The studies described in this application are designed to elucidate the mechanism of assembly and processing of intestinal apolipoprotein B (apo B). Intestinal apo B is central to the regulation of lipid transport from enterocytes, and is structurally and physiologically distinct from apo B of hepatic origin. In abetalipoproteinemia, the inability to secrete apo B containing lipoproteins may be related to faulty posttranslational processing of apo B rather than a genetically determined absence of protein synthesis. Until recently, data concerning assembly and posttranslational processing of human intestinal apo B have been difficult to obtain because of the lact of an appropriate model system. However, we have recently demonstrated that an established cell line from human colon carcinoma (Caco-2) that spontaneously differentiates in confluent culture and expresses functions characteristic of differentiated small intestinal enterocytes can be utilized to study intestinal apo B. The following posttranslational events will be examined: glycosylation, phosphorylation, sulfation and fatty acid acylation. Metabolic labeling will be performed in Caco-2 cells using appropriate radioactive precursors followed by immunoprecipitation with affinity-purified antibody, and analysis of the immunoprecipitates by SDS-PAGE. The role of glycosylation and oligosaccharide processing will be delineated by pretreating cells with antibiotics that interfere with protein glycosylation and oligosaccharide processing. Determination of oligosaccharide structure(s) will be performed utilizing endo- and exoglycosidases, lectin-affinity columns and high performance liquid chromatography. This will allow an examination of alterations in carbohydrate during posttranslational modification and initial stage of intracellular transport of the protein. It is anticipated that substantial new knowledge will be gained concerning the mechanism of apo B synthesis and processing. This should contribute to the understanding of intestinal lipid metabolism and determination of metabolic defects in various lipid disorders.
| Sasak, W V; Lown, J S; Colburn, K A (1991) Human small-intestinal apolipoprotein B-48 oligosaccharide chains. Biochem J 274 ( Pt 1):159-65 |
| Sasak, W V; Buller, H A; Reinhold, R (1989) Biosynthetic precursor (214 kDa) of apolipoprotein B-48 is not secreted by Caco-2 cells and normal human intestine. Biochem J 264:365-70 |
