Biosynthesis and transport of lysosomal α-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 × 10(3) Mr. This form is converted into a precursor of slightly higher Mr (112 × 10(3)) by the addition of complex oligosaccharide chains. Via an intermediate form of 95 × 10(3) Mr, this precursor is processed into a mature form of 76 × 10(3) Mr. Combination of metabolic labelling with subcellular fractionation showed that the 112 × 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.

Immunomodulatory activity of enzymatically synthesized glycogen and its digested metabolite in a co-culture system consisting of differentiated Caco-2 cells and RAW264.7 macrophages

“…Klumperman et al showed that a-glucosidase was secreted from the apical membrane of Caco-2 cells. 25 Meanwhile, as described in the Materials and methods section, to obtain RG, ESG was treated with a-amylase. Although a-amylase is an endo-acting enzyme and a-glucosidase is an exo-acting enzyme against glucan, aamylase seemed to trim off only the peripheral parts of the ESG molecules in the previous report.…”

Biocatalytic Synthesis of the Rare Sugar Kojibiose: Process Scale-Up and Application Testing

“…Therefore, we conclude that most probably, kojibiose is not easily taken up by enterocytes as such and therefore, has a high chance of reaching the intestinal microbiota in relatively high concentrations, which is a requirement for prebiotic activity. Yet, it needs to be noted that Caco-2 cells are able to secrete α-glucosidases at their brush-border side − and that kojibiose can be cleaved by rat rodent α-glucosidases . In our assay, the α-glucosidase activity of the Caco-2 cells was not measured, and therefore, its concentration may be too low to see effects.…”