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N J Bulleid

Publications and source records attributed to N J Bulleid.

At least 37 records · Page 2Linked to original sources
PubMed1998

A cautionary note when using pepsin as a probe for the formation of a collagen triple helix.

The ability of the collagen triple helix to resist digestion with proteases has been used as a conformational probe to ascertain whether a collagenous molecule has formed a correctly aligned helix during biosynthesis or during refolding of the protein in vitro. During our studies into the synthesis and folding of a variety of engineered procollagen polypeptide chains, we noted that resistance to digestion with proteases, in particular pepsin, could be misleading and does not necessarily indicate the formation of a collagen triple helix. These results clearly show that resistance to pepsin digestion alone should not be used to indicate correct folding and that preferably an alternative assay should be used to unequivocally demonstrate the formation of a correctly aligned triple helix.

Animals↗
PubMed1997

The C-propeptide domain of procollagen can be replaced with a transmembrane domain without affecting trimer formation or collagen triple helix folding during biosynthesis.

The folding and assembly of procollagen occurs within the cell through a series of discrete steps leading to the formation of a stable trimer consisting of three distinct domains: the N-propeptide, the C-propeptide and the collagen triple helix flanked at either end by short telopeptides. We have established a semi-permeabilized cell system which allows us to study the initial stages in the folding and assembly of procollagen as they would occur in the intact cell. By studying the folding and assembly of the C-propeptide domain in isolation, and a procollagen molecule which lacks the C-propeptide, we have shown that this domain directs the initial association event and is required to allow triple helix formation. However, the essential function of this domain does not include triple helix nucleation or alignment, since this can occur when the C-propeptide is substituted with a single transmembrane domain. Also the telopeptide region is not involved in triple helix nucleation; however, a minimum of two hydroxyproline-containing Gly-X-Y triplets at the C-terminal end of the triple helix are required for nucleation to occur. Thus, the C-propeptide is required solely to ensure association of the monomeric chains; once these are brought together, the triple helix is able to nucleate and fold to form a correctly aligned triple helix.

Amino Acid Sequence↗
PubMed1997

Calnexin and calreticulin bind to enzymically active tissue-type plasminogen activator during biosynthesis and are not required for folding to the native conformation.

The roles of the endoplasmic-reticulum lectins calnexin and calreticulin in the folding of tissue-type plasminogen activator (tPA) have been investigated using an in vitro translation system that reconstitutes these processes as they would occur in the intact cell. Using co-immunoprecipitation of newly synthesized tPA with antibodies to calnexin and calreticulin, it was demonstrated that the interaction of tPA with both lectins was dependent upon tPA glycosylation and glucosidase trimming. When tPA was synthesized in the presence of semi-permeabilized cells under conditions preventing complex formation with calnexin and calreticulin, the translation product had a specific plasminogenolytic activity identical with that when synthesized under conditions permitting interactions with both lectins. Furthermore, complexes of tPA bound to calnexin and calreticulin were shown to be enzymically active. These results demonstrate that calnexin and calreticulin can form a stable interaction with correctly folded tPA; however, such interactions are not required for the synthesis of enzymically active tPA.

Calcium-Binding Proteins↗
PubMed1997

Identification of the molecular recognition sequence which determines the type-specific assembly of procollagen.

A key question relating to procollagen biosynthesis is the way in which closely related procollagen chains discriminate between each other to assemble in a type-specific manner. Intracellular assembly of procollagen occurs via an initial interaction between the C-propeptides followed by vectorial propagation of the triple-helical domain in the C to N direction. Recognition signals within the C-propeptides must, therefore, determine the selective association of individual procollagen chains. We have used the pro alpha1 chain of type III procollagen [pro alpha1(III)] and the pro alpha2 chain of type I procollagen [pro alpha2(I)] as examples of procollagen chains that are either capable or incapable of self-assembly. When we exchanged the C-propeptides of the pro alpha1(III) chain and the pro alpha(I) chain we demonstrated that this domain is both necessary and sufficient to direct the assembly of homotrimers with correctly aligned triple-helices. To identify the sequences within this domain that determine selective association we constructed a series of chimeric procollagen chains in which we exchanged specific sequences from the pro alpha1(III) C-propeptide with the corresponding region within the pro alpha2(I) C-propeptide (and vice versa) and assayed for the ability of these molecules to form homotrimers. Using this approach we have identified a discontinuous sequence of 15 amino acids which directs procollagen self-association. By exchanging this sequence between different procollagen chains we can direct chain association and, potentially, assemble molecules with defined chain compositions.

Amino Acid Sequence↗
PubMed1997

Interaction of the thiol-dependent reductase ERp57 with nascent glycoproteins.

Calnexin and calreticulin interact specifically with newly synthesized glycoproteins in the endoplasmic reticulum (ER) and function as molecular chaperones. The carbohydrate-specific interactions between ER components and glycoproteins synthesized in isolated canine pancreatic microsomes were analyzed using a cross-linking approach. A carbohydrate-dependent interaction between newly synthesized glycoproteins, the thiol-dependent reductase ERp57, and either calnexin or calreticulin was identified. The interaction between ERp57 and the newly synthesized glycoproteins required trimming of the N-linked oligosaccharide side chain. Thus, it is likely that ERp57 functions as part of the glycoprotein-specific quality control machinery operating in the lumen of the ER.

Animals↗
PubMed1996

Intracellular dissociation and reassembly of prolyl 4-hydroxylase:the alpha-subunits associated with the immunoglobulin-heavy-chain binding protein (BiP) allowing reassembly with the beta-subunit.

Prolyl 4-hydroxylase (P4-H) consists of two distinct polypeptides; the catalytically more important alpha-subunit and the beta-subunit, which is identical to the multifunctional enzyme protein disulphide isomerase. The enzyme appears to be assembled in vivo into an alpha 2 beta 2 tetramer from newly synthesized alpha-subunits associating with an endogenous pool of beta-subunits. Using a cell-free system, we have shown previously that enzyme assembly is redox-dependent and that assembled alpha-subunits are intramolecularly disulphide-bonded [John and Bulleid (1994) Biochemistry 33, 14018-14025]. Here we have studied this assembly process within intact cells by expressing both subunits in COS-1 cells. Newly synthesized alpha-subunits were shown to assemble with the beta-subunit, to form insoluble aggregates, or to remain soluble but not associate with the beta-subunit. Treatment of cells with dithiothreitol (DTT) led to dissociation of P4-H into subunits and on removal of DTT the enzyme reassembled. This reassembly was ATP-dependent, suggesting an interaction with an ATP-dependent chaperone. This was confirmed when immunoglobulin-heavy-chain binding protein (BiP) and alpha-subunits were co-immunoprecipitated with antibodies against the alpha-subunit and BiP, respectively. These results indicate that unassembled alpha-subunits are maintained in an assembly-competent form by interacting with the molecular chaperone BiP.

Adenosine Triphosphate↗
PubMed1996

Type-III procollagen assembly in semi-intact cells: chain association, nucleation and triple-helix folding do not require formation of inter-chain disulphide bonds but triple-helix nucleation does require hydroxylation.

Procollagen assembly is initiated within the endoplasmic reticulum by three alpha-chains associating via their C-propeptides (C-terminal propeptides). To study the requirements for the association of procollagen monomers at synthesis we have reconstituted the initial stages in the folding, assembly and modification of procollagen using semi-permeabilized cells. By translating a type-III procollagen "mini-gene' which lacks part of the triple-helical domain, we demonstrate that these cells efficiently carry out the assembly of hydroxylated, triple-helical, procollagen trimers and allow the identification of specific disulphide-bonded intermediates in the folding pathway. Mutant chains, which lack the ability to form inter-chain disulphide bonds within the C-propeptide, were still able to assemble within this system. Furthermore, characterization of the trimeric molecules formed suggested that inter-chain disulphide bonds had formed within the C-telopeptide (C-terminal telopeptide). However, when hydroxylation of prolyl and lysyl residues was inhibited no inter-chain disulphide bonds were formed in the C-telopeptide, indicating that hydroxylation is required for the initial nucleation of the triple-helical domain. Mutant chains which lacked the ability to form inter-chain disulphide bonds within the C-propeptide or the C-telopeptide could still assemble to form trimeric triple-helical molecules linked by inter-chain disulphide bonds within the N-propeptide (N-terminal propeptide). These results indicate that inter-chain disulphide bond formation within the C-propeptide or the C-telopeptide is not required for chain association and triple-helix formation.

Amino Acid Sequence↗
PubMed1995

Folding of human intestinal lactase-phlorizin hydrolase.

The folding of human intestinal prolactase-phlorizin hydrolase (pro-LPH) has been analyzed in a cell-free transcription/translation system. In the presence of the thiol oxidant GSSG, disulfide bond formation in pro-LPH can be promoted concomitant with the binding of the molecule to a conformation-specific monoclonal anti-LPH antibody. Under these conditions, pro-LPH does not bind to the molecular chaperone BiP. In the absence of GSSG, on the other hand, pro-LPH does not bind to the monoclonal anti-LPH antibody, but can be immunoprecipitated with a polyclonal antibody that is directed against a denatured form of the enzyme. In this case, interaction of pro-LPH with immunoglobulin heavy chain binding protein can be discerned. The results demonstrate the existence of intramolecular disulfide bonds that are essential for the promotion of pro-LPH to a native conformation. Furthermore, BiP is involved in the folding events of pro-LPH.

Adenosine Triphosphate↗
PubMed1995

The translocation, folding, assembly and redox-dependent degradation of secretory and membrane proteins in semi-permeabilized mammalian cells.

We describe here a semi-permeabilized cell-system which reconstitutes the efficient synthesis, translocation, folding, assembly and degradation of membrane and secretory proteins. Cells grown in culture were treated with the detergent digitonin which selectively permeabilized the plasma membrane leaving the cellular organelles, such as the endoplasmic reticulum (ER) and trans-Golgi network intact. These permeabilized cells were added to an in vitro translation system, either wheatgerm or reticulocyte lysate, supplemented with RNA coding for either membrane or secretory proteins. Efficient translocation and modification of proteins by these cells was demonstrated by protease protection, photocross-linking of nascent chains to components of the translocation apparatus and by post-translational modifications such as glycosylation or hydroxylation. A comparison was made between the ability of semi-permeabilized cells and microsomal vesicles to fold and assemble proteins. The results show that the intact ER within these cells can assemble proteins much more efficiently than vesicularized ER. Furthermore, the semi-permeabilized cells carried out the redox-dependent degradation of tissue-type plasminogen activator. This system has all the advantages of conventional cell-free systems, including speed and, importantly, the ability to manipulate the components of the assay, while retaining intracellular organelles and, therefore, allowing cellular processes to occur as they would in the intact cell.

Animals↗
PubMed1995

Intracellular folding of tissue-type plasminogen activator. Effects of disulfide bond formation on N-linked glycosylation and secretion.

The addition of N-linked core oligosaccharides to membrane and secretory glycoproteins occurs co-translationally at asparagine residues in the tripeptide sequon Asn-Xaa-Ser/Thr soon after translocation of the nascent polypeptide into the lumen of the endoplasmic reticulum. However, the presence of the sequon does not automatically ensure core glycosylation, as many proteins contain sequons that remain either unglycosylated or glycosylated to a variable extent. To investigate whether intracellular protein folding can influence sequon utilization, we have expressed tissue-type plasminogen activator (t-PA) in cell culture in the presence of mild concentrations of the reducing agent dithiothreitol to prevent co-translational disulfide bond formation in the endoplasmic reticulum. We show that conditions that prevent disulfide bond formation lead to complete glycosylation of a sequon that otherwise undergoes variable glycosylation in untreated cells. This demonstrated that folding and disulfide bond formation of t-PA determines its extent of core N-linked glycosylation. When dithiothreitol was removed from the cells, the reduced and overglycosylated t-PA formed disulfide bonds, folded, and was secreted. We also show t-PA present within cells is more susceptible to reduction with low concentrations of dithiothreitol than secreted t-PA.

Amino Acid Sequence↗
PubMed1994

Prolyl 4-hydroxylase: defective assembly of alpha-subunit mutants indicates that assembled alpha-subunits are intramolecularly disulfide bonded.

The vital hydroxylation of peptidyl proline residues in collagens and protein with collagen-like amino acid sequences is catalyzed by the tetrameric enzyme prolyl 4-hydroxylase (P4-H). We have previously detailed [John et al. (1993) EMBO J. 12, 1587-1595] the redox-dependent assembly of the catalytically important alpha-subunit (64 kDa) in a cell-free system containing endogenous beta-subunits (PDI, 60 kDa). To identify the origin of this redox-dependent assembly, we have now shown directly by an electrophoretic mobility shift assay that the assembled wild-type protein possesses at least one intramolecular disulfide bond. We also analyzed five alpha-subunit mutants that have single Cys to Ser mutations in one of the five Cys residues present in the wild-type protein and found that (i) subunits mutated at Cys150 or Cys511 formed intramolecular disulfide bonds, whereas subunits mutated at Cys276, Cys293, or Cys486 did not, (ii) mutation of Cys276, Cys293, or Cys486 led to a large reduction in alpha-beta complex formation, (iii) subunits mutated at Cys276, Cys293, Cys486, or Cys511 were recognized by an antiserum raised against an alpha-subunit C-terminal peptide which failed to recognize the assembled wild-type subunit or the assembled subunit mutated at Cys150, and (iv) the assembled complexes fractionated in a similar position to the purified protein on sucrose gradients whereas the assembly-defective mutants formed higher molecular weight aggregates or complexes with other proteins. On the basis of these results, we propose that P4-H alpha-subunits possess an intramolecular disulfide bond between Cys276 and Cys293 that is essential for alpha-beta complex formation.

Animals↗
PubMed1994

The role of cysteine residues in the folding and association of the COOH-terminal propeptide of types I and III procollagen.

Procollagen chains assemble in a type-specific manner forming either homo- or heterotrimers. The molecular mechanisms underlying procollagen chain selectivity are unknown, although it is thought that the C-propeptide (COOH-terminal propeptide) is responsible for directing chain recognition and assembly. To define the processes involved in chain selection we reconstituted the initial stages of procollagen folding and assembly in a cell-free system. Using human pro-alpha 1(III) and pro-alpha 2(I) chains as prototypes of chains that are either capable or incapable of forming homotrimeric molecules, respectively, we constructed two minigenes (p alpha 1(III) delta 1 and p alpha 2(I) delta 1) that lacked most of the triple helical domains. The minigenes were transcribed in vitro and translated in a rabbit reticulocyte lysate supplemented with microsomal membranes under conditions that favored disulfide bond formation. Both pro-alpha 1(III) delta 1 and pro-alpha 2(I) delta 1 chains formed intrachain disulfide bonds within the C-propeptide. However, only pro-alpha 1(III) delta 1 chains were able to self-associate forming homotrimers stabilized by interchain disulfide bonds. The C-propeptide of the pro-alpha 1(III) chain contains 8 cysteine residues (Cys-1-8). We used a site-directed mutagenesis to investigate the role of specific cysteine residues in trimer formation and found that substitution of serine for Cys-1, Cys-2, Cys-3, and Cys-4 prevented interchain disulfide bonding and trimerization. Furthermore, mutations in Cys-1 and Cys-4 also prevented intrachain disulfide bond formation within the C-propeptide. The C-propeptide of the pro-alpha 2(I) chain contains only 7 cysteine residues, lacking cysteine at position 2. Substitution of the existing Ser residue with Cys did not produce a homotrimeric phenotype, indicating that additional recognition signals are required to determine chain selection.

Animals↗
PubMed1993

Reconstitution of the folding pathway of collagen in a cell-free system: formation of correctly aligned and hydroxylated triple helices.

We describe here a cell-free system which will carry out the initial stages in the synthesis, post-translational modification and assembly of type-X collagen. The mRNA coding for bovine type-X collagen was synthesized in vitro and translated in a rabbit reticulocyte lysate to yield a protein that was collagenase sensitive and could be immunoprecipitated with antibodies raised to purified avian type-X collagen. When type-X collagen was synthesized in the absence of added microsomes or in the presence of canine pancreas microsomes, the translation products showed partial resistance to digestion with pepsin but were completely degraded with a mixture of chymotrypsin and trypsin, suggesting that only incorrectly aligned non-native collagen molecules were synthesized under these conditions. When the protein was synthesized in the presence of microsomes derived from avian fibroblasts or a human fibrosarcoma cell line, the translocated product migrated as a diffuse band characteristic of hydroxylated collagen. The synthesized polypeptides were also resistant to both pepsin and trypsin/chymotrypsin digestion, demonstrating the formation of correctly aligned native collagen. Furthermore, the collagen polypeptides assembled into higher-order structures, possibly trimers, which were stabilized by interchain disulphide bonds. The collagen helix synthesized in vitro had a melting temperature of 41 degrees C which is comparable with the protein synthesized in vivo, further demonstrating that the polypeptides were hydroxylated and that the triple helix formed was correctly aligned.

Animals↗
PubMed1993

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.

Animals↗
PubMed1992

Cell-free synthesis of enzymically active tissue-type plasminogen activator. Protein folding determines the extent of N-linked glycosylation.

Tissue-type plasminogen activator (t-PA) is synthesized in mammalian cells as a mixture of two forms that differ in their extent of N-linked glycosylation. We have investigated the mechanism underlying this variation in glycosylation, using a cell-free system that consists of a rabbit reticulocyte lysate optimized for the formation of disulphide bonds and supplemented with dog pancreas microsomal membranes. Molecules of human t-PA synthesized in vitro are enzymically active and responsive to natural activators and inhibitors, and are glycosylated in a pattern identical with that of the protein produced in vivo. This demonstrates that t-PA synthesized in vitro folds into the same conformation as the protein synthesized in vivo. We show that the extent of glycosylation of individual t-PA molecules is dependent on the state of folding of the polypeptide chain, since the probability of addition of an oligosaccharide side chain at Asn-184 is decreased under conditions that promote the formation of enzymically active molecules. This variation in glycosylation is independent of the rate of protein synthesis.

Amino Acid Sequence↗
PubMed1990

Source of heterogeneity in secreted interferon-gamma. A study on products of translation in vitro.

A cDNA clone coding for human interferon-gamma (IFN-gamma) was subcloned into a transcription-translation vector. When the mRNA transcribed in vitro was added to a rabbit reticulocyte-lysate system, two polypeptides were synthesized: one corresponding in Mr to pre-IFN-gamma (18,000) and one with a lower Mr (12,000) which corresponds to a polypeptide arising from incorrect initiation of translation. When microsomal vesicles isolated from dog pancreas or Chinese-hamster ovary (CHO) cells were added to the translation system, translocation of the pre-IFN-gamma occurred, as judged by protection from exogenous proteinases. The resultant changes in the Mr of the translation products were indicative of signal-peptide cleavage and heterogeneous core glycosylation. When translation products were treated with N-glycanase, the higher-Mr products were no longer observed, consistent with removal of all oligosaccharide side chains, leaving a single core polypeptide. Glycosylation of the synthesized protein yielded both singly and doubly glycosylated products compatible with the glycosylation variants seen in secreted IFN-gamma. Quantitative differences were seen in the relative amounts of singly and doubly glycosylated products synthesized by dog pancreatic compared with CHO-derived microsomes. These data indicate that the relative amounts of IFN-gamma glycosylation variants are determined at an early stage in protein synthesis and that product variants may occur when IFN-gamma is expressed in cells derived from different tissues.

Animals↗