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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 15, 2004 as doi:10.1096/fj.04-2397fje. |
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* Institute of Physiology, University of Zürich; and
Functional Genomics Center Zürich, Zürich, Switzerland
1Correspondence: Institute of Physiology, Winterthurerstrasse 190, Zurich 8057, Switzerland. E-mail: thennet{at}access.unizh.ch
SPECIFIC AIMS
Congenital disorders of glycosylation (CDG) are a family of diseases characterized by defects of N-linked glycosylation. In CDG-I, several genetic defects cause a shortage of dolichol-linked oligosaccharides, which leads to underglycosylation of nascent glycoproteins. N-linked glycosylation is important for proper folding and trafficking of glycoproteins. Inhibition of glycosylation results in the buildup of misfolded proteins in the endoplasmic reticulum, which induces a protective reaction known as the unfolded protein response (UPR). To investigate whether UPR components are induced in CDG, we have performed a transcriptome analysis of primary fibroblasts from unaffected control subjects and from CDG-I patients using oligonucleotide gene expression arrays.
PRINCIPAL FINDINGS
1. Human primary fibroblasts sustain endoplasmic reticulum stress when glycosylation is impaired by tunicamycin treatment and as a result of genetic defects
Three distinct CDG-I types were analyzed to filter out isolated events and allow a reliable association between the transcriptional responses detected and the condition of abnormal glycosylation found in CDG. Nine CDG-I fibroblast samples were isolated from four CDG-Ic patients with ALG6 glucosyltransferase deficiency, three CDG-Ie patients with DPM1 dolichylphosphate-mannose synthase defect and two CDG-Ig patients with ALG12 mannosyltransferase deficiency. The three types of CDG-I applied here were characterized by distinct glycosylation defects with accumulation of different incomplete dolichylpyrophosphate-linked oligosaccharides. Human primary fibroblasts can mount a typical UPR after treatment with tunicamycin that completely blocks N-glycosylation. Transcriptional response to tunicamycin in human primary control fibroblasts was extensive and included components of the protein folding machinery, the ER-associated protein degradation (ERAD) pathway, transcription factors, and proteins regulating translation initiation, vesicular transport, and glycosylation (Fig. 1
). In contrast, glucose deprivation for 24 h did not induce a UPR in primary fibroblasts. Even chaperones like Bip and TRA1, also known as glucose-regulated proteins (GRP), were not or were only marginally overexpressed in glucose-deprived fibroblasts. The only UPR proteins significantly induced by glucose deprivation were STCH/Hsp70, the human SEL1L counterpart of the yeast ERAD protein Hrd3p, the DNAJC3/P58IPK kinase inhibitor, and two RAB27B and RAB33A vesicular transport proteins (Table 1
).
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2. The status of the UPR in CDG-I fibroblasts was moderate compared with the response observed in cells treated with tunicamycin
The expression of several chaperone proteins, including ERP70/PDI and CALR, was significantly induced in CDG-I fibroblasts, yet less than a 2-fold level. The strongest transcriptional induction in CDG-I was detected for the DNAJC3/P58IPK gene (Table 1)
. Because DNAJC3/P58IPK is implied in the regulation of protein translation via the control of PKR- and PERK-mediated eIF2
phosphorylation, we measured the rate of protein synthesis in the human control and CDG-I fibroblasts investigated in our study. Although incorporation of [35S]Met/Cys varied from sample to sample, the values obtained showed no difference between control cells and any type of CDG-I tested.
3. Cluster analysis of gene expression profiles showed a coordinated induction of amino acid metabolism and transport in the fibroblast UPR
Groups of genes showing highly correlated expression throughout the different CDG-I or stress conditions were determined and analyzed by hierarchical clustering. This analysis allowed the identification of genes that were coordinately induced by tunicamycin, glucose deprivation, and the CDG-I condition, thus defining the extent of the UPR in human primary fibroblasts. A cluster of similarly expressed genes included several components of the UPR such as XBP1, GRP58, STCH, and DNAJB9. The same cluster contained genes encoding proteins involved in the synthesis and transport of amino acids like ATA1, SLC3A2, WARS, and PSAT1. Besides being expressed in coordination with established UPR components, several genes of amino acid transport and metabolism were consistently overexpressed in tunicamycin-treated and glucose-deprived fibroblasts as well as in CDG-I fibroblasts.
4. Levels of UPR proteins were also elevated in glycosylation-impaired fibroblasts
We measured the amount of the UPR protein calreticulin, calnexin, HSP40, and protein disulfide isomerase (PDI) in fibroblasts by Western blot and immunofluorescence analyses to determine whether changes in protein amounts correlated with the mRNA induction values obtained in transcriptome analysis. Calreticulin protein levels were increased under stress condition, but the increase was modest compared with induction detected at the mRNA level. Calnexin showed the same pattern, with a significantly increased protein level in stressed cells. HSP40 was detected using a polyclonal antibody that reacted with several proteins, likely representing other members of the DNAJ family. Three members of the HSP40 family of 40, 58, and 65 kDa were overexpressed in control fibroblasts after tunicamycin treatment and glucose deprivation; a consistent increase in expression was observed in CDG-I fibroblasts. The thiol-dependent reductase ERp57/PDI mRNA was increased upon tunicamycin treatment but remained unchanged in glucose-deprived and CDG-I fibroblasts (Table 1)
. Levels of PDI protein remained rather constant throughout the conditions tested although a significant heterogeneity in expression level was observed among the control fibroblasts. A slightly decreased signal was noted in normal control or CDG-I fibroblast cells treated with tunicamycin.
CONCLUSIONS AND SIGNIFICANCE
Using global transcriptome analysis, we have determined for the first time the scope and extent of the UPR in cells isolated from CDG-I patients. Our results indicate that endoplasmic stress in CDG-I cells is similar, though moderate, to the response induced by tunicamycin-mediated inhibition of N-glycosylation. The moderate UPR detected in CDG-I cells indicates that these cells do not accumulate unfolded proteins, which may lead to the collapse of the secretory pathway. Consequently, the symptoms associated with CDG do not appear to be caused by general glycoprotein folding and secretion.
Transcriptional activation of amino acid transporters was observed after tunicamycin treatment, after glucose deprivation, and in CDG-I cells, thus defining a new stress response in human fibroblasts representing a possible component of the UPR. The significance of this amino acid response is unclear. The increased amino acid supply may be required to compensate for the production of misfolded proteins in glycosylation-stressed fibroblasts. A key role in the long-term response of CDG cells is likely played by the p58IPK protein. Future work will address the exact role of p58IPK in maintaining protein translation and cell viability and determine whether it represents a target for therapeutical approaches aimed at attenuating the pathological consequences of chronic ER stress.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2397fje;
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