RESUMEN
In this study, we identified and characterized the mouse orthologue of the human chondrolectin gene, Chodl. Chodl is located at chromosome 16C3 and consists of six exons and five introns. The putative full-length mouse cDNA of Chodl consists of 2393 bp, with an open reading frame of 839 bp, 243 bp of 5' untranslated region and 1310 bp of 3' untranslated region. The predicted Chodl protein is a type I transmembrane protein containing one carbohydrate recognition domain (CRD) of C-type lectin in its extracellular portion and shares a significant similarity (45%) with layilin, a hyaluronan receptor. Reverse transcription-polymerase chain reaction and subsequent Southern blotting analysis revealed that in adult mice, Chodl is preferentially expressed in skeletal muscle, testis, brain, and lung. Analysis of the embryonic expression of Chodl showed that during gestation (embryonic day (E) 7-15) its expression is up-regulated. In situ hybridization on E15 mouse embryo revealed that Chodl is expressed in muscle cells of heterogeneous origin, including those from tongue, trunk, and tail. Furthermore, fluorescent immunostaining on limbs of newborn mice, localized the Chodl protein to striated muscle cells. Finally, Western blot analysis demonstrated expression of Chodl protein during the proliferation as well as differentiation phases of the myoblastic C2C12 cell line.
Asunto(s)
Lectinas Tipo C/genética , Músculo Esquelético/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Secuencia de Bases , Células COS , Línea Celular , Mapeo Cromosómico , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Embrión de Mamíferos/metabolismo , Expresión Génica , Genes/genética , Hibridación in Situ , Hibridación Fluorescente in Situ , Masculino , Ratones , Datos de Secuencia Molecular , Músculo Esquelético/citología , Análisis de Secuencia de ADNRESUMEN
Chondrolectin (CHODL) is a novel type I transmembrane protein containing one carbohydrate recognition domain (CRD) of C-type lectins. Recently, data base searching revealed a variant of CHODL (AK022689) with a different 5' leader sequence derived from a new putative upstream alternative promoter (P2). The P2 promoter gives rise to at least three additional alternatively spliced isoforms, designated as CHODLf, CHODLf Delta E, and CHODL Delta E. Of all variants, the alternative exon E-splicing isoforms (CHODLf Delta E/CHODL Delta E) are expressed exclusively in the T lymphocyte lineage and are regulated during T lymphopoiesis. Peripheral T lymphocytes demonstrated a unique exon E-splicing pattern in comparison with end maturation stage thymocytes, suggesting its association with the post-thymic maturation of T cells. Since exon E encodes the transmembrane domain of CHODL, the exon E-skipping variant results in a non-transmembrane domain-containing isoform (CHODLf Delta E/CHODL Delta E) terminating in the QDEL sequence, thus suggesting different functional attributes of CHODL isoforms during the development of T cells. Double label immunofluoresence experiments demonstrated that the transmembrane-containing isoform (CHODLf) colocalizes with rBet1 to the endoplasmic reticulum-Golgi apparatus. In summary, this study describes the molecular characterization of novel members of the chondrolectin family associated with T cell maturation and a subcellular localization of CHODLf in the endoplasmic reticulum-Golgi apparatus.
Asunto(s)
Empalme Alternativo , Membrana Celular/química , Expresión Génica , Lectinas Tipo C/genética , Proteínas de la Membrana/genética , Isoformas de Proteínas/genética , Linfocitos T/fisiología , Secuencia de Aminoácidos , Animales , Células de la Médula Ósea/química , Células de la Médula Ósea/citología , Células COS , Separación Celular , Retículo Endoplásmico/química , Exones , Técnica del Anticuerpo Fluorescente , Aparato de Golgi/química , Proteínas Fluorescentes Verdes , Hematopoyesis , Células Madre Hematopoyéticas/química , Células Madre Hematopoyéticas/citología , Humanos , Lectinas Tipo C/análisis , Lectinas Tipo C/química , Proteínas Luminiscentes/genética , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Microscopía Fluorescente , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Isoformas de Proteínas/análisis , Isoformas de Proteínas/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/ultraestructura , Transfección , Células Tumorales Cultivadas , Proteína Fluorescente RojaRESUMEN
CHODL, a novel human gene encoding chondrolectin, was isolated by PCR screening. It is localized at chromosome 21q21 and consists of six exons and five introns. The open reading frame of CHODL encodes a type I transmembrane protein containing a single carbohydrate recognition domain (CRD) of C-type lectins in its extracellular portion. CHODL was detected as a 2.6-kb transcript by northern blot using enriched human testis RNA. RT-PCR analysis revealed preferential expression of CHODL in testis, prostate, and spleen. Immunohistochemistry demonstrated that the expression of CHODL is mainly limited to vascular muscle of testis, smooth muscle of prostate stroma, heart muscle, skeletal muscle, crypts of small intestine, and red pulp of spleen. Western blot analysis revealed that CHODL is an N-glycosylated protein with a molecular weight of approximately 36 kDa. In transiently transfected COS1 cells, CHODL shows a predominantly perinuclear localization. Although the predicted CHODL protein shares a significant homology (45% overall and 60% within the CRD) with layilin, a recently identified hyaluronan receptor, we failed to detect a specific interaction between CHODL and hyaluronan using cetylpyridinium chloride precipitation.