Fibrinogen Biosynthesis: In Vitro Translation. Glycosylation And Translocation Of Fibrinogen Peptide Chains

Mapping Intimacies ◽

10.1055/s-0038-1652739 ◽

1981 ◽

Author(s):

G M Fuller ◽

J M Nickerson

Keyword(s):

Signal Peptide ◽

Hepatoma Cell ◽

Temporal Analysis ◽

In Vitro Translation ◽

Translation System ◽

Hepatoma Cell Line ◽

Amino Terminal ◽

Cellular Processes ◽

Microsomal Membranes

Fibrinogen is a hepatically derived plasma glycoprotein that is composed of three pairs of nonidentical chains linked together by complex sets of disulfide bridges. In an effort to understand the molecular and cellular processes of translating and assembling this important multichained protein we have utilized an in vitro translating system using mRNA’s for rat fibrinogen. Highly specific antibodies to fibrinogen and to each chain have been developed and used to immunoprecipitate the nascent Aα, Bβ, and γ polypeptides. We have also used a rat hepatoma cell line which synthesizes and secretes fibrinogen to prepare nonglycosylated but processed fibrinogen subunits. SDS/PAGE analysis of the translation products clearly show that each polypeptide has a “signal” peptide located at its amino terminal end. The size of the signal peptide is different for each chain. These results demonstrate that separate mRNA’s exist for each of the fibrinogen subunits. Temporal analysis of the glycosylation of the Bβ and γ chain reveal that the γ chain receives its Asn-linked carbohydrate as an early cotranslational event. The Bβ chain’s core carbohydrate moiety is near the end of the polypeptide and our evidence shows that the glycosylation event likely occurs posttranslationally. When microsomal membranes are added to an on-going translation system, all three of fibrinogen's polypeptides translocate into the cisternal space, with an apparent equal stiochiometry. Additional experiments suggest that fibrinogen assembly occurs as a cotranslational process.These studies have been supported in part by NIH HL - 16445 and HL 00162.

Download Full-text

The NH2 terminus of preproinsulin directs the translocation and glycosylation of a bacterial cytoplasmic protein by mammalian microsomal membranes.

The Journal of Cell Biology ◽

10.1083/jcb.103.6.2263 ◽

1986 ◽

Vol 103 (6) ◽

pp. 2263-2272 ◽

Cited By ~ 10

Author(s):

E M Eskridge ◽

D Shields

Keyword(s):

Signal Peptide ◽

Fusion Proteins ◽

Signal Sequence ◽

Membrane Vesicles ◽

In Vitro Translation ◽

Signal Peptidase ◽

Endoplasmic Reticulum Membrane ◽

Amino Terminal ◽

Microsomal Membranes

To investigate putative sorting domains in precursors to polypeptide hormones, we have constructed fusion proteins between the amino terminus of preproinsulin (ppI) and the bacterial cytoplasmic enzyme chloramphenicol acetyltransferase (CAT). Our aim is to identify sequences in ppI, other than the signal peptide, that are necessary to mediate the intracellular sorting and secretion of the bacterial enzyme. Here we describe the in vitro translation of mRNAs encoding two chimeric molecules containing 71 and 38 residues, respectively, of the ppI NH2 terminus fused to the complete CAT sequence. The ppI signal peptide and 14 residues of the B-chain were sufficient to direct the translocation and segregation of CAT into microsomal membrane vesicles. Furthermore, the CAT enzyme underwent N-linked glycosylation, presumably at a single cryptic site, with an efficiency that was comparable to that of native glycoproteins synthesized in vitro. Partial amino-terminal sequencing demonstrated that the downstream sequences in the fusion proteins did not alter the specificity of signal peptidase, hence cleavage of the ppI signal peptide occurred at precisely the same site as in the native precursor. This is in contrast to results found in prokaryotic systems. These data demonstrate that the first 38 residues of ppI encode all the information necessary for binding to the endoplasmic reticulum membrane, translocation, and proteolytic (signal sequence) processing.

Download Full-text

four-jointed is required for intermediate growth in the proximal-distal axis in Drosophila

Development ◽

10.1242/dev.121.9.2767 ◽

1995 ◽

Vol 121 (9) ◽

pp. 2767-2777 ◽

Cited By ~ 5

Author(s):

J.L. Villano ◽

F.N. Katz

Keyword(s):

Optic Lobe ◽

Regional Growth ◽

Positional Information ◽

In Vitro Translation ◽

Translation System ◽

Regional Pattern ◽

Microsomal Membranes ◽

Position Sensitive ◽

Coordination Function

Genes capable of translating positional information into regulated growth lie at the heart of morphogenesis, yet few genes with this function have been identified. Mutants in the Drosophila four-jointed (fj) gene show reduced growth and altered differentiation only within restricted sectors of the proximal-distal (PD) axis in the leg and wing, thus fj is a candidate for a gene with this coordination function. Consistent with a position-sensitive role, we show that fj is expressed in a regional pattern in the developing leg, wing, eye and optic lobe. The fj gene encodes a novel type II membrane glycoprotein. When the cDNA is translated in an in vitro translation system in the presence of exogenous microsomal membranes, the intralumenal portion of some of the molecules is cleaved, yielding a secreted C-terminal fragment. We propose that fj encodes a secreted signal that functions as a positive regulator of regional growth and differentiation along the PD axis of the imaginal discs.

Download Full-text

Transmembrane topology of the mammalian KDEL receptor.

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6435 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6435-6441 ◽

Cited By ~ 17

Author(s):

P Singh ◽

B L Tang ◽

S H Wong ◽

W Hong

Keyword(s):

Membrane Protein ◽

Fusion Proteins ◽

Integral Membrane Protein ◽

In Vitro Translation ◽

Translation System ◽

Transmembrane Topology ◽

Amino Terminal ◽

Hydrophilic Region ◽

Kdel Receptor

The mammalian KDEL receptor is an integral membrane protein with seven hydrophobic regions. Fusion proteins comprising a 37-kDa N-glycosylation reporter fused downstream of amino-terminal fragments of the KDEL receptor with varying numbers of hydrophobic regions were synthesized in an in vitro translation system containing canine pancreatic microsomes. The luminal or cytosolic orientation of the reporter, and hence of the hydrophilic region to which it is fused, was inferred from the presence or absence of glycosylation, which occurs only in the lumen of the microsomes. The cytosolic orientation of the N and C termini was also confirmed immunocytochemically. Our results suggest that the KDEL receptor is inserted into the membrane with only six transmembrane domains and that both the amino and carboxy termini are located in the cytoplasm.

Download Full-text

The Role of Intracellular Membranes in Protein Processing

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100099052 ◽

1981 ◽

Vol 39 ◽

pp. 514-515

Author(s):

Dennis Shields ◽

Thomas G. Warren ◽

Sara E. Roth ◽

Reza F. Green

Keyword(s):

Endoplasmic Reticulum ◽

Signal Peptide ◽

Messenger Rna ◽

Polypeptide Chain ◽

Secretory Protein ◽

Free Protein ◽

Amino Terminal ◽

Free Translation ◽

Microsomal Membranes

Most polypeptides destined for secretion are synthesized on polyribosomes bound to the membrane of the endoplasmic reticulum (E.R.), in contrast, cytosolic proteins are made on free ribosomes. When the messenger RNA (mRNA) for a secretory protein is translated in a cell-free protein synthesizing system, the product is usually larger than the mature protein by about 3,000 daltons. Numerous studies have demonstrated that the higher molecular weight of the cell-free translation product can be attributed to an amino terminal extension of about 20-30 amino acids termed the “signal peptide”. This signal peptide is thought to mediate binding of ribosomes bearing the nascent polypeptide chain to the membrane of the endoplasmic reticulum. Upon interaction with the E.R., the polypeptide chain is translocated across the membrane usually resulting in proteolytic removal of the signal peptide and segregation of the “processed” polypeptide into the ER. cisternae. This series of reactions can be followed in vitro by supplementing the cell-free protein synthesizing system with heterologous microsomal membranes which have been stripped of their endogenous ribosomes.

Download Full-text

Transmembrane topology of the mammalian KDEL receptor

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6435-6441.1993 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6435-6441

Author(s):

P Singh ◽

B L Tang ◽

S H Wong ◽

W Hong

Keyword(s):

Membrane Protein ◽

Fusion Proteins ◽

Integral Membrane Protein ◽

In Vitro Translation ◽

Translation System ◽

Transmembrane Topology ◽

Amino Terminal ◽

Hydrophilic Region ◽

Kdel Receptor

Download Full-text

Fibrillin assembly: dimer formation mediated by amino-terminal sequences

Journal of Cell Science ◽

10.1242/jcs.112.20.3549 ◽

1999 ◽

Vol 112 (20) ◽

pp. 3549-3558 ◽

Cited By ~ 1

Author(s):

J.L. Ashworth ◽

V. Kelly ◽

R. Wilson ◽

C.A. Shuttleworth ◽

C.M. Kielty

Keyword(s):

Proteinase K ◽

In Vitro Translation ◽

Translation System ◽

Dimer Formation ◽

Amino Terminal ◽

Reducing Conditions ◽

Sds Page ◽

Fibrillin 1 ◽

The Stability

We have investigated recombinant fibrillin-1 (profib-1) and fibrillin-2 (glyfib-2) molecules encoding the proline- or glycine-rich regions with flanking domains (exons 9–11), in order to establish whether these sequences might mediate specific molecular recognition events important in fibrillin assembly. Our data demonstrate that both recombinant molecules can form extracellular dimers, but highlight subtle differences in the stability of these dimers. Following expression in COS-1 cells, SDS-PAGE analysis showed that glyfib-2 was present intracellularly as monomers, and extracellularly as monomers and disulphide-bonded dimers. Size fractionation in native non-reducing conditions prior to SDS-PAGE analysis highlighted that glyfib-2 also formed non-covalent associations. In contrast, profib-1 appeared monomeric in cells and medium. Using an in vitro translation system supplemented with semipermeabilised HT1080 cells together with chemical crosslinking, dimers of the fibrillin-1 and fibrillin-2 molecules were detected. Dimerisation was not cell-dependent since molecules translated in the absence of cells dimerised, and was not an intracellular event as judged by proteinase K digestions. A crosslinking and coimmunoprecipitation strategy provided a means of investigating whether molecular chaperones might be involved in preventing dimerisation of translocated molecules. Proteinase K-resistant recombinant molecules associated rapidly with BiP, and thereafter with protein disulphide isomerase and calreticulin. Differences between the two fibrillin isoforms in ability to form stable dimers prompted investigation of the proline- and glycine-rich sequences. Differences in solubility and pI were apparent that may contribute to reduced stability of proline-rich region interactions. These studies suggest that extracellular dimer formation mediated by interactions of the proline- and glycine-rich regions may be a crucial early step in the extracellular assembly of fibrillin into microfibrils.

Download Full-text

Pyrrolizidine Alkaloids Induce Cell Death in Human HepaRG Cells in a Structure-Dependent Manner

International Journal of Molecular Sciences ◽

10.3390/ijms22010202 ◽

2020 ◽

Vol 22 (1) ◽

pp. 202

Author(s):

Josephin Glück ◽

Julia Waizenegger ◽

Albert Braeuning ◽

Stefanie Hessel-Pras

Keyword(s):

Cell Death ◽

Cell Viability ◽

Pyrrolizidine Alkaloids ◽

Defense Mechanism ◽

Hepatoma Cell ◽

Hepatoma Cell Line ◽

Dependent Manner ◽

Human Hepatoma Cell

Pyrrolizidine alkaloids (PAs) are a group of secondary metabolites produced in various plant species as a defense mechanism against herbivores. PAs consist of a necine base, which is esterified with one or two necine acids. Humans are exposed to PAs by consumption of contaminated food. PA intoxication in humans causes acute and chronic hepatotoxicity. It is considered that enzymatic PA toxification in hepatocytes is structure-dependent. In this study, we aimed to elucidate the induction of PA-induced cell death associated with apoptosis activation. Therefore, 22 structurally different PAs were analyzed concerning the disturbance of cell viability in the metabolically competent human hepatoma cell line HepaRG. The chosen PAs represent the main necine base structures and the different esterification types. Open-chained and cyclic heliotridine- and retronecine-type diesters induced strong cytotoxic effects, while treatment of HepaRG with monoesters did not affect cell viability. For more detailed investigation of apoptosis induction, comprising caspase activation and gene expression analysis, 14 PA representatives were selected. The proapoptotic effects were in line with the potency observed in cell viability studies. In vitro data point towards a strong structure–activity relationship whose effectiveness needs to be investigated in vivo and can then be the basis for a structure-associated risk assessment.

Download Full-text