A soluble form of Sda-beta 1,4-N-acetylgalactosaminyltransferase is released by differentiated human colon carcinoma CaCo-2 cells.
We have previously shown that human colon carcinoma CaCo-2 cells express the Sda-beta 1,4-N-acetylgalactosaminyltransferase (Sda-beta GalNAc-transferase) and that the enzyme activity correlates with the degree of enterocytic differentiation. Here we report that a large amount of this glycosyltransferase is released in soluble form, particularly when CaCo-2 cells are maintained in culture for more than 3 weeks in order to ensure an higher degree of enterocyte differentiation. The soluble enzyme was concentrated and partially purified by Blue-Sepharose and fetuin-Sepharose chromatography. The substrate specificity of the partially purified enzyme was similar to that of Sda-enzyme from epithelial cells of colon mucosa, and for its activity strictly required the presence in acceptors of NeuAc in alpha 2,3-linkage to subterminal galactose. Among the low molecular glycans tested, NeuAc alpha 2,3Gal beta 1,4GlcNAc appeared to be the best acceptor, whereas sialyl-Lewisx and sialyl-Lewisa did not serve as acceptors, indicating that the fucosylation of sub-terminal GlcNAc hindered the transferase activity. Contrary to this, the activity towards a disialylated acceptor such as di-sialyl-lacto-N-tetraose was reduced but not abolished. When CaCo-2 cells were cultured on porous membranes and the transferase activity assayed in medium collected from chambers corresponding to either the apical or basolateral face of highly differentiated CaCo-2 cells, a preferential release from the basolateral surface was found. Considering that Sda-beta GalNAc-transferase is mainly located in the large intestine, current results support the notion that colonic cells largely contribute to the presence of the enzyme in human plasma.
Serafini-Cessi
F
F
Dipartimento di Patologia sperimentale, Università di Bologna, Italy.
Malagolini
N
N
Guerrini
S
S
Turrini
I
I
D018938:Caco-2 Cells; D002240:Carbohydrate Sequence; D002454:Cell Differentiation / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000378:metabolism*; D012995:Solubility; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D017350:N-Acetylgalactosaminyltransferases; D000097763:Polypeptide N-acetylgalactosaminyltransferase