Secretion of human apoA-I lipoprotein by transfected MDCK cells




Sinclair-Worley, Leslie

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Human apoA-I was selected to study the process of secretion in Madin-Darby canine kidney (MDCK) cells, a polarized epithelial cell line. Permanently secreting transfected cells, chosen on the basis of antibiotic resistance, were screened for the secretion of normal human apoA-I. The clone ApoAIN2A was grown on filter inserts to analyze the dynamics of secretion by pulse-chase labeling. Immunoprecipitation of samples with time of secreted apoA-I revealed that it was predominantly released basolaterally. However, if the cells were labeled continuously for longer time periods, increasing amounts of apoA-I were detected apically. To rule out the possibility of transcytosis of apoA-I to the apical surface, cells were incubated in the presence of radiolabeled apoA-I in the basal chamber. No significant radiolabeled apoA-I was found in the apical chamber. Immunofluorescence photomicroscopy of the clone revealed perinuclear labeling consistent with the presence of apoA-I in the Golgi complex. The secretion of altered forms of apoA-I by other MDCK clones transfected with mutated apoA-I constructs was predominantly through the basolateral surface in all cases. To test the effects of nutritional factors on the secretion of apoA-I, N2A cells were cultured in 2.0% FBS or in serum free medium containing both 100 μM Zn\sp2+, 80 μM Fe\sp2+. In both cases, the pattern of secretion was not altered. An enhancement in the amount of apoA-I secreted was only observed for cells incubated in 2.0% FBS. Numerous ultrastructural similarities were observed between N2A and CaCo-2 cells which secret human apoA-I endogenously. These included translucent intracellular inclusions, that most likely consist of glycogen and whorls of membrane, that suggest increased lipid synthesis. The N2A cell also possessed vesicles containing small spherical particles that resemble precursor HDL particles. Thus, the transfected MDCK cells have all of the machinery necessary for intracellular HDL assembly and can be used to study this process.



Biological sciences, Antibiotic resistance, Secretion of proteins