A Family of Human β4-Galactosyltransferases

We found a novel human glycosyltransferase gene carrying a hypothetical ␤1,4-glycosyltransferase motif during a BLAST search, and we cloned its full-length open reading frame by using the 5 -rapid amplification of cDNA ends method. It encodes a type II transmembrane protein of 999 amino acids with homology to chondroitin sulfate synthase in its C-terminal region (GenBank TM accession number AB089940). Its putative orthologous gene was also found in mouse (accession number AB114826). The truncated form of the human enzyme was expressed in HEK293T cells as a soluble protein. The recombinant enzyme transferred GalNAc to GlcNAc ␤-benzyl. The product was deduced to be GalNAc␤1-4GlcNAc␤-benzyl based on mass spectrometry and NMR spectroscopy. We renamed the enzyme ␤1,4-N-acetylgalactosaminyltransferase-III (␤4GalNAc-T3). ␤4GalNAc-T3 effectively synthesized N,N -diacetylgalactosediamine, GalNAc␤1-4GlcNAc, at non-reducing termini of various acceptors derived not only from N-glycans but also from O-glycans. Quantitative real time PCR analysis showed that its transcript was highly expressed in stomach, colon, and testis. As some glycohormones contain N,N -diacetylgalactosediamine structures in their N-glycans, we examined the ability of ␤4GalNAc-T3 to synthesize N,N -diacetylgalactosediamine structures in N-glycans on a model protein.When fetal calf fetuin treated with neuraminidase and ␤1,4-galactosidase was utilized as an acceptor protein, ␤4GalNAc-T3 transferred GalNAc to it. Furthermore, the majority of the signal from GalNAc disappeared on treatment with glycopeptidase F. These results suggest that ␤4GalNAc-T3 could transfer GalNAc residues, producing N,N -diacetylgalactosediamine structures at least in Nglycans and probably in both N-and O-glycans.

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confidence: 99%

Molecular Cloning and Characterization of a Novel Human β1,4-N-Acetylgalactosaminyltransferase, β4GalNAc-T3, Responsible for the Synthesis of N,N′-Diacetyllactosediamine, GalNAcβ1–4GlcNAc

Sato

Gotoh

Kiyohara

et al. 2003

“…The six ␤4Gal-Ts have highly conserved sequence motifs in the putative catalytic domain including four conserved cysteine residues. The genomic organization of the first four genes is similar and includes conservation of spacing for five intron/exon boundaries in the coding regions (4,7,9,10). This suggests that these genes arose late in evolutionary terms as a result of gene duplication and subsequent sequence divergence.…”

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confidence: 79%

“…The effects of these substitutions are in agreement with predictions based on x-ray crystallography data on the catalytic unit of ␤4Gal-T1. 4 The region around Ala 186 corresponds to a ␤4Gal-T7, Seventh Member of ␤4-Galactosyltransferase Familysegment in ␤4Gal-T1 that is included in the protein core. The adjacent Pro and His residues at positions 182 and 184 in ␤4Gal-T7 are strictly conserved among all human ␤4GalT sequences.…”

Section: Discussionmentioning

confidence: 99%

“…Identification and Cloning of ␤4Gal-T7-tBLASTn analysis of the dbEST data base at the National Center for Biotechnology Information (National Institutes of Health, Bethesda, MD) with sequences from human ␤4Gal-T1 to ␤4Gal-T6, performed as described previously (4,8), revealed several ESTs covering a total of 931 base pairs of the 3Ј coding sequence of ␤4Gal-T7. Additional sequence was obtained by 5Ј-rapid amplification of cDNA ends with a fetal brain cDNA library (CLON-TECH) using antisense primer EBER1218 (5Ј-CTGAAGTGGTCCAC-CTGGTTG-3Ј) and sequencing on PAC genomic DNA.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Cloning and Expression of a Proteoglycan UDP-Galactose:β-Xylose β1,4-Galactosyltransferase I

Almeida¹,

Levery²,

Mandel³

et al. 1999

Self Cite

218

A seventh member of the human ␤4-galactosyltransferase family, ␤4Gal-T7, was identified by BLAST analysis of expressed sequence tags. The coding region of ␤4Gal-T7 depicts a type II transmembrane protein with sequence similarity to ␤4-galactosyltransferases, but the sequence was distinct in known motifs and did not contain the cysteine residues conserved in the other six members of the ␤4Gal-T family. The genomic organization of ␤4Gal-T7 was different from previous ␤4Gal-Ts. Expression of ␤4Gal-T7 in insect cells showed that the gene product had ␤1,4-galactosyltransferase activity with ␤-xylosides, and the linkage formed was Gal␤1-4Xyl. Thus, ␤4Gal-T7 represents galactosyltransferase I enzyme (xylosylprotein ␤1,4-galactosyltransferase; EC 2.4. Six members of a family of human UDP-galactose:␤-Nacetylglucosamine/␤-glucosylceramide ␤1,4-galactosyltransferases (␤4Gal-Ts) 1 have previously been characterized (1-7).These six ␤4Gal-Ts catalyze biosynthesis of Gal␤1-4GlcNAc and/or Gal␤1-4Glc linkages in different glycoconjugates and free saccharides (for a review see Ref. 8). The six ␤4Gal-Ts have highly conserved sequence motifs in the putative catalytic domain including four conserved cysteine residues. The genomic organization of the first four genes is similar and includes conservation of spacing for five intron/exon boundaries in the coding regions (4, 7, 9, 10). This suggests that these genes arose late in evolutionary terms as a result of gene duplication and subsequent sequence divergence. Detailed analysis of the kinetic properties of these enzymes clearly show that each has a distinct function in biosynthesis of different glycoconjugates and saccharide structures, but in accordance with their close evolutionary relationships the linkages formed are similar. In the present study, a seventh homologue of the ␤4Gal-T gene family was characterized. The gene was identified by sequence analysis of the EST data base. The coding region of the novel gene, designated ␤4Gal-T7, exhibited distinct substitutions in the sequence motifs highly conserved among ␤4Gal-T1 to ␤4Gal-T6. Notably, none of the four cysteines conserved among other ␤4Gal-Ts were found in the ␤4Gal-T7 sequence. It was predicted that the enzymatic properties of ␤4Gal-T7 were different from other ␤4Gal-Ts. Analysis of the substrate specificity of recombinant ␤4Gal-T7 revealed that this enzyme formed the Gal␤1-4Xyl␤1-R linkage found in the linkage region of proteoglycans (GlcA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser). ␤4Gal-T7 was proposed to encode a galactosyltransferase I (xylosylprotein ␤1,4-galactosyltransferase; EC 2.4.1.133) gene (11)(12)(13). 15) showed that partial inactivation of galactosyltransferase I represented the primary defect in one patient with progeroidal appearance and symptoms of the Ehlers-Danlos syndrome. As a consequence of the enzyme deficiency, only about half of the core proteins of the small proteoglycans decorin and biglycan were linked with glycosaminoglycan chains (16), 2 whereas no abnormality in the biosynthesis of large dermatan sulf...

“…Some of them are able to synthesize N-acetyllactosamine on glycoproteins. which is potentially further modified by GlcAT-P. We recently reported that ␤4GalT-II-deficient mice showed similar phenotypes to GlcAT-P-deficient mice with a reduction in HNK-1 expression and impaired spatial learning and memory (19), although HNK-1 expression was unaltered in the ␤4GalT-I-deficient mouse brain (20) despite the similarity in primary structure and acceptor specificity of these two enzymes (21,22). These results indicated that ␤4GalT-II has specific roles in the biosynthesis of HNK-1 carbohydrate.…”

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confidence: 99%

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

Kouno

Kizuka

Nakagawa

et al. 2011

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.