Production of Functional Human Vitamin A Transporter/RBP Receptor (STRA6) for Structure Determination

STRA6 is a plasma membrane protein that mediates the transport of vitamin A, or retinol, from plasma retinol binding protein (RBP) into the cell. Mutations in human STRA6 are associated with Matthew-Wood syndrome, which is characterized by severe developmental defects. Despite the obvious importance of this protein to human health, little is known about its structure and mechanism of action. To overcome the difficulties frequently encountered with the production of membrane proteins for structural determination, STRA6 has been expressed in Pichia pastoris as a fusion to green fluorescent protein (GFP), a strategy which has been a critical first step in solving the crystal structures of several membrane proteins. STRA6-GFP was correctly targeted to the cell surface where it bound RBP. Here we report the large-scale expression, purification and characterisation of STRA6-GFP. One litre of culture, corresponding to 175 g cells, yielded about 1.5 mg of pure protein. The interaction between purified STRA6 and its ligand RBP was studied by surface plasmon resonance-based binding analysis. The interaction between STRA6 and RBP was not retinol-dependent and the binding data were consistent with a transient interaction of 1 mole RBP/mole STRA6.     

RBP4 disrupts vitamin A uptake homeostasis in a STRA6-deficient animal model for Matthew-Wood syndrome

The cellular uptake of vitamin A from its RBP4-bound circulating form (holo-RBP4) is a homeostatic process that evidently depends on the multidomain membrane protein STRA6. In humans, mutations in STRA6 are associated with Matthew-Wood syndrome, manifested by multisystem developmental malformations. Here we addressed the metabolic basis of this inherited disease. STRA6-dependent transfer of retinol from RBP4 into cultured NIH 3T3 fibroblasts was enhanced by lecithin:retinol acyltransferase (LRAT). The retinol transfer was bidirectional, strongly suggesting that STRA6 acts as a retinol channel/transporter. Loss-of-function analysis in zebrafish embryos revealed that Stra6 deficiency caused vitamin A deprivation of the developing eyes. We provide evidence that, in the absence of Stra6, holo-Rbp4 provokes nonspecific vitamin A excess in several embryonic tissues, impairing retinoic acid receptor signaling and gene regulation. These fatal consequences of Stra6 deficiency, including craniofacial and cardiac defects and microphthalmia, were largely alleviated by reducing embryonic Rbp4 levels by morpholino oligonucleotide or pharmacological treatments.     

Lung hypoplasia in the nitrofen model of congenital diaphragmatic hernia occurs early in development

The teratogen nitrofen produces a congenital diaphragmatic hernia (CDH) and pulmonary hypoplasia in rodent fetuses that closely parallel observations made in humans. We hypothesized that these changes may be due to primary pulmonary hypoplasia and not herniation of the abdominal contents. Timed-pregnant rats were given nitrofen on day 9, and fetuses were harvested on days 13 through 21. Initial evagination of lung buds on gestational day 11 was not delayed in nitrofen-treated fetuses. On gestational day 13, however, there was a significant decrease in the number of terminal end buds in the lungs of nitrofen-exposed fetuses vs. controls. Thymidine-labeled lung epithelial and mesenchymal cells were significantly decreased in nitrofen-treated lungs. Lungs from nitrofen-treated fetuses exhibited wide septae with disorganized, compacted tissue, particularly around the air spaces. Expression of surfactant protein B and C mRNAs was significantly decreased in the nitrofen litters. In situ hybridization of fetal lung tissue at all gestational ages showed no difference in the expression of vascular endothelial growth factor, Flk-1, or Flt-1 mRNAs. Because closure of the diaphragm is completed on gestational day 16 in the rat, our results suggest that lung hypoplasia in this model of CDH is due at least in part to a primary effect of nitrofen on the developing lung.     

A homozygous mutation in LTBP2 causes isolated microspherophakia

Microspherophakia is an autosomal-recessive congenital disorder characterized by small spherical lens. It may be isolated or occur as part of a hereditary systemic disorder, such as Marfan syndrome, autosomal dominant and recessive forms of Weill-Marchesani syndrome, autosomal dominant glaucoma–lens ectopia–microspherophakia–stiffness–shortness syndrome, autosomal dominant microspherophakia with hernia, and microspherophakia-metaphyseal dysplasia. The purpose of this study was to map and identify the gene for isolated microspherophakia in two consanguineous Indian families. Using a whole-genome linkage scan in one family, we identified a likely locus for microspherophakia (MSP1) on chromosome 14q24.1–q32.12 between markers D14S588 and D14S1050 in a physical distance of 22.76 Mb. The maximum multi-point lod score was 2.91 between markers D14S1020 and D14S606. The MSP1 candidate region harbors 110 reference genes. DNA sequence analysis of one of the genes, LTBP2, detected a homozygous duplication (insertion) mutation, c.5446dupC, in the last exon (exon 36) in affected family members. This homozygous mutation is predicted to elongate the LTBP2 protein by replacing the last 6 amino acids with 27 novel amino acids. Microspherophakia in the second family did not map to this locus, suggesting genetic heterogeneity. The present study suggests a role for LTBP2 in the structural stability of ciliary zonules, and growth and development of lens.     

Prenatal administration of retinoic acid upregulates insulin-like growth factor receptors in the nitrofen-induced hypoplastic lung

BACKGROUND : Pulmonary hypoplasia (PH) is the main cause of mortality in newborns with congenital diaphragmatic hernia (CDH). Prenatal administration of retinoic acid (RA) stimulates alveologenesis in the nitrofen‐induced pulmonary hypoplasia. Insulin‐like growth factor receptors (IGFRs) play a crucial role in alveologenesis during lung development. We recently demonstrated that IGFRs were downregulated in later stages of lung development in the nitrofen CDH model. Several studies suggest the ability of RA to regulate insulin‐like growth factor signaling. We hypothesized that IGFRs pulmonary gene expression is upregulated after the administration of RA in the nitrofen‐induced CDH model. METHODS : Pregnant rats were exposed to either olive oil or nitrofen on day 9 (D9) of gestation. RA was given intraperitoneally on days D18, D19, and D20. Fetal lungs were dissected on D21 and divided into control, control + RA, CDH, and CDH + RA group. IGFRs gene and protein expression were determined using RT‐PCR and immunohistochemistry. RESULTS : mRNA expression levels of IGFRs were significantly increased in control + RA and CDH + RA compared with CDH group. Immunoreactivity of IGFRs was markedly increased in control + RA and CDH + RA compared with CDH lungs. CONCLUSIONS : Upregulation of pulmonary gene and protein expression of IGFRs after prenatal RA treatment in the nitrofen model suggests that RA may promote lung growth by stimulating IGFRs mediated alveologenesis. Birth Defects Res (Part B) 92:148–151, 2011. © 2011 Wiley‐Liss, Inc.     

Grouping of multiple-lentigines/LEOPARD and Noonan syndromes on the PTPN11 gene

Multiple-lentigines (ML)/LEOPARD (multiple lentigines, electrocardiographic-conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth, and sensorineural deafness) syndrome is an autosomal dominant condition--characterized by lentigines and café au lait spots, facial anomalies, cardiac defects--that shares several clinical features with Noonan syndrome (NS). We screened nine patients with ML/LEOPARD syndrome (including a mother-daughter pair) and two children with NS who had multiple café au lait spots, for mutations in the NS gene, PTPN11, and found, in 10 of 11 patients, one of two new missense mutations, in exon 7 or exon 12. Both mutations affect the PTPN11 phosphotyrosine phosphatase domain, which is involved in <30% of the NS PTPN11 mutations. The study demonstrates that ML/LEOPARD syndrome and NS are allelic disorders. The detected mutations suggest that distinct molecular and pathogenetic mechanisms cause the peculiar cutaneous manifestations of the ML/LEOPARD-syndrome subtype of NS.     

The Dominant white, Dun and Smoky color variants in chicken are associated with insertion/deletion polymorphisms in the PMEL17 gene

Dominant white, Dun, and Smoky are alleles at the Dominant white locus, which is one of the major loci affecting plumage color in the domestic chicken. Both Dominant white and Dun inhibit the expression of black eumelanin. Smoky arose in a White Leghorn homozygous for Dominant white and partially restores pigmentation. PMEL17 encodes a melanocyte-specific protein and was identified as a positional candidate gene due to its role in the development of eumelanosomes. Linkage analysis of PMEL17 and Dominant white using a red jungle fowl/White Leghorn intercross revealed no recombination between these loci. Sequence analysis showed that the Dominant white allele was exclusively associated with a 9-bp insertion in exon 10, leading to an insertion of three amino acids in the PMEL17 transmembrane region. Similarly, a deletion of five amino acids in the transmembrane region occurs in the protein encoded by Dun. The Smoky allele shared the 9-bp insertion in exon 10 with Dominant white, as expected from its origin, but also had a deletion of 12 nucleotides in exon 6, eliminating four amino acids from the mature protein. These mutations are, together with the recessive silver mutation in the mouse, the only PMEL17 mutations with phenotypic effects that have been described so far in any species.     

Analysis of two Arab families reveals additional support for a DFNB2 nonsyndromic phenotype of MYO7A

Variants in the head and tail domains of the MYO7A gene, encoding myosin VIIA, cause Usher syndrome type 1B (USH1B) and nonsyndromic deafness (DFNB2, DFNA11). In order to identify the genetic defect(s) underling profound deafness in two consanguineous Arab families living in UAE, we have sequenced a panel of 19 genes involved in Usher syndrome and nonsyndromic deafness in the index cases of the two families. This analysis revealed a novel homozygous insertion of AG (c.1952_1953insAG/p.C652fsX11) in exon 17 of the MYO7A gene in an Iraqi family, and a homozygous point mutation (c.5660C>T/p.P1887L) in exon 41 affecting the same gene in a large Palestinian family. Moreover, some individuals from the Palestinian family also harbored a novel heterozygous truncating variant (c.1267C>T/p.R423X) in the DFNB31 gene, which is involved in autosomal recessive nonsyndromic deafness type DFNB31 and Usher syndrome type II. Assuming an autosomal recessive mode of inheritance in the two inbred families, we conclude that the homozygous variants in the MYO7A gene are the disease-causing mutations in these families. Furthermore, given the absence of retinal disease in all affected patients examined, particularly a 28 year old patient, suggests that at least one family may segregate a DFNB2 presentation rather than USH1B. This finding further supports the premise that the MYO7A gene is responsible for two distinct diseases and gives evidence that the p.P1887L mutation in a homozygous state may be responsible for nonsyndromic hearing loss.     

Molecular prenatal diagnosis of autosomal recessive childhood spinal muscular atrophies (SMAs)

Autosomal recessive childhood spinal muscular atrophy (SMAs) is the second most common neuromuscular disorder and a common cause of infant disability and mortality. SMA patients are classified into three clinical types based on age of onset, and severity of symptoms. About 94% of patients have homozygous deletion of exon 7 in survival motor neuron (SMN1) gene. The neuronal apoptosis inhibitory protein (NAIP) gene was found to be more frequently deleted in the severest form of the disease. This study aimed to comment on the implementation of genetic counseling and prenatal diagnosis of SMAs for 85 fetuses from 75 Egyptian couples at risk of having an affected child. The homozygous deletion of exon 7 in SMN1 gene and the deletion of exon 5 of the NAIP gene were detected using PCR-REFLP and multiplex PCR methods respectively. Eighteen fetuses showed homozygous deletion of exon 7 in SMN1 gene and deletion of exon 5 in NAIP gene. In conclusion prenatal diagnosis is an important tool for accurate diagnosis and genetic counseling that help decision making in high risk families.     

De novo alu-element insertions in FGFR2 identify a distinct pathological basis for Apert syndrome.

Apert syndrome, one of five craniosynostosis syndromes caused by allelic mutations of fibroblast growth-factor receptor 2 (FGFR2), is characterized by symmetrical bony syndactyly of the hands and feet. We have analyzed 260 unrelated patients, all but 2 of whom have missense mutations in exon 7, which affect a dipeptide in the linker region between the second and third immunoglobulin-like domains. Hence, the molecular mechanism of Apert syndrome is exquisitely specific. FGFR2 mutations in the remaining two patients are distinct in position and nature. Surprisingly, each patient harbors an Alu-element insertion of approximately 360 bp, in one case just upstream of exon 9 and in the other case within exon 9 itself. The insertions are likely to be pathological, because they have arisen de novo; in both cases this occurred on the paternal chromosome. FGFR2 is present in alternatively spliced isoforms characterized by either the IIIb (exon 8) or IIIc (exon 9) domains (keratinocyte growth-factor receptor [KGFR] and bacterially expressed kinase, respectively), which are differentially expressed in mouse limbs on embryonic day 13. Splicing of exon 9 was examined in RNA extracted from fibroblasts and keratinocytes from one patient with an Alu insertion and two patients with Pfeiffer syndrome who had nucleotide substitutions of the exon 9 acceptor splice site. Ectopic expression of KGFR in the fibroblast lines correlated with the severity of limb abnormalities. This provides the first genetic evidence that signaling through KGFR causes syndactyly in Apert syndrome.     

Acidic retinoids synergize with vitamin A to enhance retinol uptake and STRA6, LRAT, and CYP26B1 expression in neonatal lung

Vitamin A (VA) is essential for fetal lung development and postnatal lung maturation. VA is stored mainly as retinyl esters (REs), which may be mobilized for production of retinoic acid (RA). This study was designed 1) to evaluate several acidic retinoids for their potential to increase RE in the lungs of VA-supplemented neonatal rats, and 2) to determine the expression of retinoid homeostatic genes related to retinol uptake, esterification, and catabolism as possible mechanisms. When neonatal rats were treated with VA combined with any one of several acidic retinoids (RA, 9-cis-RA, or Am580, a stable analog of RA), lung RE increased ∼5–7 times more than after an equal amount of VA alone. Retinol uptake and esterification during the period of absorption correlated with increased expression of both STRA6 (retinol-binding protein receptor) and LRAT (retinol esterification), while a reduction in RE after 12 h in Am580-treated, VA-supplemented rats correlated with a strong and persistent increase in CYP26B1 (RA hydroxylase). We conclude that neonatal lung RE can be increased synergistically by VA combined with both natural and synthetic acidic retinoids, concomitant with induction of the dyad of STRA6 and LRAT. However, the pronounced and prolonged induction of CYP26B1 by Am580 may counteract lung RE accumulation after the absorption process is completed.     

Aberrant splicing of phenylalanine hydroxylase mRNA: the major cause for phenylketonuria in parts of southern Europe.

We report a mutation within the phenylalanine hydroxylase (PAH) gene that causes aberrant splicing of the mRNA and that is in tight association with chromosomal haplotypes 6, 10, and 36. Because of the high frequency of these particular haplotypes in Bulgaria, Italy, and Turkey, it appears to be one of the more frequent defects in the PAH gene causing classical phenylketonuria in this part of Europe. The mutation is a G to A transition at position 546 in intron 10 of the PAH gene, 11 bp upstream from the intron 10/exon 11 boundary. It activates a cryptic splice site and results in an in-frame insertion of 9 nucleotides between exon 10 and exon 11 of the processed mRNA. Normal amounts of liver PAH protein are present in homozygous patients, but no catalytic activity can be detected. This loss of enzyme activity is probably caused by conformational changes resulting from the insertion of three additional amino acids (Gly-Leu-Gln) between the normal sequences encoded by exon 10 and exon 11.     

New splicing-site mutations in the SURF1 gene in Leigh syndrome patients

The gene SURF1 encodes a factor involved in the biogenesis of cytochrome c oxidase, the last complex in the respiratory chain. Mutations of the SURF1 gene result in Leigh syndrome and severe cytochrome c oxidase deficiency. Analysis of seven unrelated patients with cytochrome c oxidase deficiency and typical Leigh syndrome revealed different SURF1 mutations in four of them. Only these four cases had associated demyelinating neuropathy. Three mutations were novel splicing-site mutations that lead to the excision of exon 6. Two different novel heterozygous mutations were found at the same guanine residue at the donor splice site of intron 6; one was a deletion, whereas the other was a transition [588+1G>A]. The third novel splicing-site mutation was a homozygous [516-2_516-1delAG] in intron 5. One patient only had a homozygous polymorphism in the middle of the intron 8 [835+25C>T]. Western blot analysis showed that Surf1 protein was absent in all four patients harboring mutations. Our studies confirm that the SURF1 gene is an important nuclear gene involved in the cytochrome c oxidase deficiency. We also show that Surf1 protein is not implicated in the assembly of other respiratory chain complexes or the pyruvate dehydrogenase complex.     

Developmental toxicity evaluation of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in Wistar rats

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is a genotoxic chlorination by-product in drinking water. There is some evidence that it has developmental toxic effects in vitro but its potential to cause developmental effects in vivo is not known. The developmental effects were evaluated in Wistar rats. Rats (22-26 dams per dose group) were administered MX by gavage at the dose levels of 3, 30, or 60 mg/kg in water on gestation days 6-19. Control animals received plain water. Clinical signs, body weight, and food and water consumption were recorded for the dams. On gestation day 20, a cesarean section was performed and the ovaries anduterine contents of the dams were examined and the liver, kidneys, spleen, and thyroid glands weighed. The fetuses of all dose groups were weighed, sexed, and observed for external and skeletal malformations and the fetuses of the two highest dose groups were evaluated for visceral malformations. The highest dose, 60 mg/kg of MX, was slightly toxic to the dams. It decreased the corrected body weight gain of dams by 32% and the water consumption by 16-17%. Kidney and liver weights were slightly increased. MX did not affect the number of implantations nor did it cause any resorptions. The body weights of fetuses were not significantly affected. MX did not cause external malformations or skeletal anomalies. Two fetuses at 60 mg/kg and one fetus at 30 mg/kg had major visceral malformations (persistent truncus arteriosus, diaphragmatic hernia, dilated aorta with a stenosis of pulmonary arteries) and two minor artery abnormalities were observed in those animals. The frequency of unilateral displaced testis was slightly higher (9.2%) in the 60-mg/kg dose group than in controls (1.6%). Since the abnormalities did not form a consistent pattern and occurred most at maternally toxic dose, we conclude that MX can be regarded as non-teratogenic.     

Adaptive Evolution of the STRA6 Genes in Mammalian

Stimulated by retinoic acid 6 (STRA6) is the receptor for retinol binding protein and is relevant for the transport of retinol to specific sites such as the eye. The adaptive evolution mechanism that vertebrates have occupied nearly every habitat available on earth and adopted various lifestyles associated with different light conditions and visual challenges, as well as their role in development and adaptation is thus far unknown. In this work, we have investigated different aspects of vertebrate STRA6 evolution and used molecular evolutionary analyses to detect evidence of vertebrate adaptation to the lightless habitat. Free-ratio model revealed significant rate shifts immediately after the species divergence. The amino acid sites detected to be under positive selection are within the extracellular loops of STRA6 protein. Branch-site model A test revealed that STRA6 has undergone positive selection in the different phyla of mammalian except for the branch of rodent. The results suggest that interactions between different light environments and host may be driving adaptive change in STRA6 by competition between species. In support of this, we found that altered functional constraints may take place at some amino acid residues after speciation. We suggest that STRA6 has undergone adaptive evolution in different branch of vertebrate relation to habitat environment.     

The STRA6 Receptor Is Essential for Retinol-binding Protein-induced Insulin Resistance but Not for Maintaining Vitamin A Homeostasis in Tissues Other Than the Eye

The plasma membrane protein STRA6 is thought to mediate uptake of retinol from its blood carrier retinol-binding protein (RBP) into cells and to function as a surface receptor that, upon binding of holo-RBP, activates a JAK/STAT cascade. It was suggested that STRA6 signaling underlies insulin resistance induced by elevated serum levels of RBP in obese animals. To investigate these activities in vivo, we generated and analyzed Stra6-null mice. We show that the contribution of STRA6 to retinol uptake by tissues in vivo is small and that, with the exception of the eye, ablation of Stra6 has only a modest effect on retinoid homeostasis and does not impair physiological functions that critically depend on retinoic acid in the embryo or in the adult. However, ablation of Stra6 effectively protects mice from RBP-induced suppression of insulin signaling. Thus one biological function of STRA6 in tissues other than the eye appears to be the coupling of circulating holo-RBP levels to cell signaling, in turn regulating key processes such as insulin response.