β-1,4-Galactosylation of N-glycans is a complex process

Furukawa, Kiyoshi; Satô, Takeshi

doi:10.1016/s0304-4165(99)00169-5

Cited by 107 publications

(61 citation statements)

References 56 publications

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“…The knowledge that mammals have six ␤4GalTs with overlapping in vitro acceptor specificities (8,36,37) presents the challenge of sorting out their unique biological functions. An important question is the degree of redundancy between different members of the ␤4GalT family in transferring Gal to complex glycoprotein and glycolipid acceptors.…”

Section: Discussionmentioning

confidence: 99%

Chinese Hamster Ovary (CHO) Cells May Express Six β4-Galactosyltransferases (β4GalTs)

Lee

Sundaram

Shaper

et al. 2001

Journal of Biological Chemistry

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Six ␤4-galactosyltransferase (␤4GalT) genes have been cloned from mammalian sources. We show that all six genes are expressed in the Gat ؊ 2 line of Chinese hamster ovary cells (Gat ؊ 2 CHO). Two independent mutants termed Pro ؊ 5Lec20 and Gat ؊ 2Lec20, previously selected for lectin resistance, were found to have a galactosylation defect. Radiolabeled biantennary Nglycans synthesized by Pro ؊ 5Lec20 were proportionately less ricin-bound than similar species from parental CHO cells, and Lec20 cell extracts had a markedly reduced ability to transfer Gal to GlcNAc-terminating acceptors. Northern blot analysis revealed a severe reduction in ␤4GalT-1 transcripts in Pro ؊ 5Lec20 cells. The Gat ؊ 2Lec20 mutant expressed ␤4GalT-1 transcripts of reduced size due to a 311-base pair deletion in the ␤4GalT-1 gene coding region. Northern analysis with probes from the remaining five ␤4GalT genes revealed that Gat ؊ 2 CHO and Gat ؊ 2Lec20 cells express all six ␤4GalT genes. Unexpectedly, the ␤4GalT-6 gene is not expressed in either Pro ؊ 5 or Pro ؊ 5Lec20 cells. Thus, in addition to a deficiency in ␤4GalT-1, Pro ؊ 5Lec20 cells lack ␤4GalT-6. Nevertheless, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry data of N-glycans released from cellular glycoproteins showed that both the ␤4GalT-1 ؊ (Gat ؊ 2Lec20) and ␤4GalT-1 ؊ /␤4GalT-6 ؊ (Pro ؊ 5Lec20) mutants have a similar Gal deficiency, affecting neutral and sialylated bi-, tri-, and tetraantennary N-glycans. By contrast, glycolipid synthesis was normal in both mutants. Therefore, ␤4GalT-1 is a key enzyme in the galactosylation of Nglycans, but is not involved in glycolipid synthesis in CHO cells. ␤4GalT-6 contributes only slightly to the galactosylation of N-glycans and is also not involved in CHO cell glycolipid synthesis. These CHO glycosylation mutants provide insight into the variety of in vivo substrates of different ␤4GalTs. They may be used in glycosylation engineering and in investigating roles for ␤4GalT-1 and ␤4GalT-6 in generating specific glycan ligands.

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Section: Discussionmentioning

confidence: 99%

Chinese Hamster Ovary (CHO) Cells May Express Six β4-Galactosyltransferases (β4GalTs)

Lee

Sundaram

Shaper

et al. 2001

Journal of Biological Chemistry

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“…The biochemical activity and structure of the vertebrate enzymes have been well studied (Lo et al 1998;Amado et al 1999;Furukawa and Sato 1999;Gastinel et al 1999;Ramakrishnan et al 2002) and all are able to transfer galactose (Gal) to N-acetylglucosamine (GlcNAc), generating LacNAc (Galb1,. This structure is a common feature of mammalian glycoproteins and glycolipids (Amado et al 1999;Varki et al 1999), and it is also a major substrate for sialyltransferases (Varki et al 1999), thus making it essential for the generation of a diverse array of sialylated glycans.…”

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confidence: 99%

Functional Roles for β1,4-N-Acetlygalactosaminyltransferase-A in Drosophila Larval Neurons and Muscles

Haines

Stewart

2007

Genetics

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Adult Drosophila mutant for the glycosyltransferase b1,4-N-acetlygalactosaminyltransferase-A (b4Gal-NAcTA) display an abnormal locomotion phenotype, indicating a role for this enzyme, and the glycan structures that it generates, in the neuromuscular system. To investigate the functional role of this enzyme in more detail, we turned to the accessible larval neuromuscular system and report here that larvae mutant for b4GalNAcTA display distinct nerve and muscle phenotypes. Mutant larvae exhibit abnormal backward crawling, reductions in nerve terminal bouton number, decreased spontaneous transmitterrelease frequency, and short, wide muscles. This muscle shape change appears to result from hypercontraction since the individual sarcomeres are shorter in mutant muscles. Analysis of muscle calcium signals showed altered calcium handling in the mutant, suggesting a mechanism by which hypercontraction could occur. All of these phenotypes can be rescued by a transgene carrying the b4GalNAcTA genomic region. Tissue-specific expression, using the Gal4-UAS system, reveals that neural expression rescues the mutant crawling phenotype, while muscle expression rescues the muscle defect. Tissuespecific expression did not appear to rescue the decrease in neuromuscular junction bouton number, suggesting that this defect arises from cooperation between nerve and muscle. Altogether, these results suggest that b4GalNAcTA has at least three distinct functional roles.

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“…In addition, the molecular sizes of many glycosyltransferases are around 40 kDa. 48,49) Furthermore, the molecular size of bacterial chondroitin synthase, which has both GlcA-T and GalNAc-T activities 50) is 80 kDa: this value is reasonable when it is considered that the synthase consists of GlcA-T and GalNAc-T protein parts, each of which could have a size of about 40 kDa.…”

Section: Discussionmentioning

confidence: 92%

Purification and Characterization of Chick Corneal .BETA.-D-Glucuronyltransferase Involved in Chondroitin Sulfate Biosynthesis.

Ogata

Takahashi

Nakazawa

2002

Biological & Pharmaceutical Bulletin

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β-1,4-Galactosylation of N-glycans is a complex process

Cited by 107 publications

References 56 publications

Chinese Hamster Ovary (CHO) Cells May Express Six β4-Galactosyltransferases (β4GalTs)

Chinese Hamster Ovary (CHO) Cells May Express Six β4-Galactosyltransferases (β4GalTs)

Functional Roles for β1,4-N-Acetlygalactosaminyltransferase-A in Drosophila Larval Neurons and Muscles

Purification and Characterization of Chick Corneal .BETA.-D-Glucuronyltransferase Involved in Chondroitin Sulfate Biosynthesis.

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