α-Lactalbumin (LA) Stimulates Milk β-1,4-Galactosyltransferase I (β4Gal-T1) to Transfer Glucose from UDP-glucose to N-Acetylglucosamine

Abstract

␤-1,4-Galactosyltransferase 1 (Gal-T1) transfers galactose (Gal) from UDP-Gal to N-acetylglucosamine (GlcNAc), which constitutes its normal galactosyltransferase (Gal-T) activity. In the presence of ␣-lactalbumin (LA), it transfers Gal to Glc, which is its lactose synthase (LS) activity. It also transfers glucose (Glc) from UDPGlc to GlcNAc, constituting the glucosyltransferase (Glc-T) activity, albeit at an efficiency of only 0.3-0.4% of Gal-T activity. In the present study, we show that LA increases this activity almost 30-fold. It also enhances the Glc-T activity toward various N-acyl substituted glucosamine acceptors. Steady state kinetic studies of Glc-T reaction show that the K m for the donor and acceptor substrates are high in the absence of LA. In the presence of LA, the K m for the acceptor substrate is reduced 30-fold, whereas for UDP-Glc it is reduced only 5-fold. In order to understand this property, we have determined the crystal structures of the Gal-T1⅐LA complex with UDP-Glc⅐Mn 2؉ and with N-butanoyl-glucosamine (N-butanoyl-GlcN), a preferred sugar acceptor in the Glc-T activity. The crystal structures reveal that although the binding of UDP-Glc is quite similar to UDPGal, there are few significant differences observed in the hydrogen bonding interactions between UDP-Glc and Gal-T1. Based on the present kinetic and crystal structural studies, a possible explanation for the role of LA in the Glc-T activity has been proposed.

Molecular Cloning and Characterization of a Human Multisubstrate Specific Nucleotide-sugar Transporter Homologous to Drosophila fringe connection

“…HFRC1 is the first UDP-Glc transporter to be reported in a mammal, although the physiological significance of UDPGlc transport in the Golgi apparatus is obscure. Possibly, it is involved in some glycosylation process in the Golgi apparatus, such as the addition of O-linked glucose (34) or the glucosyltransferase reaction by ␤4GalT1 in the presence of ␣-lactalbumin (35). On the other hand, we found that HFRC1 transported GDP-Man alone in the yeast, not in the mammalian cells.…”

A novel β1,3‐N‐acetylglucosaminyltransferase (β3Gn‐T8), which synthesizes poly‐N‐acetyllactosamine, is dramatically upregulated in colon cancer

“…The 1194-bp (397 amino acids) ORF of b3Gn-T8 encoded a typical type II membrane protein, the same as in other b3Gn-Ts, consisting of an N-terminal cytoplasmic domain of 4 residues, a transmembane segment of 19 residues, and a stem region and a putative catalytic domain of 374 residues. A comparison of the amino acid sequence with that of other b3-glycosyltransferases revealed that this enzyme lacks the first aspartic acid of the DXD motif, identified as one of the b3Gn-T motifs and a divalent cationbinding site by a crystallization study on other glycosyltransferases [27,28]. Though b3Gn-T8 has an incomplete DXD motif, a divalent cation, Mn 2+ , is essential for the enzymatic activity like for other members of the family (data not shown).…”

“…b3Gn-T8 has QDD in its amino acid sequence instead of the DDD motif conserved in the other b3Gn-T members. It has been reported that the third aspartic acid in the DXD sequence in b4Gal-T1 binds to divalent metal ions [28]. b3Gn-T8 showed an absolute requirement for divalent metal ions, such as Mn 2+ , and the optimum concentration of Mn 2+ was between 2.5 and 10 mM.…”

Proteoglycan UDP-Galactose:β-Xylose β1,4-Galactosyltransferase I Is Essential for Viability inDrosophila melanogaster

“…2A) (25). The crystal structure of the bovine ␤4GalT1 has already been reported (24,27,28). The DXD motif is a Mn 2ϩ -binding site, and the other two motifs expose the surface of the catalytic pocket.…”