RESUMO
OBJECTIVE@#To investigate distribution specificity of human fucosyltransferase 5 (FUT5) as well as its expression and localization in spermatids.@*METHODS@#Human semen, vaginal swab, saliva and venous blood from healthy individuals were collected. The spermatids were isolated and the spermatid membrane protein was then extracted. Expression levels of FUT5 from human spermatid membrane, seminal plasma, vaginal fluid, saliva and serum were detected by immunoblotting technique. The expression and localization of FUT5 in spermatids were analyzed by immunofluorescent method.@*RESULTS@#Immunoblotting technique showed that FUT5 was expressed on spermatid membranes and in serum, but not in seminal plasma, vaginal fluid and saliva. The expressed FUT5 on spermatids was mostly localized on head of spermatids by fluorescent microscopy, suggesting that there was certain amount of FUT5 on human spermatid membrane, and the spermatids might be isolated from mixed stains with vaginal fluid by antigen-antibody reaction.@*CONCLUSION@#Human FUT5 shows a characteristic distribution specificity, and this feature may be used for identification of mixed stain involved in criminal sexual offence in future forensic practice.
Assuntos
Feminino , Humanos , Masculino , Membrana Celular/metabolismo , Imunofluorescência/métodos , Genética Forense/métodos , Fucosiltransferases/metabolismo , Immunoblotting , Saliva/metabolismo , Sêmen/metabolismo , Espermátides/metabolismo , Vagina/metabolismoRESUMO
The Notch signaling pathway plays an important role in development and physiology. In Drosophila, Notch is activated by its Delta or Serrate ligands, depending in part on the sugar modifications present in its extracellular domain. O-fucosyltransferase-1 (OFUT1) performs the first glycosylation step in this process, O-fucosylating various EGF repeats at the Notch extracellular domain. Besides its O-fucosyltransferase activity, OFUT1 also behaves as a chaperone during Notch synthesis and is able to down regulate Notch by enhancing its endocytosis and degradation. We have reevaluated the roles that O-fucosylation and the synthesis of GDP-fucose play in the regulation of Notch protein stability. Using mutants and the UAS/Gal4 system, we modified in developing tissues the amount of GDP-mannose-deshydratase (GMD), the first enzyme in the synthesis of GDP-fucose. Our results show that GMD activity, and likely the levels of GDP-fucose and O-fucosylation, are essential to stabilize the Notch protein. Notch degradation observed under low GMD expression is absolutely dependent on OFUT1 and this is also observed in Notch Abruptex mutants, which have mutations in some potential O-fucosylated EGF domains. We propose that the GDP-fucose/OFUT1 balance determines the ability of OFUT1 to endocytose and degrade Notch in a manner that is independent of the residues affected by Abruptex mutations in Notch EGF domains.
Assuntos
Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Fucosiltransferases/metabolismo , Guanosina Difosfato Fucose/metabolismo , Guanosina Difosfato Manose/metabolismo , Receptores Notch/metabolismo , Asas de Animais/metabolismo , Alelos , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/metabolismo , Endocitose/genética , Fucosiltransferases/genética , Guanosina Difosfato Fucose/genética , Guanosina Difosfato Manose/genética , Imuno-Histoquímica , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores Notch/genética , Transdução de Sinais , Asas de Animais/anatomia & histologiaRESUMO
This report concerns the stepwise biosynthesis in vitro of Sialyl Lewis X, (SA-Le(x)), a carcinoembryonic antigen, in human colon carcinoma KM12 cells exhibiting different metastatic behaviors. The significance of SA-Le(x) has become even more apparent since the detection of its terminal epitope NeuAc(alpha 2-3)Gal beta 1-4(Fuc alpha 1-3)GlcNAc-, as the binding ligand of the selectin family member ELAM-1. The activity level of galactosyltransferase GalT-4 which catalyzes the formation of core nLcOse4Cer (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) is very high in all the metastatic lines tested with highly metastatic lines (KM12-SM) exhibiting the highest activity. The same activity pattern for galactosyltransferase is also observed when tested with iLcOse5Cer (GlcNAc beta 1-3nLcOse4Cer), the precursor for polylactosamine glycolipid. Sialyltransferase SAT-3 which catalyzes the formation of LM1 (NeuAc alpha 2-3nLcOse4Cer), the precursor for SA-Le(x), is also present in all the metastatic cell lines although the activity levels are much lower compared to galactosyltransferase. The fucosyltransferase FucT-3, which catalyzes the formation of R'-Gal-Fuc(alpha 1-3)GlcNAc-R linkage, is active with both nonsialylated substrate, nLcOse4Cer, and sialylated substrate, LM1 (NeuAc alpha 2-3nLcOse4Cer) with the formation of either Le(x) (Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc-Cer) or SA-Le(x) (NeuAc alpha 2-3nLcOse4Cer). However, the sialylated substrate LM1 is preferred to enzymatic activity since it exhibited lower Km (46 microM) than that of nLcOse4Cer (67 microM).(ABSTRACT TRUNCATED AT 250 WORDS)