Protein disulfide isomerase acts as a molecular chaperone during the assembly of procollagen.

Protein-disulfide isomerase (PDI) has been shown to be a multifunctional enzyme catalyzing the formation of disulfide bonds, as well as being a component of the enzymes prolyl 4-hydroxylase (P4-H) and microsomal triglyceride transfer protein. It has also been proposed to function as a molecular chaperone during the refolding of denatured proteins in vitro. To investigate the role of this multifunctional protein within a cellular context, we have established a semi-permeabilized cell system that reconstitutes the synthesis, folding, modification, and assembly of procollagen as they would occur in the cell. We demonstrate here that P4-H associates transiently with the triple helical domain during the assembly of procollagen. The release of P4-H from the triple helical domain coincides with assembly into a thermally stable triple helix. However, if triple helix formation is prevented, P4-H remains associated, suggesting a role for this enzyme in preventing aggregation of this domain. We also show that PDI associates independently with the C-propeptide of monomeric procollagen chains prior to trimer formation, indicating a role for this protein in coordinating the assembly of heterotrimeric molecules. This demonstrates that PDI has multiple functions in the folding of the same protein, that is, as a catalyst for disulfide bond formation, as a subunit of P4-H during proline hydroxylation, and independently as a molecular chaperone during chain assembly.

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.

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.

Immunisation of common marmosets with vaccinia virus expressing Epstein-Barr virus (EBV) gp340 and challenge with EBV.

Epstein-Barr virus (EBV) is the cause of infectious mononucleosis and is associated with a variety of life-threatening diseases in humans. Therefore the development of an effective vaccine is an important objective. Many of the initial studies of vaccine efficacy analyse the ability of vaccine preparations to prevent the induction of lymphomas in cottontop tamarins by the B95-8 strain of EBV. We used a vaccinia virus recombinant expressing gp340, vMA1, tested previously in the cotton-top tamarin, to evaluate a common marmoset model in which the challenge virus, M81, resembles more closely the wild-type strains of EBV in the general population than does the standard B95-8 strain. We characterised the M81 strain of EBV with respect to the sequence of its gp340/220 gene and in regard to the presence of a region deleted in B95-8. Replication of the challenge virus in the group vaccinated with vMA1 was decreased when compared to control groups.

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.

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.

The Epstein-Barr virus candidate vaccine antigen gp340/220 is highly conserved between virus types A and B.

Anti-Epstein-Barr Virus (EBV) vaccines are being developed which are based on the gp340/220 membrane antigen (MA) gene products from the B95-8 strain. Some proteins are known to be immunologically quite different between type-A (1) and type-B (2) strains of EBV and therefore from a vaccine point of view it was critical to evaluate the degree of conservation of gp340/220. The complete MA coding sequence was determined for two B-type viruses, AG876 and P3HR-1, for comparison with the A-type B95-8. A variable region within MA was sequenced from several other strains. In addition the other open reading frames within the MA-containing BamHI-L fragment of AG876 were sequenced and compared. The results show that there is a high degree of homology between all strains examined. Although some differences were found within the MA coding sequence the only major site of variation was within the repeat region and no consistent A/B changes were found. Monoclonal antibodies generated against A-type MA and representing six epitope groups along the length of the gp340 molecule were found to recognize B-type gp340, thereby demonstrating functional homology. We conclude that, as a vaccine antigen, B95-8 gp340/220 should be equally effective against both types of EBV.

Detection of EBV DNA in post-nasal space biopsy tissue from asymptomatic EBV-seropositive individuals.

The association between EBV and nasopharyngeal carcinoma (NPC) has been well documented although the precise role of the virus in the genesis of the tumour is not understood. We undertook this study to examine the prevalence of EBV infection in nasopharyngeal tissue obtained from 33 healthy individuals not considered to be at risk of developing NPC. Using polymerase chain amplification (PCR) and in situ hybridization we have identified EBV DNA in 70% (23/33) of the tissues examined. Our data demonstrate that EBV is present at the site of tumour development in the low-risk population and by inference that the virus is also present before the onset of disease in the high-risk group. This survey supports the concept of NPC pathogenesis as a multifactorial process.

The structure of the largest murine neurofilament protein (NF-H) as revealed by cDNA and genomic sequences.

The complete primary structure of the largest mammalian neurofilament component, NF-H, is predicted from mouse cDNA and genomic clones, revealing a protein of molecular weight ca. 115,000. A central filament-forming domain structurally typical of all intermediate filament proteins is present, but anomalies are noted which may place constraints on the mechanism of NF-H assembly into filaments. The COOH-terminal portion of the protein is extremely long (661 amino acids) by comparison to non-neuronal intermediate filament components and has a remarkably monotonous, highly charged composition (Glu and Lys at 20% each). Its most remarkable feature is a tandem repeat of a 6 amino acid sequence containing the motif Lys-Ser-Pro that extends for more than half the length of the COOH-terminus. The Lys-Ser-Pro motif appears 48 times and since it is now known that the serine therein is a target for in vivo kinases, the massive axonal phosphorylation of NF-H is explained. Comparison of mouse and human NF-H reveals that otherwise conserved proteins have been subjected to evolutionary mutation within their multiphosphorylation repeat domains, although the Lys-Ser-Pro motif has been conserved.

The structure and organization of the human heavy neurofilament subunit (NF-H) and the gene encoding it.

Genomic clones for the largest human neurofilament protein (NF-H) were isolated, the intron/exon boundaries mapped and the entire protein-coding regions (exons) sequenced. The predicted protein contains a central region that obeys the structural criteria identified for alpha-helical 'rod' domains typically present in all IF protein components: it is approximately 310 amino acids long, shares amino acid sequence homology with other IF protein rod domains and displays the characteristic heptad repeats of apolar amino acids which facilitate coiled-coil interaction. Nevertheless, anomalies are noted in the structure of the NF-H rod which could explain observations of its poor homopolymeric assembly in vitro. The protein segment on the carboxy-terminal side of the human NF-H rod is uniquely long (greater than 600 amino acids) compared to other IF proteins and is highly charged (greater than 24% Glu, greater than 25% Lys), rich in proline (greater than 12%) and impoverished in cysteine, methionine and aromatic amino acids. Its most remarkable feature is a repetitive sequence that covers more than half its length and includes the sequence motif, Lys-Ser-Pro (KSP) greater than 40 times. Together with the recent identification of the serine in KSP as the main target for NF-directed protein kinases in vivo, this repetitive character explains the massive phosphorylation of the NF-H subunit that can occur in axons. The human NF-H gene has three introns, two of which interrupt the protein-coding sequence at identical points to introns in the genes for the two smaller NF proteins, NF-M and NF-L.(ABSTRACT TRUNCATED AT 250 WORDS)

mRNA levels of all three neurofilament proteins decline following nerve transection.

The control of neurofilament (NF) protein gene expression was studied by determining and comparing the levels of mRNA to the heavy (NF-H), mid-sized (NF-M) and light (NF-L) NF protein subunits in rat dorsal root ganglia (DRG) following sciatic nerve transection. mRNA to NF-H (4.5 kb), to NF-M (3.4 kb) and to NF-L (2.5 and 4.0 kb) were identified in Northern blots and quantitated in dot blot analyses, using specific cDNA probes for each NF protein. Following transection and continuing for at least 28 days. The early and co-terminal fall in mRNAs suggests that the 3 NF genes are regulated by common factor(s) and that the function of these factor(s) is influenced by the state of axonal continuity with the target organ.

Nucleotide sequence of the Bunyamwera virus M RNA segment: conservation of structural features in the Bunyavirus glycoprotein gene product.

The complete nucleotide sequence of the Bunyamwera virus M RNA segment was determined from four overlapping cDNA clones and by primer extension. The RNA segment is 4458 bases in length, and encodes a single gene product in the viral complementary RNA. The predicted protein is 1433 amino acids long (mol wt 162,065), contains four potential glycosylation sites, and is relatively cysteine rich. It is presumed that the three proteins G1, G2, and NSM which have been mapped to the M RNA segment are synthesized as a precursor polyprotein which is subsequently proteolytically cleaved. A putative hydrophobic signal sequence at the amino terminus and a hydrophobic anchor sequence at the carboxy terminus of the predicted protein have been identified, in addition to internal regions of hydrophobicity of unknown function. The nucleotide and amino acid sequences of the Bunyamwera virus M segment have been compared with those of the snowshoe hare virus M segment (Y. Eshita and D. H. L. Bishop, Virology 137, 227-240, 1984). Common features include the overall architecture of the RNAs, single cysteine-rich primary gene products, and conservation of hydrophobic domains in the gene products. When aligned the amino acid sequences are 43% homologous, and 66 of 70 cysteine residues can be matched. The evolutionary significance of these findings is discussed.

[Genome analysis of bunyavirus recombinants by dot hybridization]. / Analiz genoma rekombinantov bun'iavirusov metodom tochechnoi gibridizatsii.

A simple method of the so-called pinpoint hybridization for the detection of genome RNAs and individual fragments of genome of the Bunyamwera family viruses is described. The method established linking of 2 out of 3 fragments of genome RNA of different members of the family.

Genome subunit reassortment among Bunyaviruses analysed by dot hybridization using molecularly cloned complementary DNA probes.

A simple and rapid procedure for determining the genotypes of viruses has been applied to analysis of genome subunit reassortment in heterologous crosses of Batai virus, Bunyamwera virus, and Maguari virus, three members of the Bunyamwera serogroup of bunyaviruses. The procedure for determining genotype made use of specific molecular probes to identify the parental origin of the L and M RNA subunits. Complementary DNA copies of the three RNA segments of Bunyamwera virus were prepared by reverse transcription using synthetic oligonucleotide primers for first and second strand synthesis. The cDNA transcripts were inserted into a pBR322 vector and gene-specific probes prepared from nick-translated plasmid DNA. L and M gene-specific probes were identified which could unequivocally discriminate Bunyamwera virus genome subunits in a dot-hybridization test using cytoplasmic RNA extracts immobilised on nitrocellulose filters. None of the S gene-specific probes were sufficiently discriminatory for use in this test. Instead the parental origin of the S RNA subunit was inferred from the electrophoretic mobility of the virion N protein. It was observed that reassortment did not occur at random in heterologous crosses of is mutants of the three viruses, and only the M RNA subunit appeared to segregate freely. However, unrestricted reassortment was observed when recombinant viruses with nonhomologous subunit combinations were used as the parental viruses. It was concluded, therefore, that restriction was mediated at the gene product level and that nonrandom reassortment was not due to incompatibility of genome subunits.

Effects of chronic treatment of intact and hypophysectomized rats with thyroid-stimulating hormone on the metabolism of [35S]methimazole and [35S]propylthiouracil.

Chronic treatment of intact rats with various doses of TSH increased the thyroidal 35S accumulation after single doses of [35S]methimazole (MMI) and [35S]propylthiouracil (PTU). However, no effect on the intrathyroidal breakdown of the drugs was observed. Thus absolute thyroidal levels of unmetabolized MMI and PTU were increased by factors of up to 2 and 3, respectively, compared to the control groups. Simultaneous decreases in the levels of thyroidal total iodine were observed. Hypophysectomized rats showed a marked inhibition of both thyroidal accumulation and oxidation of [35S]-MMI but TSH treatment of hypophysectomized rats restored the accumulation and oxidation to sham-operated and control group levels. The results show that in rats TSH has an important role in the control of thyroidal levels of antithyroid drugs currently used in the treatment of hyperthyroidism.

Role of TSH in the changes in thyroidal metabolism of [35S]methimazole in phenobarbital and thyroxine-treated rats.

The effect of phenobarbital (PB) and/or thyroxine on the thyroidal accumulation and oxidation of [35S]methimazole (MMI) and serum TSH levels was studied in rats. PB treatment increased the accumulation of MMI and the serum TSH levels, but concurrent administration of T4 reversed these effects. It was concluded that increased TSH secretion in PB-treated animals was likely to be the major mechanism involved in the increased MMI accumulation. PB also increased the intrathyroidal oxidation of MMI to sulphate. However, in contrast to the PB effect on accumulation, concurrent T4 administration only partially reversed the effect on oxidation. The results suggested that the increased oxidation of MMI in PB-treated animals was due to a direct effect of PB or possibly a combination of this direct effect and the indirect TSH effect. Possible mechanisms postulated for a direct effect were thyroidal microsomal enzyme induction and/or changes in thyroidal protein binding of MMI.