Jesús Cruces

Characterization of human PA2.26 antigen (T1α-2, podoplanin), a small membrane mucin induced in oral squamous cell carcinomas

We report the full cDNA sequence encoding the human homologue of murine PA2.26 (T1α‐2, podoplanin), a small mucin‐type transmembrane glycoprotein originally identified as a cell‐surface antigen induced in keratinocytes during mouse skin carcinogenesis. The human PA2.26 gene is expressed as 2 transcripts of 0.9 and 2.7 kb in several normal tissues, such as the placenta, skeletal muscle, heart and lung. Using a specific polyclonal antibody raised against a synthetic peptide of the protein ectodomain, PA2.26 was immunohistochemically detected in about 25% (15/61) of human early oral squamous cell carcinomas. PA2.26 distribution in the tumours was heterogeneous and often restricted to the invasive front. Double immunofluorescence and confocal microscopy analysis showed that PA2.26 colocalized with the membrane cytoskeleton linker ezrin at the surface of tumour cells and that its presence in vivo was associated with downregulation of membrane E‐cadherin protein expression. Ectopic expression of human PA2.26 in HeLa carcinoma cells and immortalized HaCaT keratinocytes promoted a redistribution of ezrin to the cell edges, the formation of cell‐surface protrusions and reduced Ca2+‐dependent cell‐cell adhesiveness. These results point to PA2.26 as a novel biomarker for oral squamous cell carcinomas that might be involved in migration/invasion.

Function of partially duplicated human α77 nicotinic receptor subunit CHRFAM7A gene: potential implications for the cholinergic anti-inflammatory response.

The neuronal α7 nicotinic receptor subunit gene (CHRNA7) is partially duplicated in the human genome forming a hybrid gene (CHRFAM7A) with the novel FAM7A gene. The hybrid gene transcript, dupα7, has been identified in brain, immune cells, and the HL-60 cell line, although its translation and function are still unknown. In this study, dupα7 cDNA has been cloned and expressed in GH4C1 cells and Xenopus oocytes to study the pattern and functional role of the expressed protein. Our results reveal that dupα7 transcript was natively translated in HL-60 cells and heterologously expressed in GH4C1 cells and oocytes. Injection of dupα7 mRNA into oocytes failed to generate functional receptors, but when co-injected with α7 mRNA at α7/dupα7 ratios of 5:1, 2:1, 1:1, 1:5, and 1:10, it reduced the nicotine-elicited α7 current generated in control oocytes (α7 alone) by 26, 53, 75, 93, and 94%, respectively. This effect is mainly due to a reduction in the number of functional α7 receptors reaching the oocyte membrane, as deduced from α-bungarotoxin binding and fluorescent confocal assays. Two additional findings open the possibility that the dominant negative effect of dupα7 on α7 receptor activity observed in vitro could be extrapolated to in vivo situations. (i) Compared with α7 mRNA, basal dupα7 mRNA levels are substantial in human cerebral cortex and higher in macrophages. (ii) dupα7 mRNA levels in macrophages are down-regulated by IL-1β, LPS, and nicotine. Thus, dupα7 could modulate α7 receptor-mediated synaptic transmission and cholinergic anti-inflammatory response.The neuronal α7 nicotinic receptor subunit gene (CHRNA7) is partially duplicated in the human genome forming a hybrid gene (CHRFAM7A) with the novel FAM7A gene. The hybrid gene transcript, dupα7, has been identified in brain, immune cells, and the HL-60 cell line, although its translation and function are still unknown. In this study, dupα7 cDNA has been cloned and expressed in GH4C1 cells and Xenopus oocytes to study the pattern and functional role of the expressed protein. Our results reveal that dupα7 transcript was natively translated in HL-60 cells and heterologously expressed in GH4C1 cells and oocytes. Injection of dupα7 mRNA into oocytes failed to generate functional receptors, but when co-injected with α7 mRNA at α7/dupα7 ratios of 5:1, 2:1, 1:1, 1:5, and 1:10, it reduced the nicotine-elicited α7 current generated in control oocytes (α7 alone) by 26, 53, 75, 93, and 94%, respectively. This effect is mainly due to a reduction in the number of functional α7 receptors reaching the oocyte membrane, as deduced from α-bungarotoxin binding and fluorescent confocal assays. Two additional findings open the possibility that the dominant negative effect of dupα7 on α7 receptor activity observed in vitro could be extrapolated to in vivo situations. (i) Compared with α7 mRNA, basal dupα7 mRNA levels are substantial in human cerebral cortex and higher in macrophages. (ii) dupα7 mRNA levels in macrophages are down-regulated by IL-1β, LPS, and nicotine. Thus, dupα7 could modulate α7 receptor-mediated synaptic transmission and cholinergic anti-inflammatory response.

Two new patients bearing mutations in the fukutin gene confirm the relevance of this gene in Walker-Warburg syndrome

Walker–Warburg syndrome (WWS) is an autosomal recessive disorder characterized by congenital muscular dystrophy, brain malformations and structural abnormalities of the eye. We have studied two WWS patients born to non‐consanguineous parents, and in both cases, we identified mutations in the fukutin gene responsible for this syndrome. One of the patients carries a homozygous‐single nucleotide insertion that produces a frameshift, being this the first time that this insertion has been described in homozygosis and causing a WWS phenotype. The other patient carries two novel mutations, one being a point mutation that produces an amino acid substitution, while the other is a deletion in the 3′UTR that affects the polyadenylation signal of the fukutin gene. This deletion would probably result in the complete loss of the fukutin transcripts from this allele. This is the first time a mutation localized outside of the fukutin coding region has been identified as a cause of WWS.

O-Mannose and O–N-acetyl galactosamine glycosylation of mammalian α-dystroglycan is conserved in a region-specific manner

Defects in the O-linked glycosylation of the peripheral membrane protein α-dystroglycan (α-DG) are the main cause of several forms of congenital muscular dystrophies and thus the characterization of the glycosylation of α-DG is of great medical importance. A detailed investigation of the glycosylation pattern of the native α-DG protein is essential for the understanding of the biological processes related to human disease in which the protein is involved. To date, several studies have reported novel O-glycans and attachment sites on the mucin-like domain of mammalian α-DG with both similar and contradicting glycosylation patterns, indicating the species-specific O-glycosylation of mammalian α-DG. By applying a standardized purification scheme and subsequent glycoproteomic analysis of native α-DG from rabbit and human skeletal muscle biopsies and from cultured mouse C2C12 myotubes, we show that the O-glycosylation patterns of the mucin-like domain of native α-DG are conserved among mammalians in a region-specific manner.

Promoter alteration causes transcriptional repression of the POMGNT1 gene in limb-girdle muscular dystrophy type 2O.

Limb-girdle muscular dystrophy type 2O (LGMD2O) belongs to a group of rare muscular dystrophies named dystroglycanopathies, which are characterized molecularly by hypoglycosylation of α-dystroglycan (α-DG). Here, we describe the first dystroglycanopathy patient carrying an alteration in the promoter region of the POMGNT1 gene (protein O-mannose β-1,2-N-acetylglucosaminyltransferase 1), which involves a homozygous 9-bp duplication (-83_-75dup). Analysis of the downstream effects of this mutation revealed a decrease in the expression of POMGNT1 mRNA and protein because of negative regulation of the POMGNT1 promoter by the transcription factor ZNF202 (zinc-finger protein 202). By functional analysis of various luciferase constructs, we localized a proximal POMGNT1 promoter and we found a 75% decrease in luciferase activity in the mutant construct when compared with the wild type. Electrophoretic mobility shift assay (EMSA) revealed binding sites for the Sp1, Ets1 and GATA transcription factors. Surprisingly, the mutation generated an additional ZNF202 binding site and this transcriptional repressor bound strongly to the mutant promoter while failing to recognize the wild-type promoter. Although the genetic causes of dystroglycanopathies are highly variable, they account for only 50% of the cases described. Our results emphasize the importance of extending the mutational screening outside the gene-coding region in dystroglycanopathy patients of unknown aetiology, because mutations in noncoding regions may be the cause of disease. Our findings also underline the requirement to perform functional studies that may assist the interpretation of the pathogenic potential of promoter alterations.

Restriction mapping of the rRNA genes from Artemia larvae

Abstract A restriction endonuclease analysis of the genes coding for the ribosomal RNA from Artemia larvae has shown that these genes consist of a repeat unit of 16.2 kilobase pairs (10.7 Mdaltons) and that the repeat unit seems to be homogeneous in size.

A double homozygous mutation in the POMT1 gene involving exon skipping gives rise to Walker-Warburg syndrome in two Spanish Gypsy families.

To the Editor : Alpha-dystroglycanopathies are a large group of autosomal recessive muscular dystrophies that involve defects in α-dystroglycan (α-DG) Oglycosylation. Walker–Warburg syndrome (WWS, OMIM 236670) is the most severe alpha-dystroglycanopathy, and affected infants rarely survive past the first year of life. The phenotype mainly involves muscle, eye and brain abnormalities associated with anomalous neuronal migration, and it overlaps with Fukuyama congenital muscular dystrophy (FCMD, OMIM 253800) and muscleeye-brain (MEB) disease (OMIM 253280), other severe alpha-dystroglycanopathies (1, 2). Mutations in six genes are known to cause alphadystroglycanopathies (3), all encoding known or putative glycosyltransferases: POMT1, POMT2, POMGnT1, fukutin, FKRP and LARGE (4–10). Mutations in POMT1 are responsible for approximately 25% of WWS cases and its targeted disruption in mice provokes embryonic lethality (11). Murine Pomt1 is predominantly expressed in the tissues most severely affected in WWS patients, this expression persisting in the muscles, eyes, brain and cerebellum (11, 12). Here, we describe a double homozygous mutation in the POMT1 gene in two unrelated Gypsy families, reflecting the often higher incidence of recessive diseases in endogamic populations. Family 1. Two affected male siblings, patients 1.1 and 1.2, were born to second-degree cousins in a Spanish Gypsy family. Patient 1.1 had profound muscular hypotonia at birth and a creatine kinase (CK) value of 8,400 IU/l. Muscular dystrophy was later confirmed by biopsy although merosin appeared normal. The infant showed retromicrognatia, low implanted ears, microphthalmia, cataract, microcornia and retinal dysplasia. Brain MRI identified type II lyssencephaly, hydrocephalus, agenesis of the corpus callosum, hypomyelination, cerebellar and brain stem hypoplasia, and Dandy–Walker malformation. He died at 4 months. Patient 1.2 displayed similar symptoms and had a similar appearance to his brother, but there are no biological samples and his age of death was not determined. Family 2. This affected male sibling, patient 2.1, was born to double first-degree cousins in a Spanish Gypsy family. The patient had profound paralysing muscle hypotonia and a CK value of 40,851 IU/L at birth. Muscle biopsy confirmed muscular dystrophy although merosin appeared normal. Ocular examination revealed microphthalmia and bilateral retinal detachment. The patient also exhibited hypertelorism, frontal bossing and low implanted ears. Brain MRI showed lissencephaly, hydrocephalus, agenesis of the septum pellucidum and of the posterior corpus callosum, and cerebellar hypoplasia. The patient died 10 days after birth. Analysis of the complete coding region of the POMT1 gene revealed a double homozygous mutation in both patients in reference to the most common POMT1 splice variant (NM_001077 365.1) (13). The first homozygous mutation, g.3553G>T (NC_000009.10), was found at the beginning of intron 4 at the exon–intron boundary (Fig. 1Aa). Indeed, the mutation produced the loss of the entire exon 4 in mRNA transcripts from patients 1.1 and 2.1 (Fig. 1Ab). As this loss of 17 amino acids did not alter the reading frame, the ensuing protein has 708 instead of 725 amino acids. Exon 4 is located at Loop 1 of the POMT1 protein, within the conserved pfam02366 domain (protein mannosyltransferase, PMT) that potentially influences the protein’s

Ribosomal ribonucleic acids from the crustacean Artemia

Ribosomal RNAs (rRNAs) in eukaryotic organisms comprise 4 well-defined molecular entities, with standard sedimentation coefficients of 28 S, 18 S, 5.8 S and 5 S. 5.8 S rRNA is attached to 28 S rRNA by hydrogen bonding [l]. Since the discovery [2] that some insect 28 S rRNA is dissociated into two 18 S products after different denaturing treatments (heat, urea or dimethylsulfoxide, Me,SO), a large number of other organisms have been shown to have a hidden break in the 28 S rRNA, most of them being protostomes [3]. We are interested in the control of gene expression during the embryogenesis of the crustacean Artemiu, particularly of the rRNA genes. The restriction map of these genes has been reported [4] and we now present the molecular species which are present in the ‘28 S rRNA complex’ and the other rRNAs from Artemia embryos.

Expression pattern in retinal photoreceptors of POMGnT1, a protein involved in muscle-eye-brain disease

Purpose The POMGNT1 gene, encoding protein O-linked-mannose β-1,2-N-acetylglucosaminyltransferase 1, is associated with muscle-eye-brain disease (MEB) and other dystroglycanopathies. This gene’s lack of function or expression causes hypoglycosylation of α-dystroglycan (α-DG) in the muscle and the central nervous system, including the brain and the retina. The ocular symptoms of patients with MEB include retinal degeneration and detachment, glaucoma, and abnormal electroretinogram. Nevertheless, the POMGnT1 expression pattern in the healthy mammalian retina has not yet been investigated. In this work, we address the expression of the POMGNT1 gene in the healthy retina of a variety of mammals and characterize the distribution pattern of this gene in the adult mouse retina and the 661W photoreceptor cell line. Methods Using reverse transcription (RT)–PCR and immunoblotting, we studied POMGNT1 expression at the mRNA and protein levels in various mammalian species, from rodents to humans. Immunofluorescence confocal microscopy analyses were performed to characterize the distribution profile of its protein product in mouse retinal sections and in 661W cultured cells. The intranuclear distribution of POMT1 and POMT2, the two enzymes preceding POMGnT1 in the α-DG O-mannosyl glycosylation pathway, was also analyzed. Results POMGNT1 mRNA and its encoded protein were expressed in the neural retina of all mammals studied. POMGnT1 was located in the cytoplasmic fraction in the mouse retina and concentrated in the myoid portion of the photoreceptor inner segments, where the protein colocalized with GM130, a Golgi complex marker. The presence of POMGnT1 in the Golgi complex was also evident in 661W cells. However, and in contrast to retinal tissue, POMGnT1 additionally accumulated in the nucleus of the 661W photoreceptors. Colocalization was found within this organelle between POMGnT1 and POMT1/2, the latter associated with euchromatic regions of the nucleus. Conclusions Our results indicate that POMGnT1 participates not only in the synthesis of O-mannosyl glycans added to α-DG in the Golgi complex but also in the glycosylation of other yet-to-be-identified proteins in the nucleus of mouse photoreceptors.Esta investigacion ha sido financiada por los proyectos GRE12-03 y GRE14-05 (Universidad de Alicante).

Clinical Features and Molecular Characterization of a Patient With Muscle-Eye-Brain Disease A Novel Mutation in the POMGNT1 Gene

Muscle-eye-brain disease is a congenital muscular dystrophy characterized by structural brain and eye defects. Here, we describe a 12-year-old boy with partial agenesis of corpus callosum, ventriculomegaly, flattened brain stem, diffuse pachygyria, blindness, profound cognitive deficiencies, and generalized muscle weakness, yet without a clear dystrophic pattern on muscle biopsy. There was no glycosylation of α-dystroglycan and the genetic screening revealed a novel truncating mutation, c.1545delC (p.Tyr516Thrfs*21), and a previously identified missense mutation, c.1469G>A (p.Cys490Tyr), in the protein O-mannose beta-1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) gene. These findings broaden the clinical spectrum of muscle-eye-brain disease to include pronounced hypotonia with severe brain and eye malformations, yet with mild histopathologic changes in the muscle specimen, despite the absence of glycosylated α-dystroglycan.