THE IMMUNOCYTOCHEMICAL LOCALIZATION OF APOPROTEIN A-I IN RAT JEJUNUM

Abstract

It is well established that the epithelial cells of the small intestine have the ability to absorb products of intraluminal lipolysis, resynthesize triglycerides, synthesize phospholipids and apoproteins, and combine these to form lipoprotein complexes. This association of protein with lipid apparently permits its secretion and subsequent transport in the vascular system.;In order to identify the intracellular structures which are involved in the synthesis and secretion of apoproteins, I have utilized an immunocytochemical method for the detection of apoprotein A-I. Apo A-I comprises (TURN)50% of rat lymph chylomicron protein and 40% of high density lipoprotein (HDL). SDS-PAGE bands of apo A-I isolated from a delipidated HDL fraction were homogenized and injected into rabbits. Antisera and IgG fractions gave single immunoprecipitin lines with isolated apo A-I or with rat serum. To test binding, as distinct from precipitating specificity, 'Western blotting' techniques were used for the apoproteins. Control and specific IgG were digested with pepsin to yield F(ab')(,2) fragments and conjugated to horseradish peroxidase (HRP). Indirect immunofluorescence stainings with F(ab')(,2) on rat jejunum showed specific localization in supranuclear vacuolar elements in the absorptive cells. Nonimmune controls gave no analogous staining. When HRP conjugated fragments were used to stain fixed tissue sections, reaction product was additionally seen within the intercellular spaces of the absorptive cells and those of the lamina propria. At the ultrastructural level, reaction product was visible in rough ER, smooth ER, elements of the Golgi apparatus, some portions of GERL, secretory vacuoles, the intercellular spaces of the epithelial cells whose basolateral membranes exhibit clathrin coats, and between the connective tissue elements of the lamina propria. The specific binding activity of the HRP-F(ab')(,2) anti-rat apo A-I conjugate was removed by preadsorption with apo HDL. Nonimmune reagent showed no specific binding. Specific staining was not seen in other organelles, such as mitochondria or microperoxisomes. Thus, direct visualization showed the secretory pathway of apo A-I to involve the Golgi apparatus and portions of GERL. While apoprotein A-I is not glycosylated, it appears to take the same secretory pathway followed by glycoproteins of chylomicra.