ERp57 functions as a subunit of specific complexes formed with the ER lectins calreticulin and calnexin.

ERp57 is a lumenal protein of the endoplasmic reticulum (ER) and a member of the protein disulfide isomerase (PDI) family. In contrast to archetypal PDI, ERp57 interacts specifically with newly synthesized glycoproteins. In this study we demonstrate that ERp57 forms discrete complexes with the ER lectins, calnexin and calreticulin. Specific ERp57/calreticulin complexes exist in canine pancreatic microsomes, as demonstrated by SDS-PAGE after cross-linking, and by native electrophoresis in the absence of cross-linking. After in vitro translation and import into microsomes, radiolabeled ERp57 can be cross-linked to endogenous calreticulin and calnexin while radiolabeled PDI cannot. Likewise, radiolabeled calreticulin is cross-linked to endogenous ERp57 but not PDI. Similar results were obtained in Lec23 cells, which lack the glucosidase I necessary to produce glycoprotein substrates capable of binding to calnexin and calreticulin. This observation indicates that ERp57 interacts with both of the ER lectins in the absence of their glycoprotein substrate. This result was confirmed by a specific interaction between in vitro synthesized calreticulin and ERp57 prepared in solution in the absence of other ER components. We conclude that ERp57 forms complexes with both calnexin and calreticulin and propose that it is these complexes that can specifically modulate glycoprotein folding within the ER lumen.

Evaluation of cardiovascular risk-lowering health benefits accruing from laboratory-based, community-based and exercise-referral exercise programmes.

BACKGROUND: To evaluate the ability of community-based exercise programmes to facilitate public participation in exercise and hence improved cardiovascular health, we assessed the respective impacts of: a continuously monitored exercise programme based within our university (study 1); a Valleys Regional Park-facilitated community-based outdoor exercise programme (study 2); a Wales National Exercise Referral Scheme-delivered exercise-referral programme (study 3). METHODS: Biomolecular (monocytic PPARγ target gene expression), vascular haemodynamic (central/peripheral blood pressure, arterial stiffness), clinical (insulin sensitivity, blood lipids) and anthropometric (body mass index, waist circumference, heart rate) parameters were investigated using RT-PCR, applanation tonometry, chemical analysis and standard anthropometric techniques. RESULTS: In studies 1-3, 22/28, 32/65 and 11/14 participants adhered to their respective exercise programmes, and underwent significant increases in physical activity levels. Importantly, beneficial effects similar to those seen in our previous studies (eg, modulations in expression of monocytic PPARγ target genes, decreases in blood pressure/arterial stiffness, improvements in blood lipids/insulin sensitivity) were observed (albeit to slightly differing extents) only in participants who adhered to their respective exercise programmes. While study 1 achieved more intense exercise and more pronounced beneficial effects, significant cardiovascular risk-lowering health benefits related to biomolecular markers, blood pressure, arterial stiffness and blood lipids were achieved via community/referral-based delivery modes in studies 2 and 3. CONCLUSIONS: Because cardiovascular health benefits were observed in all 3 studies, we conclude that the majority of benefits previously reported in laboratory-based studies can also be achieved in community-based/exercise-referral settings. These findings may be of use in guiding policymakers with regard to introduction and/or continued implementation of community/referral-based exercise programmes.

Role of calreticulin in regulating intracellular Ca2+ storage and capacitative Ca2+ entry in HeLa cells.

Calreticulin is a Ca2+ binding protein located primarily in the endoplasmic reticulum (ER) lumen of non-excitable cells, where it is considered to be involved mainly in Ca2+ storage and buffering. However, there is increasing evidence to implicate the protein in other facets of Ca2+ signalling. In this study, we sought to establish more clearly the role of the protein in the regulation of intracellular Ca2+ signalling. Generating HeLa cells stably transfected with GFP-tagged calreticulin (GFPCRT) allowed to us to select cells by FACS in which calreticulin was expressed at ten times its endogenous levels. Using transiently expressed aequorin as a Ca2+ indicator in these cells, we investigated the role of calreticulin in intracellular Ca2+ storage, IP3-mediated Ca2+ release, and capacitative Ca2+ entry. The data showed that the capacity of the ionomycin-sensitive Ca2+ store was doubled in over-expressing cells, indicating that although calreticulin has a role in Ca2+ storage within the lumen, other lumenal proteins are also likely to be involved. No difference was observed in the release of Ca2+ from the IP3-sensitive store in response to prolonged single stimulation with histamine in the absence of extracellular Ca2+, but use of short, sequential pulses of histamine and ATP revealed that calreticulin may exert an effect upon IP3-mediated Ca2+ release. Two different experimental approaches indicated that calreticulin participates in the regulation of capacitative Ca2+ entry. In the presence of extracellular Ca2+, the histamine-generated cytosolic Ca2+ signal was significantly lower in GFPCRT cells than those in control cells. Induction of capacitative Ca2+ entry by complete emptying of the store using the SERCA pump inhibitor, cyclopiazonic acid also showed that the influx component was significantly reduced in the GFPCRT cells. Use of ER-targeted apoaequorin acting as a luciferase demonstrated that the resting ER free [Ca2+] in the GFPCRT cells was lower than that in control cells. These data implicate calreticulin in the control of IP3-mediated Ca2+ release and capacitative Ca2+ entry, which may involve direct interaction with Ca2+ signalling components or control of ER free [Ca2+].

Development of a luminescent bioassay for thyroid stimulating antibodies.

The hyperthyroidism of Graves Disease (GD) is due to thyroid stimulating antibodies (TSAb) which are thyrotropin (TSH) agonists. They are detected routinely by measuring their ability to inhibit TSH binding to the receptor (TBII), which does not reflect their true biological activity. Current bioassays which measure cAMP by RIA, are not suitable for routine use. We have developed a luminescent bioassay for TSAb, by introducing a cAMP responsive luciferase construct into CHO cells stably expressing the human TSH receptor (TSHR). Clone lulul displays dose dependent TSH response detectable from 10 microU/ml and maximal at 10 mU/ml when a >25 fold increase in light output is obtained. 34 euthyroid sera were tested to determine a reference range, with values >1.5 relative light units (R.L.U.) being considered positive. An international TSAb standard responded in a dose dependent manner with 10 mIU/ml giving an R.L.U. of >10. The assay was adapted to a 96 well format for automatic readout and 100 treated GD samples (50 TBII negative and 50 TBII positive) were tested, 73% being positive. In contrast only 4% of 79 control sera from individuals with Hashimoto's, non-thyroid autoimmunity or multinodular goitre produced R.L.U. >1.5. When 44 of the GD sera were compared in a traditional salt-free bioassay, 61% were positive compared with 75% in the new luminescent assay. In conclusion, we have developed a luminescent bioassay for TSAb, using unfractionated serum which is capable of high throughput suitable for routine use.

Nuclear localisation of calreticulin in vivo is enhanced by its interaction with glucocorticoid receptors.

The multi-functional protein calreticulin (CRT) is normally found within the lumen of the endoplasmic reticulum (ER). However, some of its proposed functions require it to be located within the nucleus, where its presence is contentious. We have investigated this in live COS7, HeLa and LM(TK-) cells using green fluorescent protein (GFP)-fusion proteins. GFP-CRT, and GFP, with an ER signal peptide and a KDEL sequence (ER-GFP), were localised to the ER. In addition, GFP-CRT was located in the nucleus of all the cell types at low levels. The higher levels of nuclear fluorescence in LM(TK-) and HeLa cells suggested that glucocorticoid receptors might enhance nuclear localisation of calreticulin. Dexamethasone treatment of LM(TK-) cells doubled the amount of nuclear GFP-CRT, but did not affect the localisation of a GFP-CRT fusion in which the glucocorticoid receptor-binding N-domain of calreticulin had been deleted. Thus, despite ER targeting and retention signals, calreticulin is also located within the nucleus where its presence increases due to its interaction with glucocorticoid receptors.

Overexpression of calreticulin fails to abolish its induction by perturbation of normal ER function.

Along with other endoplasmic reticulum (ER) Ca2+-binding proteins, notably the glucose-response proteins grp78 and grp94, expression of calreticulin is induced in response to perturbation of normal ER function. It has yet to be clearly defined how this stress is signaled from the ER to the nucleus in mammalian cells, particularly with regard to its initiation. Using a GFP-calreticulin fusion protein, we have generated and selected stably transfected HeLa cells that overexpress calreticulin to investigate whether the protein might be involved in signaling its own induction. Basal levels of endogenous calreticulin mRNA and protein were unaffected in these cells, indicating that overexpression alone does not induce a stress response. ER stress induced calreticulin expression in response to either thapsigargin or tunicamycin was equivalent in these cells to that seen in control, nontransfected cells, leading us to conclude that calreticulin is unlikely be involved in its own induction. Levels of the mRNA encoding the fusion protein were also increased by tunicamycin, but not thapsigargin, suggesting that, in agreement with our previous observations, inhibition of N-linked glycosylation may increase the stability of calreticulin mRNA. This indicates that in mammalian cells, there is more than one signaling pathway for the ER stress response.

Pathophysiological roles of calreticulin in autoimmune disease.

Autoantibodies against the endoplasmic reticulum (ER) luminal protein, calreticulin are often present in sera from patients with systemic lupus erythematosus, rheumatic disease and various parasitic diseases including onchocerciasis. New information has revealed that calreticulin is implicated in a number of autoimmune processes, including molecular mimicry, epitope spreading, complement inactivation and stimulation of inflammatory mediators, such as nitric oxide production. Calreticulin also binds to the Ro/SS-A antigen complex, which is composed of at least three immunologically distinct proteins bound to a group of small cytoplasmic RNAs that together form a common target for autoimmune responses. Up-regulation of calreticulin at the protein and RNA levels can be triggered by cell stresses, including heat shock, exposure to heavy metals and perturbation of normal ER function, which may in some cases lead to its secretion from cells. Calreticulin is targeted by autoantibodies following its release into the extracellular environment, possibly as a result of cell death, or its presence at the cell surface in response to insults such as viral infection or ultraviolet irradiation. These findings suggest that calreticulin is not just an autoantigen, but plays an active role in the pathology of various autoimmune disease through determinant spreading.

Differences in HAC1 mRNA processing and translation between yeast and mammalian cells indicate divergence of the eukaryotic ER stress response.

Perturbation of normal endoplasmic reticulum (ER) function induces a stress response found throughout eukaryotes, sometimes termed the unfolded protein response (UPR). In yeast, auxotrophic mutants have identified two genes, IRE1 and HAC1, whose products are key components. Normally HAC1 mRNA is not translated owing to a 252-nt "intron." Disruption of ER function activates Ire1p to remove this intron through endogenous endoribonuclease activity. Together with tRNA ligase, cleavage and splicing produces a translatable HAC1 mRNA to give Hac1p, a transcription factor that upregulates the expression of genes responsive to ER stress. No Hac1p homologue has been identified in mammalian cells, but Ire1p homologues exist with endoribonuclease activity required for a fully functional UPR, raising the possibility that the key features of the yeast UPR might be conserved in higher eukaryotic cells. To address this, we expressed yeast HAC1 in HeLa and HEK 293T human cell lines, both on its own and as fusions with yellow fluorescent protein (YFP) to investigate its processing and translation. HAC1 mRNA was not processed, but efficiently translated irrespective of whether the cells were subjected to ER stress. Expression of exogenous HAC1 mRNA constructs in yeast showed UPR-induced splicing required the presence of its 3' UTR. These results suggest that the mammalian ER stress response has diverged from the yeast UPR.

KDEL receptor expression is not coordinatedly up-regulated with ER stress-induced reticuloplasmin expression in HeLa cells.

Perturbation of the normal functioning of the endoplasmic reticulum (ER) increases the expression of lumenal proteins, such as grp78, and calreticulin. These proteins are retained within the compartment by a salvage mechanism involving the recognition of a C-terminal tetra-peptide sequence by the KDEL receptor. We have investigated whether disrupting normal ER function concomitantly increases the expression of the mRNAs encoding the two mammalian isoforms of the receptor, erd2.1 and erd2.2. Inhibition of N-linked glycosylation of proteins by tunicamycin had no effect upon the levels of the single mRNA species recognized by the erd2.1 probe, or the multiple transcripts detected with the erd2.2 cDNA probe. ER Ca2+ store depletion by thapsigargin did not increase erd2.1 mRNA, but actually caused a decrease in erd2.2 mRNA. Both thapsigargin, and tunicamycin, increased calreticulin secretion from the cells, although this might be due to more than simply saturation of KDEL receptor binding.

Induction of calreticulin expression in HeLa cells by depletion of the endoplasmic reticulum Ca2+ store and inhibition of N-linked glycosylation.

Calreticulin is now considered to be a multifunctional Ca(2+)-binding protein. Its primary role is as a Ca2+ storage protein within the lumen of the endoplasmic reticulum (ER), where it also seems to assist in the correct folding and assembly of proteins. We have investigated whether agents that affect these processes can alter calreticulin expression in HeLa cells. Perturbation of intracellular Ca2+ levels by prolonged exposure to either thapsigargin or ionomycin induced calreticulin mRNA, both in the presence and absence of extracellular Ca2+, consistent with the proposal that sustained depletion of the ER Ca2+ store can trigger these increases. The mechanism underlying the induction seems to be transcriptional up-regulation as both agents increased calreticulin promoter-driven firefly luciferase expression in transfected cells to the same degree as the observed increases in calreticulin mRNA. Experiments with a truncated promoter construct showed that the sequences that confer this inducibility reside within the 225 bp immediately upstream of the putative major transcriptional start site. We also examined the effect of tunicamycin, which inhibits N-linked glycosylation in the ER thereby interfering with protein processing. This caused increases in calreticulin mRNA greater than those with either thapsigargin or ionomycin, but failed to transactivate the calreticulin promoter. Thus either additional cis sequences that reside outside our promoter region are necessary for transcriptional activation by tunicamycin, or the increases in calreticulin mRNA occur post-transcriptionally. This suggests that there are probably different mechanisms by which calreticulin expression can be induced in response to agents that affect normal ER functioning.

Homozygous acute intermittent porphyria: compound heterozygosity for adjacent base transitions in the same codon of the porphobilinogen deaminase gene.

A sister and brother with severe porphobilinogen (PBG) deaminase deficiency are described. Each of their parents carries a different mutation for acute intermittent porphyria and the children are homozygous for the PBG-deaminase deficiency that causes this disorder. Both are compound heterozygotes for adjacent base transitions in the same codon in exon 10 of the PBG deaminase gene.

Non-erythroid form of acute intermittent porphyria caused by promoter and frameshift mutations distant from the coding sequence of exon 1 of the HMBS gene.

Acute intermittent porphyria (AIP) is a low-penetrant, autosomal dominant disorder caused by mutations in the HMBS gene. The gene is transcribed from two promoters to produce ubiquitous and erythroid isoforms of porphobilinogen deaminase, which differ only at their NH2 ends. In the classical form of AIP, both isoforms are deficient, but about 5% of families have the non-erythroid variant in which only the ubiquitous isoform is affected. Previously identified mutations in this variant have been within or close to the coding region of exon 1 of the HMBS gene, the only exon that is expressed solely in the ubiquitous isoform. Here, we describe mutations in the ubiquitous promoter (-154delG) and in exon 3 (41delA) that cause the non-erythroid variant. Reporter gene and electrophoretic mobility shift assays show that the G nucleotide at position -154, the most 5' of several transcription-initiation sites in the ubiquitous HMBS promoter, which lies immediately 3' to a transcription-factor IIB binding motif, is essential for normal transcription. The frameshift mutation in exon 3 introduces a stop codon into mRNA for the ubiquitous isoform only. Our investigations identify two new mechanisms for production of the non-erythroid variant of AIP and demonstrate that mutational analysis for diagnosis of this variant needs to include wider regions of the HMBS gene than indicated by previous reports. Furthermore, they show that deletion of one of several transcription initiation sites in the promoter of a housekeeping gene that lacks both TATA and initiator elements can produce disease.

Linkage disequilibrium between DNA polymorphisms within the porphobilinogen deaminase gene.

Three restriction fragment length polymorphisms (RFLPs) (MspI, PstI, ScrFI/BstNI) within the human porphobilinogen deaminase (PBG-D) gene have been studied in 47 unrelated patients with the autosomal dominant disorder, acute intermittent porphyria (AIP), and in 92 control subjects. Each enzyme identified a two-allele polymorphism with allele frequencies close to 0.50: however, marked linkage disequilibrium limited the number of observed haplotypes to four, of which one is uncommon. No association was detected between any haplotype and AIP.

Acute intermittent porphyria caused by a C----T mutation that produces a stop codon in the porphobilinogen deaminase gene.

A mutation of the porphobilinogen (PBG) deaminase gene that produces the cross-reacting immunological material (CRIM)-negative type of acute intermittent porphyria (AIP) has been identified in one of 43 unrelated patients with this form of the disorder. The mutation is a C----T transition that abolishes a PstI recognition site in exon 9 of the gene and converts a codon for glutamine to a stop codon.

Acute intermittent porphyria caused by defective splicing of porphobilinogen deaminase RNA: a synonymous codon mutation at -22 bp from the 5' splice site causes skipping of exon 3.

Acute intermittent porphyria (AIP) results from mutations in the porphobilinogen deaminase (PBG) gene. Three of 14 randomly selected, unrelated patients with the cross reacting immunological material (CRIM) negative form of AIP were found to have previously undescribed RNA splicing defects. Defective splicing of exons 12 and 13 was caused by a C-->G transversion at position -3 of the 3' splice site of intron 11 and a G-->A transition at the first position of intron 13, respectively. Defective splicing of exon 3 was associated with a synonymous codon mutation (CGC-->CGG, R28R) at position -22 from the 5' splice site. Our findings are consistent with previous reports indicating that about 15% of mutations in the PBG deaminase gene that cause AIP affect RNA splicing and add to the evidence that synonymous intraexonic codon mutations may cause disease.

DNA polymorphism of human porphobilinogen deaminase gene in acute intermittent porphyria.

A common two-allele MspI restriction fragment length polymorphism of the human erythroid porphobilinogen (PBG)-deaminase gene was investigated in 33 unrelated patients with acute intermittent porphyria (AIP) and 20 controls. The polymorphism was tightly linked (lod score 3.14; no recombinants) to the locus for AIP as identified by measurement of erythrocyte PBG-deaminase activity. The frequency of the polymorphism in the AIP patients did not differ significantly from that in the controls. No common polymorphisms for eight other restriction endonucleases were found in either group. In 30 of the AIP patients no crossreacting immunological material (CRIM) was produced by the mutant PBG-deaminase allele. The MspI polymorphism enabled each PBG-deaminase allele to be distinguished in subjects heterozygous for the polymorphism; thus a major gene deletion was excluded as the cause of the CRIM-negative mutation in all of the 18 families that contained an affected CRIM-negative individual heterozygous for the polymorphism. In suitable families, the MspI polymorphism provides a more certain way of identifying carriers of the AIP gene than current enzymatic methods and major gene deletions are unlikely to be present in more than a small proportion of the commonest type of AIP, the CRIM-negative form.