Efectos del inhibidor de glicosilación benzil GalNAc sobre las rutas secretora y endocítica de células epiteliales

Abstract

El benzil GalNAc fue descrito inicialmente como un inhibidor de la O-glicosilación de tipo mucina (Kuan et al., 1989). El tratamiento crónico de células mucosecretoras HT-29 M6 con este fármaco produjo una serie de efectos entre los que destacan: i) reducción de la proliferación, ii) inhibición de la secreción de mucinas, iii) acumulación de glicoproteínas apicales en vesículas citoplasmáticas electronlúcidas y, iv) reducción de la sialilación de glicoproteínas (Huet et al., 1998; (Hennebicq-Reig et al., 1998). El benzil GalNAc también inducía estos efectos sobre la población HT-29 parental y sobre otras poblaciones celulares derivadas de ésta, independientemente de su fenotipo. El benzil GalNAc es metabolizado a benzil GalNAc-Gal, el cual a su vez inhibe competitivamente la actividad ?2,3 sialiltransferasa de células HT-29, la actividad sialiltransferasa mayoritariamente expresada en estas células. Como resultado de ésto, se sintetizan compuestos sialilados como benzil GalNAc-Gal?2,3Neu5Ac (Huet et al., 1995; Delannoy et al., 1996). Por otro lado, el tratamiento con benzil GalNAc no afectaba sensiblemente a las células Caco-2, las cuales expresan fundamentalmente ?2,6 sialiltransferasas, enzimas que no son inhibidas por los metabolitos derivados del benzil GalNAc (Huet et al., 1998).<br/>El objetivo de este trabajo ha sido determinar el factor(es) responsable(s) del origen del fenotipo de células HT-29 tratadas crónicamente con benzil GalNAc. Más concretamente, nos interesó determinar cómo un inhibidor de glicosilación produce tantos efectos diferentes y si el efecto de este inhibidor estaba restringido o no a células derivadas de HT-29. La hipótesis que se manejó inicialmente proponía que los efectos globales del tratamiento con benzil GalNAc podrían estar causados por la hiposialilación de tardíos como, Rab7 y LBPA, pero no de endosomas primarios (Rab5, EEA1) y de Golgi (GRASP65, TGN46), colocalizaban con la ?1 integrina de las vesículas. El conjunto de los resultados obtenidos indica que las vesículas-BG corresponden a una población heterogénea de endosomas aberrantes, relacionados con endosomas tardíos.<br/>Las características fenotípicas de las células tratadas con benzil GalNAc reproducen parcialmente los defectos descritos en células de algunas enfermedades de depósito lisosomal (EDL), concretamente las que se producen por la acumulación de sacáridos en compartimentos degradativos. Así, la morfología de las vesículas-BG es similar a la de las que se originan en células tratadas con sacarosa (sacarosomas) y varias EDL como la sialidosis o la infantile sialic acid storage disease (ISSD). Análisis del procesamiento de la AAG en fibroblastos de pacientes con ISSD mostraron defectos similares a los encontrados en células HT-29 M6 tratadas con benzil GalNAc. Además, las células tratadas con benzil GalNAc también acumulan LBPA y colesterol, hallazgos característicos de algunas EDL. Confirmando la idea de que el benzil GalNAc induce un fenotipo de depósito lisosomal, el tratamiento con sacarosa en células IMIM-PC-1 produjo cambios sobre la morfología de las células y la acumulación intracelular de MUC1 y ?1 integrina, de manera semejante a lo observado en las células tratadas con benzil GalNAc. La sialilación de glicoproteínas en células IMIM-PC-1 tratadas con sacarosa no estaba alterada, resultado que indica que la hiposialilación no es un requisito para la acumulación intracelular de glicoproteínas de membrana.<br/>Dado que células tratadas con benzil GalNAc acumulan una gran cantidad de oligosacáridos derivados de esta droga, situación análoga a la que ocurre en células tratadas con sacarosa y algunas EDL, proponemos que la acumulación de estos metabolitos, y no la hiposialilación de glicoproteínas, es la que origina el fenotipo global de células HT-29 M6 e IMIM-PC-1 tratadas con este fármaco.

Benzyl GalNAc was initially described as an inhibitor of mucin type O-glycosilation (Kuan et al., 1989). Long term exposure with this pharmac on mucosecretor HT-29 M6 colon cancer cells induced several effects: i) reduction of proliferation, ii) inhibition of the mucins secretion, iii) accumulation of apical glycoproteins into electron-lucid cytoplasmic vesicles (BG-vesicles) and iv) reduction of glycoprotein sialylation (Huet et al., 1998; Hennebicq-Reig et al., 1998). Moreover, these effects were induced in HT-29 parental and derived cells irrespectively of their phenotype. In HT-29 cells, benzyl GalNAc is metabolized to benzyl GalNAc-Gal, which in turn inhibits ?2,3 sialyltransferase activity, the most common in these cells. As a consequence of this, diverse sialylated compounds like benzyl GalNAc-Gal ?2,3 Neu5Ac are synthesised (Huet et al., 1995; Delannoy et al., 1996). On the other hand, benzyl GalNAc treatment did not affect Caco-2 colon cancer cells, which express fundamentally ?2,6 sialyltransferases, enzymes that are not inhibited by benzyl GalNAc (Huet et al., 1998).<br/>The aim of this work has been to determine the factor(s) responsible for the origin of the phenotype of HT-29 cells treated chronically with benzyl GalNAc. More in detail, we were interested in determine how a glycosylation inhibitor could produce a spectra of different effects and whether these effects were restricted only to HT-29 cells or not. Initially we worked with the hypothesis that the global effects of benzylGalNAc were derived from the glycoprotein hiposialylation. This hypothesis presumed a requirement of sialic acid for the apical glycoprotein transport/sorting. Thus, in the absence of sialic acid, this glycoproteins would be accumulated in TGN derived exocitic carriers, corresponding to the electron-lucid cytoplasmic vesicles (BG-vesicles).<br/>Consistent with this idea, we found through lectin analysis that ?2,3 linked sialic acid was distributed mainly in the apical membrane of epithelial cells both in culture and in tissues. Notoriously, ?2,6 linked sialic acid show a broad distribution and was located both in the apical membrane or in the basolateral membrane.<br/>The analysis of the treatment with benzyl GalNAc on a panel of cell lines indicated that its effects and severity were cell type depending but were not restricted only to HT-29 cells. Thus, benzyl GalNAc treated IMIM-PC-1 pancreas cancer cells shown a similar phenotype than benzyl GalNAc treated HT-29 cells. Notoriously, in these cells both apical (eg. MUC1) and basolateral (eg. ?1-integrin) glycoproteins were accumulated and colocalised into cytoplasmic vesicles. <br/>Benzyl GalNAc treatment affected the processing of apical and lysosomal glycoproteins in HT-29 M6 cells. We found that this pharmac induced a delay in maturation of cathepsin D and the blockage -in a post-TGN compartment- of the maturation of the lysosomal ?-glucosidasa.<br/>The endosomal route was affected in IMIM-PC-1 and HT-29 cells treated with benzyl GalNAc. Thus, benzyl GalNAc induced a reduction in the endocytosis rate and an accumulation of internalised material in IMIM-PC-1 cells. This material colocalised with vesicles positive for MUC1 or ?1 integrin. Late endosomes markers (Rab7, LBPA) but not early endosomes markers (Rab5, EEA1) or Golgi markers (GRASP65, TGN46) colocalised with vesicles accumulating ?1 integrin. Altogether, these results indicate that BG-vesicles correspond to an heterogenous population of aberrant endosomes related with late endosomes.<br/>The phenotype of benzyl GalNAc treated cells partially reproduces the defects seen in cells where saccharides are accumulated in the degradative compartiments. Thus, the morphology of the BG-vesicles is similar to those originated in sucrose treated cells (sucrosomes) and in cells from lysosome storage diseases (LSD) patients. The analysis of the processing of the lysosomal ?-glucosidase in fibroblasts of Infantile Sialic acid Storage Disease patients shown similar defects to those found in benzyl GalNAc HT-29 M6 treated cells. Moreover, benzyl GalNAc treated cells accumulate LBPA and cholesterol, commonly found in some LSD patients. Consistent with the idea that benzyl GalNAc induces a lysosomal storage defect, the sucrose treatment on IMIM-PC-1 cells induced the cytoplasmic accumulation of MUC1 and ?-1 integrin. In these cells, the glycoprotein sialylation was not affected, suggesting that in benzyl GalNAc treated cells the hiposialytion of membrane glycoproteins may not originate their intracelullar accumulation.<br/>In benzyl GalNAc treated cells a huge amount of benzyl GalNAc derived metabolites are accumulated, a scenario analogous to the sucrose treated or LSD cells. Therefore, we propose that is the metabolite accumulation, but not the glycoprotein hiposialylation, the responsible for the induction of the overall phenotype seen in benzyl GalNAc HT-29 and IMIM-PC-1 treated cells.