Specific Enzyme Complex of β-1,4-Galactosyltransferase-II and Glucuronyltransferase-P Facilitates Biosynthesis of N-linked Human Natural Killer-1 (HNK-1) Carbohydrate

Abstract

Human natural killer-1 (HNK-1) carbohydrate is highly expressed in the nervous system and is involved in synaptic plasticity and dendritic spine maturation. This unique carbohydrate, consisting of a sulfated trisaccharide (HSO 3 -3GlcA␤1-3Gal␤1-4GlcNAc-), is biosynthesized by the successive actions of ␤-1,4-galactosyltransferase (␤4GalT), glucuronyltransferase (GlcAT-P and GlcAT-S), and sulfotransferase (HNK-1ST). A previous study showed that mice lacking ␤4GalT-II, one of seven ␤4GalTs, exhibited a dramatic loss of HNK-1 expression in the brain, although ␤4GalT-I-deficient mice did not. Here, we investigated the underlying molecular mechanism of the regulation of HNK-1 expression. First, focusing on a major HNK-1 carrier, neural cell adhesion molecule, we found that reduced expression of an N-linked HNK-1 carbohydrate caused by a deficiency of ␤4GalT-II is not likely due to a general loss of the ␤1,4-galactose residue as an acceptor for GlcAT-P. Instead, we demonstrated by co-immunoprecipitation and endoplasmic reticulum-retention analyses using Neuro2a (N2a) cells that ␤4GalT-II physically and specifically associates with GlcAT-P. In addition, we revealed by pulldown assay that Golgi luminal domains of ␤4GalT-II and GlcAT-P are sufficient for the complex to form. With an in vitro assay system, we produced the evidence that the kinetic efficiency k cat /K m of GlcAT-P in the presence of ␤4GalT-II was increased about 2.5-fold compared with that in the absence of ␤4GalT-II. Finally, we showed that co-expression of ␤4GalT-II and GlcAT-P increased HNK-1 expression on various glycoproteins in N2a cells, including neural cell adhesion molecule. These results indicate that the specific enzyme complex of ␤4GalT-II with GlcAT-P plays an important role in the biosynthesis of HNK-1 carbohydrate.

Bisecting GlcNAc Is a General Suppressor of Terminal Modification of N-glycan*[S]

“…Expression and purification of recombinant glycosyltransferases were performed basically as described previously (28,31,35). For preparing FUT1-, FUT2-, FUT4-, or FUT9-myc, COS7 cells were transfected with each plasmid with Polyethyleneimine MAX.…”

Enzyme Module Systems for the Synthesis of Uridine 5′‐Diphospho‐α‐D‐glucuronic Acid and Non‐Sulfated Human Natural Killer Cell‐1 (HNK‐1) Epitope

“…The activity of β4Gal‐T1 to generate the N ‐acetyllactosamine (LacNAc) type 2, Gal(β1‐4)GlcNAc, acceptor on N‐glycans has been shown to be essential for the expression of the HNK‐1 epitope in the brain 8c. Eukaryotic glucuronyltransferase‐P (GlcAT‐P) from rat, mouse and man were cloned and expressed in different hosts 9.…”

Regulated expression and neural functions of human natural killer-1 (HNK-1) carbohydrate

“…Usually the glycan terminus is capped with another acidic monosaccharide, sialic acid, in mammals [11,12]. HNK-1 epitope is mainly expressed in N-glycan and in O-mannose glycan [13][14][15], and is not found in other types of O-glycan under physiological conditions. In addition, HNK-1 glycan has some structural varieties such as non-sulfated form or 6-sulfation of the inner GlcNAc residue [16,17].…”