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

Abstract

The assembly of complex structures bearing the H determinant was examined by characterizing the specificities of a cloned blood group H gene-specified alpha 1,2-L-fucosyltransferase (FT) toward a variety of sulfated, sialylated, or fucosylated Gal beta 1,3/4GlcNAc beta- or Gal beta 1,3GalNAc alpha-based acceptor structures. (a) As compared to the basic type 2, Gal beta 1,4GlcNAc beta-(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-4 O-methylation of GlcNAc. (f) Type 1 retained 18.8% activity on alpha 2,6-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 alpha 1,3-linked Gal were inactive. (j) Gal beta 1,3GalNAc alpha- (the T-hapten) was approximately 0.4-fold as active as Gal beta 1,4GlcNAc beta-. (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) beta 1,6 branching from GalNAc of the T-hapten by a GlcNAc residue or by units such as Gal beta 1, 4GlcNAc-, Gal beta 1,4(Fuc alpha 1,3)GlcNAc-, or 3-sulfoGal beta 1,4GlcNAc- 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 (approximately 5-fold) in the K(m) for Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn in presence of 6-sulfoGal beta 1,- 4GlcNAc beta-O-Me (3.0 mM). (p) Among the two sites in Gal beta 1, 4GlcNAc beta 1,6(Gal beta 1,3) GalNAc alpha-O-Bn, the enzyme had a higher affinity ( > 3-fold) for the Gal linked to GlcNAc. (q) With respect to Gal beta 1,- 3GlcNAc beta-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 Gal beta 1,3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, Gal beta 1,3GalNAc alpha-, Gal beta 1,3Gal beta-, or Gal alpha 1,3Gal beta- units were 153.6%, 43.0%, 6.2%, 52.5%, 94.9%, 14.7%, 23.6%, and 15.6% active, respectively. (r) Fucosylation by alpha 1,2-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 alpha 1,6-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 alpha 1,2-L-FT toward bovine IgG glycopeptide after approximately 5% removal of alpha ...

Hormonal Control of H-Type α(1-2)Fucosyltransferase Messenger Ribonucleic Acid in the Mouse Uterus1

“…In order to determine whether FUT2, or other as yet unidentified ␣(1-2)FUTs, played a role in the generation of the H epitope during pregnancy, a kinetic analysis of ␣(1-2)FUT enzyme activity in the uterus was carried out with respect to two previously defined specific acceptors for ␣(1-2)FUT activity. Although no kinetic analysis of mouse ␣(1-2)FUTs have been carried out in the past, isoforms of these enzymes are well defined and display distinct differences in their affinity for specific acceptor substrates across species [43,46,47,51,52].…”

Novel interactions of complex carbohydrates with peanut (PNA), Ricinus communis (RCA-I), Sambucus nigra (SNA-I) and wheat germ (WGA) agglutinins as revealed by the binding specificities of these lectins towards mucin core-2 O-linked and N-linked glycans and related structures

“…C 35 : Fetuin triantennary asialoglycopeptide (400 μg) was α1-3 [ 14 C] fucosylated under the conditions as above using cloned FT IV (Calbiochem) and then isolated by Biogel P2 column chromatography [39]. C 36 : Fetuin triantennary asialoglycopeptide (400 μg) was α1-2 [ 14 C] fucosylated under the conditions as above using cloned α1, 2-FT (Glycomed, Alameda, CA) and then isolated by Biogel P2 column chromatography [42]. C 37 : Fetuin triantennary asialoglycopeptide (400 μg) was α1-3 [ 14 C] galactosylated under the conditions as above for C 1 , C 2 , C 3 and then isolated by Biogel P2 column chromatography.…”

Sulphotransferases acting on mucin-type oligosaccharides

“…Since a number of glycosyltransferases can act on sulphated substrates, e.g. β4-Gal-transferase [58] and α2-and α3/4-Fuc-transferases [86,88,89], sulphation is not necessarily the last step of biosynthesis.…”