The stepwise assembly of N-linked glycans requires dolichol, a polyisoprenoid. In the last step of dolichol biosynthesis, the terminal alpha isoprene unit of the polyprenol is reduced.
SRD5A3 was initially presumed to be a 5α-steroid reductase. It was identified by a genome-wide expression profile analysis in human prostate cancer cells [20
]. It consists of 318 amino acids. Recent studies idenified SRD5A3 as the polyprenol reductase involved in the final step of dolichol biosynthesis [19
The clinical features of SRD5A3 deficiency were first described in a consanguineous Emirati family with four affected children [21
]. The children had dysmorphic features, coloboma of the iris, retina or optic disc, short stature, feeding difficulties, hypertrichosis and hyperkeratosis of the skin. Brain MRI revealed frontal polymicrogyria. One of the children had a transposition of the great arteries. Five out of seven patients had cerebellar vermis hypoplasia [19
]. Genome-wide linkage analysis revealed a homozygous region on chromosome 4 and the SRD5A3 gene was identified by mutation analysis of candidate genes within this region [19
]. Since abnormal Tf glycosylation was found in these children, CDG-Ix patients were screened for SRD5A3 mutations revealing a total of 11 patients with mutations in this gene [19
]. Nystagmus was reported in nearly all of them.
Morava et al. reported 12 patients from nine families with SRD5A3-CDG [22
]. Two of the affected children were from two different consanguineous Turkish families and were originally reported by Prietsch [23
] and Assman [24
]. They had the same mutation as our patient (c.57G>A [W19X]) and showed a similar picture of clinical symptoms: Muscle hypertonia, psychomotor retardation and nystagmus. By reason of the geographic distance a potential founder effect between the two Turkish children and our patient is unlikely. Morava et al. also compared the clinical symptoms and biochemical findings of these 12 SRD5A3-CDG-patients and found no genotype–phenotype correlation [22
SRD5A3 deficiency was also identified as the cause of Kahrizi syndrome [25
]. The syndrome was described in three adult Iranian siblings with severe mental retardation. The patients were unable to speak. Two of the three patients had iris coloboma. The patients had short stature, cataracts, kyphosis and joint contractures developed later in life. No seizures occurred. Homozygosity mapping revealed a single interval of homozygosity that was unique to the patients and was localized to the pericentrometric region of chromosome 4.
SRD5A3 is the human ortholog of the yeast DFG10 gene. The phenotype of the yeast DFG10 mutant can be rescued by human SRD5A3 [19
]. Disruption of the SRD5A3 gene in mice is lethal at embryonic day 12.5 [19
In SRD5A3 fibroblasts we found dolichol concentrations that were comparable to healthy controls suggesting that there must be an alternative pathway for dolichol biosynthesis. In addition, polyprenol could serve as the acceptor molecule for the biosynthesis of N-glycans working with the lower efficiency.
Although the amount of dolichol was not reduced in SRD5A3 fibroblasts, hypoglycosylation occurred in the patient. Several explanations seem possible:
- Accumulation of intermediate products of the dolichol pathway might be toxic and inhibit glycosylation reactions. A change of isoprenoid levels could influence the cell's metabolism as it has been associated with Alzheimer's disease [27–30] and neuronal ceroid lipofuscinosis . Recently a mutation in DHDDS (OMIM ID: 613861), encoding dehydrodolichol diphosphate synthase, was discovered in an ethnic group called Ashkenazi Jews . DHDDS (syn. cis-prenyltransferase) is located two steps earlier in dolichol de novo biosynthesis than the polyprenol reductase, creating polyprenol by cis-prenyl elongation of farnesyl pyrophosphate (FPP) . Interestingly, symptoms were not as severe as in our patient and restricted to retinitis pigmentosa. No other organs were affected. Referring to toxic accumulation, FPP might have a less fatal impact than polyprenol.
- The 3-fold increase of polyprenol found in SRD5A3 fibroblasts could compete with dolichol as the lipid-anchor for N-glycosylation. In the first step of N-glycosylation, dolichol kinase transfers phosphate from CTP to dolichol () [11,12]. Substrate specificity of dolichol kinase was tested in rat liver and demonstrated a preference for the saturation of the alpha unit with 2.5-fold more activity concerning dolichol-16 and -19 compared to the corresponding polyprenols . Furthermore, for the transfer of mannosyl residues from GDP-mannose in rats, polyprenol was identified as a weak acceptor while dolichol showed good results . In contrast to bacteria, glycosylation with polyprenol in human seems to be reduced if not impossible. Nevertheless, polyprenol might compete with dolichol in early N-glycan biosynthesis reactions thereby reducing the amount of dolichol-linked oligsaccharides used for protein glycosylation.
- The operating alternative pathway cannot create the necessary amount of dolichol at the right location. One dolichol phosphate is required as the carrier while seven others import glucose and mannose into the ER . Therefore, a locally decreased pool of dolichol can lead to reduced glycosylation. A dolichol-pyrophosphate-phosphatase in yeast (CWH8) and mammalian cells (DolPP1) is known to recycle Dol-P-P after OST has transferred Glc3Man9GlcNAc2 to the protein, keeping up a steady pool of dolichol for glycosylation . Whether this is enough in patients with SRD5A3-mutations or whether this dolichol is at the right location still needs to be verified.
- The amount of dolichol-linked oligosaccharides might be sufficient for glycosylation in SRD5A3 fibroblasts but might be insufficient in cells with higher rates of N-glycan biosynthesis like liver cells.
It is intriguing to speculate that improvement of the dolichol/polyprenol ratio could be the key for a successful therapy for SRD5A3-deficient patients. Very little is known about the dolichol contents of food. Polyprenols are the dominant polyisoprenoids in plant photosynthetic tissues accompanied by the traces of dolichols. Only plant roots, yeast or the animal tissues could be considered as a possible source of dolichols [36
]. However, the dolichol-amount absorbed by nutrition is known to be insignificant in rats [37
]. Further research on dolichol content in plants or food and its absorption in human will be necessary.
Pharmacological interventions might also be possible. In CDG fibroblasts with a dolichol-phosphate-mannose synthase deficiency (CDG-Ie) the amount of dolichol-phosphate-mannose was increased by using the squalene synthase inhibitor zaragosic acid A (ZGA) [38
In contrast to statins, squalene synthase inhibitors inhibit cholesterol biosynthesis later-on in the pathway, i.e. after the step where dolichol biosynthesis is separated from the cholesterol biosynthesis pathway. In SRD5A3, directing the metabolic flux towards polyisoprenoid biosynthesis might have contrary effects and might worsen the patient's symptoms, e.g. if hypothesis #2 is correct. Alternatively it might be useful if e.g. hypothesis #4 is correct.
On the other hand, statins inhibit the cholesterol and dolichol biosynthesis by interfering with mevalonate biosynthesis at the level of HMG-CoA reductase and are used for treatment of patients at risk for secondary cardiovascular events [39
]. The mevalonate pathway is the early common part of the dolichol and cholesterol biosynthesis. Its interruption would therefore reduce not only cholesterol but polyisoprenoids as well. If accumulation of polyprenol was deleterious as discussed, this might improve the patient's symptoms. Otherwise, hypoglycosylation of proteins might increase due to reduced dolichol production [40
Statins have been used in Smith–Lemli–Opitz syndrome (SLOS) treatment. SLOS is caused by a defect of sterol-Δ7
-reductase that leads to an accumulation of the cholesterol precursor 7-dehydrocholesterol [41
]. Its production can be inhibited by statin treatment [42
]. Pappu, Connor et al. [43
] described a 7-fold higher urinary excretion rate of dolichol and ubiquinone in SLOS children than in control children. This again shows a shift in the mevalonate/cholesterol pathway towards other products such as nonsterol isoprenoids. Furthermore Pappu, Connor et al. used a high-cholesterol diet, decreasing the urinary excretion rate of dolichol by 70% and ubiquinone by 67%, possibly due to a negative feedback mechanism. In patients with polyprenol reductase deficiency a cholesterol supplementation might have similar effects on the polyprenol excretion and possibly on the polyprenol content in cells.
Metazoans protect themselves from potential toxic substrate accumulation in cells with a mechanism called translation attenuation. High ER stress rates, for example due to improperly glycosylated proteins, induce the protein kinase R-like ER kinase (PERK). This kinase then phosphorylates the eukaryotic initiation factor 2α (eIEF2α), which reduces the number of translated proteins in the ER. Hence, fewer proteins are glycosylated but regularly [44
]. Translation balancing still needs to be verified in human cells. Effects on fibroblasts from CDG-Ia patients showed variable results [44
] but hold promise for a future therapeutic option.