Mucin biosynthesis revisited: The enzymatic transfer of gal in β1,3 linkage to the GalNAc moiety of the core structure R₁-GlcNAcβ1,6GalNAcα-O-R₂

Synthetic glycosides containing the core, -Glc-NAcβ1,6GalNAcα-, acted as acceptors for β-galactosyltransferase of human ovarian tumor. A significant amount of Gal was transferred from UDP-Gal (100 nmol) to the α-benzylglycoside of LacNAcβ1,6GalNAc (LGBn) (25.1 nmol of Gal) and the α-ortho-nitrophenylglycosides of LacNAcβ1,6GalNAc (22.0 nmol of Gal), GlcNAcβ1,6GalNAc (15.5 nmol of Gal), and Fucα1,3GlcNAcβ1,6GalNAc (25.9 nmol of Gal); LacNAcβ1,6(Galβ1,3)GalNAcα-O-Bn (where Bn is benzyl) was almost inactive (only 1.2 nmol of Gal), indicating the Gal transfer to the α-GalNAc moiety. The product from LGBn was isolated in microgram quantities and identified by fast atom bombardment mass spectrometry as LacNAcβ1,6(Galβ1,3)GalNAcα-O-Bn. The αGalNAc:β1,3Gal transferase was present in high concentration in ovarian tumor tissue (ovarian cancer serum → 1.4; ascitic fluid → 0.9; tumor → 17.4). Asialo Cowper's gland mucin (ACGM) at 5 mg/ml reaction mixture inhibited the transfer of Gal to LGBn (25.2 and 53.4% respectively for 2 and 18 h incubation at 37°C); inhibition by LGBn was 13.4 and 24.5%, respectively. In contrast to the inhibition by ACGM (25.2-31.6%), there was substantial increase (13.4-35.7%) in the inhibition by LGBn, when the incubation for 2 h at 37°C was continued for 40 h at 4°C, indicating the high affinity of LGBn for the enzyme at lower temp. K_m for LGBn in presence of ACGM was 7.6 mM and in absence, 2.7 mM; K_m for ACGM (M_r 200,000) in presence of LGBn was 16.1 μM and K_i for ACGM (as the inhibitor) was 41.7 μM. In comparison with two normal ovarian tissues, the enzyme was found to be low (55-67%) in three ovarian tumors and high (146-260%) in two ovarian and one uterus tumors, as measured with ACGM; the synthetic acceptors showed similar activities. The enzyme had nearly the same extent of activity in the pH range 6-8. Fucα1,3GlcNAcβ1,6GalNAcα-O-ONP had the highest affinity for the enzyme. The present study demonstrates the feasibility of β1,3Gal attachment on αGalNAc, which has already been substituted by β1,6GlcNAc, then elongated by β1,4Gal and also terminated by α1,3Fuc.