfide bond formation yielded a product that was not associated with BiP but formed a complex with the endogenous beta-subunit (PDI). Complex formation was detected by co-precipitation of the newly synthesized alpha-subunit with antibodies raised against PDI, by sucrose gradient centrifugation and by chemical cross-linking. When microsomal vesicles were depleted of PDI, BiP and other soluble endoplasmic reticulum proteins, no complex formation was observed and the alpha-subunit aggregated even under conditions that promote disulfide bond formation. We have therefore demonstrated that the enzyme P4-H can be assembled at synthesis in a cell-free system and that the solubility of the alpha-subunit is dependent upon its association with PDI.     

Isolation and preliminary characterization of epithelial-specific messenger ribonucleic acids and their products during embryonic tooth development.

Experiments were designed to identify and characterize tissue-specific proteins involved in the process of tooth organogenesis. Epithelial and mesenchymal proteins were extracted from intact molar organs or mechanically separated tissues obtained from 25-day New Zealand White rabbit embryos. Labelling experiments with [35S]methionine followed by radioautography or gel electrophoresis and fluorography showed the presence of label only in epithelial proteins. Most of these proteins range from 43 000 mol.wt. and higher, except for one band of approx. 16 000 mol.wt. A mRNA fraction of 16--26S was isolated by ultracentrifugation on sucrose gradients. When translated in a reticulocyte-lysate cell-free system, the mRNA obtained from intact molar organs resulted in the synthesis of three proteins, of mol.wts. 65 000, 58 000 and 43 000. A similar mRNA fraction obtained from dental-pulp mesenchyme gave only the 43 000-mol.wt. protein, indicating that the 65 000- and 58 000-mol.wt. proteins are derived from epithelial cells.     

Effects of procollagen peptides on the translation of type II collagen messenger ribonucleic acid and on collagen biosynthesis in chondrocytes

Type II procollagen messenger ribonucleic acid (mRNA) was isolated from chick sternum and rat chondrosarcoma cells and translated in a reticulocyte lysate cell-free system. A high molecular weight band was identified as type II procollagen by gel electrophoresis, collagenase digestion, and specific immunoprecipitation. The translation of type II mRNA was specifically inhibited by addition of type I procollagen amino-terminal extension peptide. When this peptide was added to the media of cultured fetal calf chondrocytes, chick sternal chondrocytes, or chick tendon fibroblasts, no inhibition of collagen synthesis was evident. These data suggest a general regulation of collagen biosynthesis by these peptides in the cell-free translation system. However, as indicated by the cell culture experiments, cellular characteristics and evolutionary divergence of animal species seem to restrict the effect of the peptides.