Biosynthesis and transport of lysosomal alpha-glucosidase in the human colon carcinoma cell line Caco-2 : secretion from the apical surface

Abstract

The human adenocarcinoma cell line Caco-2 was used for studies on the biosynthesis and transport of lysosomal acid alpha-glucosidase in polarized epithelial cells. Metabolic labelling revealed that in Caco-2 cells alpha-glucosidase is synthesized as a precursor form of 110 x 10(3) Mr. This form is converted into a precursor of slightly higher Mr (112 x 10(3)) by the addition of complex oligosaccharide chains. Via an intermediate form of 95 x 10(3) Mr, this precursor is processed into a mature form of 76 x 10(3) Mr. Combination of metabolic labelling with subcellular fractionation showed that the 112 x 10(3) Mr precursor of alpha-glucosidase is transported to the lysosomes. However, the same form is secreted into the culture medium (20% of newly synthesized enzyme after 4 h of chase). Immunoprecipitation of alpha-glucosidase from culture medium derived from either the apical or basolateral site of radiolabelled Caco-2 cells, showed that 70-80% of the total amount of precursor form present in the medium is secreted from the apical membrane. Measurement of enzyme activities also showed that alpha-glucosidase, unlike other lysosomal enzymes, is mainly secreted via the apical pathway. Furthermore, immunocytochemistry showed the presence of a precursor form of alpha-glucosidase on the apical, but not the basolateral, membrane of the Caco-2 cells. We conclude that alpha-glucosidase is, unlike all other secretory proteins studied so far, secreted preferentially from the apical membrane of Caco-2 cells