RESUMO
Olfactomedin-like (OLFML) proteins are members of the olfactomedin domain-containing secreted glycoprotein (OLF) family. OLFML2A and OLFML2B are representative molecules of these glycoproteins. Olfactomedins are critical for the development and functional organization of the nervous system and retina, which is a highly conserved structure in vertebrates, having almost identical anatomical and physiological characteristics in multiple taxa. Spotted gar, a member of the Lepisosteidae family, is a freshwater fish that inhabits rivers, bayous, swamps, and brackish waters. Recently, the complete genome has been sequenced, providing a unique bridge between fish medical models to human biology, making it an excellent animal model. This study was aimed to understanding the evolution OLFML2A and OLFML2B in the retina of spotted gar through looking for the expression of these genes. Spotted gar retina was analyzed with hematoxylin-eosin staining assays to provide an overall view of the retina structure and an immunofluorescence assay to identify OLFML2A and OLFML2B protein expression. A phylogenetic tree was created using the neighbor-joining method. Forces that direct the evolution of the fish genes were tested. Spotted gar retina, as in other vertebrates, is made of several layers. OLFML2A and OLFML2B proteins were detected in the rod and cone photoreceptor layer (PRL), outer nuclear layer (ONL), and inner nuclear layer (INL). Phylogenetic tree analysis confirms the orthology within the OLFML2A gene. Purifying selection is the evolutionary force that directs the OLFML2A genes. OLFML2A genes have a well-conserved function over time and species.
Assuntos
Biologia Computacional/métodos , Mineração de Dados/métodos , Proteínas da Matriz Extracelular/metabolismo , Peixes/metabolismo , Glicoproteínas/metabolismo , Retina/metabolismo , Animais , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica , Glicoproteínas/genética , Proteínas de Membrana , TranscriptomaRESUMO
In humans, the polygenic growth hormone (GH) locus is located on chromosome 17 and contributes with three types of proteins: pituitary GH which consists of at least two isoforms one of 22â¯kDa and the other of 20â¯kDa, placental GH, which also exhibits isoforms, and chorionic somatomammotropin hormone (CSH). While pituitary GH results from the expression of the GH-1 (GH-N) gene, placental GH is produced by the expression of the GH-2 (GH-V) gene and CSH is contributed by expression of the CSH-1 and CSH-2 genes. The location where GH-1 is expressed is the anterior pituitary and the rest of the genes in the locus are expressed in placenta. On the other hand, expression and synthesis of GH in extra-pituitary tissues, including the eye, has been recently described. However, the physiological role of GH in the eye has not yet been elucidated, although a possible neuroprotective role has been hypothesized. Thus, we analyzed GH-1, GH-2, CSH1/2, Pit-1, GHR, GHRH, GHRHR, SST, SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 to elucidate the expression and regulation of the GH locus in the human eye. Through qPCR analysis, we only found evidence of GH-1 expression in retina, choroid and trabecular meshwork; its transcript turned out to be the same as pituitary GH mRNA found in major species, and no splicing variants were detected. PIT1 was absent in all the ocular tissues implying an independent GH-1 expression mechanism. We found evidence of GHR in the cornea, choroid coat and retina. These results suggest autocrine and/or paracrine regulation, possibly exerted by GHRH and SSTs (since their mRNAs and receptors were found predominantly in retinal, choroidal and corneal tissues) since expression of both molecules was detected in different ocular tissues, as well as in the same tissues where GH-1 expression was confirmed. Our results add solid evidence about the existence of a regulatory local system for GH expression and release in the human eye.
Assuntos
Olho/metabolismo , Hormônio do Crescimento/metabolismo , Receptores da Somatotropina/metabolismo , Somatostatina/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Hormônios Placentários , Adulto JovemRESUMO
BACKGROUND: The olfactomedin-like domain (OLFML) is present in at least four families of proteins, including OLFML2A and OLFML2B, which are expressed in adult rat retina cells. However, no expression of their orthologous has ever been reported in human and baboon. OBJECTIVE: The aim of this study was to investigate the expression of OLFML2A and OLFML2B in ocular tissues of baboons (Papio hamadryas) and humans, as a key to elucidate OLFML function in eye physiology. METHODS: OLFML2A and OLFML2B cDNA detection in ocular tissues of these species was performed by RT-PCR. The amplicons were cloned and sequenced, phylogenetically analyzed and their proteins products were confirmed by immunofluorescence assays. RESULTS: OLFML2A and OLFML2B transcripts were found in human cornea, lens and retina and in baboon cornea, lens, iris and retina. The baboon OLFML2A and OLFML2B ORF sequences have 96% similarity with their human's orthologous. OLFML2A and OLFML2B evolution fits the hypothesis of purifying selection. Phylogenetic analysis shows clear orthology in OLFML2A genes, while OLFML2B orthology is not clear. CONCLUSIONS: Expression of OLFML2A and OLFML2B in human and baboon ocular tissues, including their high similarity, make the baboon a powerful model to deduce the physiological and/or metabolic function of these proteins in the eye.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Olho/metabolismo , Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Animais , Código de Barras de DNA Taxonômico , Evolução Molecular , Proteínas da Matriz Extracelular/análise , Proteínas da Matriz Extracelular/genética , Olho/química , Imunofluorescência/métodos , Glicoproteínas/análise , Glicoproteínas/genética , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Fenômenos Fisiológicos Oculares , Papio , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Reversa , Análise de Sequência de ProteínaRESUMO
BACKGROUND: The olfactomedin-like domain (OLFML) is present in at least four families of proteins, including OLFML2A and OLFML2B, which are expressed in adult rat retina cells. However, no expression of their orthologous has ever been reported in human and baboon. OBJECTIVE: The aim of this study was to investigate the expression of OLFML2A and OLFML2B in ocular tissues of baboons (Papio hamadryas) and humans, as a key to elucidate OLFML function in eye physiology. METHODS: OLFML2A and OLFML2B cDNA detection in ocular tissues of these species was performed by RT-PCR. The amplicons were cloned and sequenced, phylogenetically analyzed and their proteins products were confirmed by immunofluorescence assays. RESULTS: OLFML2A and OLFML2B transcripts were found in human cornea, lens and retina and in baboon cornea, lens, iris and retina. The baboon OLFML2A and OLFML2B ORF sequences have 96% similarity with their human's orthologous. OLFML2A and OLFML2B evolution fits the hypothesis of purifying selection. Phylogenetic analysis shows clear orthology in OLFML2A genes, while OLFML2B orthology is not clear. CONCLUSIONS: Expression of OLFML2A and OLFML2B in human and baboon ocular tissues, including their high similarity, make the baboon a powerful model to deduce the physiological and/or metabolic function of these proteins in the eye.
