Characterization of the specificities of human blood group H gene- specified α1,2-L-fucosyltransferase toward sulfated/sialylated/fucosylated acceptors: Evidence for an inverse relationship between α1,2-L-fucosylation of Gal and α1,6-L-fucosylation of asparagine-linked GlcNAc

The assembly of complex structures bearing the H determinant was examined by characterizing the specificities of a cloned blood group H gene- specified α1,2-L-fucosyltransferase (FT) toward a variety of sulfated, sialylated, or fucosylated Galβ1,3/4GlcNAcβ- or Galβ1,3GalNAcα-based acceptor structures. (a) As compared to the basic type 2, Galβ1,4GlcNAcβ- (K(m) = 1.67 mM), the basic type 1 was 137% active (K(m) = 0.83 mM). (b) On C-6 sulfation of Gal, type 1 became 142.1% active and type 2 became 223.0% active (K(m) = 0.45 mM). (c) On C-6 sulfation of GlcNAc, type 2 showed 33.7% activity. (d) On C-3 or C-4 fucosylation of GlcNAc, both types 1 and 2 lost activity. (e) Type 1 showed 70.8% and 5.8% activity, respectively, on C-6 and C-40-methylation of GlcNAc. (f) Type 1 retained 18.8% activity on α2,6- sialylation of GlcNAc. (g) Terminal type 1 or 2 of extended chain had lower activity. (h) With Gal in place of GlcNAc in type 1, the activity became 43.2%. (i) Compounds with terminal α1,3-linked Gal were inactive. (j) Galβ1,3GalNAcα- (the T-hapten) was ~0.4-fold as active as Galβ1,4GlcNAcβ-. (k) C-6 sulfation of Gal on the T-hapten did not affect the acceptor activity. (l) C-6 sulfation of GalNAc decreased the activity to 70%, whereas on C-6 sulfation of both Gal and GalNAc the T-hapten lost the acceptor ability. (m) C-6 sialylation of GalNAc also led to inactivity. (n) β1,6 branching from GalNAc of the T-hapten by a GlcNAc residue or by units such as Galβ1,4GlcNAc-, Galβ1,4(Fucα1,3)GlcNAc-, or 3- sulfoGalβ1,4GlcNAc- resulted in 111.9%, 282.8%, 48.3%, and 75.3% activities, respectively. (o) The enhancement of enzyme affinity by a sulfo group on C-6 of Gal was demonstrated by an increase (~5-fold) in the K(m) for Galβ1,4GlcNAcβ1,6(Galβ1,3)GalNAcα-O-Bn in presence of 6-sulfoGalβ1- 4GlcNAcβ-O-Me (3.0 mM). (p) Among the two sites in Galβ1,4GlcNAcβ1,6(Galβ1,3)GalNAcα-O-Bn, the enzyme had a higher affinity (>3-fold) for the Gal linked to GlcNAc. (q) With respect to Galβ1,- 3GlcNAcβ-O-Bn (3.0 mM), fetuin triantennary asialo glycopeptide (2.4 mM), bovine IgG diantennary glycopeptide (2.8 mM), asialo Cowper's gland mucin (0.06 mM), and the acrylamide copolymers (0.125 mM each) containing Galβ1,3GlcNAcβ-, Galβ1,3(6-sulfo)GlcNAcβ-, Galβ1,3GalNAcα-, Galβ1,3Galβ-, or Galα1,3Galβ- units were 153.6%, 43.0%, 6.2%, 52.5%, 94.9%, 14.7%, 23.6%, and 15.6% active, respectively. (r) Fucosylation by α1,2-L-FT of the galactosyl residue which occurs on the antennary structure of the bovine IgG glycopeptide was adversely affected by the presence of an α1,6-L-fucosyl residue located on the distant glucosaminyl residue that is directly attached to the asparagine of the protein backbone. This became evident from the 4-fold activity of α1,2-L-FT toward bovine IgG glycopeptide after ~50% removal of α1,6-linked Fuc.