Biochemical characterization and mutational analysis of silkworm Bombyx mori β-1,4-N-acetylgalactosaminyltransferase and insight into the substrate specificity of β-1,4-galactosyltransferase family enzymes

Miyazaki, Takatsugu; Miyashita, Ryunosuke; Nakamura, Shuntaro; Ikegaya, Marina; Kato, Tatsuya; Park, Enoch Y.

doi:10.1016/j.ibmb.2019.103254

Cited by 9 publications

(5 citation statements)

References 72 publications

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“…In addition to GalNAc and Gal, BmGalNAcT transferred GlcNAc to the sugar chain and pNP-sugar, but not Glc and glucuronic acid GlcUA. This broad substrate specificity was not observed in the previous report 34 . Since this GlcNAc transfer to N-glycan has not been reported, it could be a unique feature of BmGalNAcT.…”

Section: Discussioncontrasting

confidence: 74%

“…Besides, to date, the LacdiNAc structure has also been found not only on human glycoproteins 41 , but in insects i.e., in Drosophila, mosquito, honeybees, and lepidopteran [15][16][17][18][19][20][21] and in glycolipids 42 . Recently, B. mori GalNAcT was identified 34 , and therefore, BmGalNAcT might synthesize uncharacterized and unique N-glycan structure(s). These pieces of evidence encouraged us to further characterize B. mori GalNAcT as also the enzyme was already identified.…”

Section: Discussionmentioning

confidence: 99%

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Bombyx mori β1,4-N-acetylgalactosaminyltransferase possesses relaxed donor substrate specificity in N-glycan synthesis

Kajiura

Miyauchi

Kakudo

et al. 2021

Sci Rep

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N-Glycosylation is one of the most important post-translational protein modifications in eukaryotic cells. Although more than 200 N-glycogenes contributing to N-glycan biosynthesis have been identified and characterized, the information on insect N-glycosylation is still limited. Here, focusing on insect N-glycosylation, we characterized Bombyx mori N-acetylgalactosaminyltransferase (BmGalNAcT) participating in complex N-glycan biosynthesis in mammals. BmGalNAcT localized at the Golgi and was ubiquitously expressed in every organ and in the developmental stage of the middle silk gland of fifth instar larvae. Analysis of recombinant BmGalNAcT expressed in Sf9 cells showed that BmGalNAcT transferred GalNAc to non-reducing terminals of GlcNAcβ1,2-R with β1,4-linkage. In addition, BmGalNAcT mediated transfer of galactose and N-acetylglucosamine residues but not transfer of either glucose or glucuronic acid from the UDP-sugar donor substrate to the N-glycan. Despite this tri-functional sugar transfer activity, however, most of the endogenous glycoproteins of insect cells were present without GalNAc, Gal, or GlcNAc residues at the non-reducing terminal of β1,2-GlcNAc residue(s). Moreover, overexpression of BmGalNAcT in insect cells had no effect on N-acetylgalactosaminylation, galactosylation, or N-acetylglucosaminylation of the major N-glycan during biosynthesis. These results suggested that B. mori has a novel multifunctional glycosyltransferase, but the N-glycosylation is highly and strictly regulated by the endogenous N-glycosylation machineries.

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Section: Discussioncontrasting

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Bombyx mori β1,4-N-acetylgalactosaminyltransferase possesses relaxed donor substrate specificity in N-glycan synthesis

Kajiura

Miyauchi

Kakudo

et al. 2021

Sci Rep

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“…First-strand cDNA was synthesized by reverse transcription with total RNA from fifth-instar larvae (Ehime Sanshu, Ehime, Japan) as described previously (76). A transmembrane region of BmSUH (GenBank TM BAP18683.1) was predicted by the TMHMM server (RRID:SCR_014935).…”

Section: Cloning Expression Purification and Mutagenesismentioning

confidence: 99%

Structure–function analysis of silkworm sucrose hydrolase uncovers the mechanism of substrate specificity in GH13 subfamily 17 exo-α-glucosidases

Miyazaki

Park

2020

Journal of Biological Chemistry

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The domestic silkworm Bombyx mori expresses two sucrose-hydrolyzing enzymes, BmSUH and BmSUC1, belonging to glycoside hydrolase family 13 subfamily 17 (GH13_17) and GH32, respectively. BmSUH has little activity on maltooligosaccharides, whereas other insect GH13_17 α-glucosidases are active on sucrose and maltooligosaccharides. Little is currently known about the structural mechanisms and substrate specificity of GH13_17 enzymes. In this study, we examined the crystal structures of BmSUH without ligands; in complexes with substrates, products, and inhibitors; and complexed with its covalent intermediate at 1.60–1.85 Å resolutions. These structures revealed that the conformations of amino acid residues around subsite −1 are notably different at each step of the hydrolytic reaction. Such changes have not been previously reported among GH13 enzymes, including exo- and endo-acting hydrolases, such as α-glucosidases and α-amylases. Amino acid residues at subsite +1 are not conserved in BmSUH and other GH13_17 α-glucosidases, but subsite −1 residues are absolutely conserved. Substitutions in three subsite +1 residues, Gln191, Tyr251, and Glu440, decreased sucrose hydrolysis and increased maltase activity of BmSUH, indicating that these residues are key for determining its substrate specificity. These results provide detailed insights into structure–function relationships in GH13 enzymes and into the molecular evolution of insect GH13_17 α-glucosidases.

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“…B4GALNT1 is an enzyme encoded by B4GALNT1 ( 8 ). In recent years, a mounting number of researches have shown that B4GALNT1 plays an essential role in the development and normal function of the central nervous system.…”

Section: Discussionmentioning

confidence: 99%

“…Beta-1,4-N-Acetyl-Galactosaminyltransferase 1 (B4GALNT1) is an enzyme encoded by the B4GALNT ( 7 , 8 ) in human cells. B4GALNT1 has been found to widely express in various organs among which the central nervous system contains B4GALNT1 much more than the other parts of the body ( 9 ).…”

Section: Introductionmentioning

confidence: 99%

B4GALNT1 enhances cell proliferation and growth in oral squamous cell carcinoma via p38 and JNK MAPK pathway

Jing¹,

Deng²,

Liang³

et al. 2020

Transl Cancer Res TCR

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Background Beta-1,4-N-Acetyl-Galactosaminyltransferase 1 (B4GALNT1) was reported to play an important role in the development of the central nervous systems. We found higher expression of B4GALNT1 in oral squamous cell carcinoma (OSCC) tissues compared to the paired normal adjacent tissues in the TCGA database. This study aimed to investigate whether there was a potential relationship between B4GALNT1 and OSCC tumorigenesis and further explored the possible regulation mechanism. Methods Gene expression level was analyzed by means of real-time quantitative PCR and further cell function experiments were performed including cell proliferation and apoptosis test, cell cycle distribution detection after silencing B4GALNT1 by transfection with B4GALNT1-shRNA lentivirus. Western Blotting was carried out to explore the possible molecular mechanism. Results The present study confirmed the overexpression of B4GALNT1 in OSCC. Compared to the control group, cell proliferation after silencing B4GALNT1 was significantly inhibited and cell apoptosis percentage was significantly higher. Besides, the knockdown of B4GALNT1 resulted in cell cycle arrest at G1 phase in our experiment. Conclusions B4GALNT1 enhances the proliferation and suppress the apoptosis of OSCC cells probably through JNK and p38 signaling pathway.

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Biochemical characterization and mutational analysis of silkworm Bombyx mori β-1,4-N-acetylgalactosaminyltransferase and insight into the substrate specificity of β-1,4-galactosyltransferase family enzymes

Cited by 9 publications

References 72 publications

Bombyx mori β1,4-N-acetylgalactosaminyltransferase possesses relaxed donor substrate specificity in N-glycan synthesis

Bombyx mori β1,4-N-acetylgalactosaminyltransferase possesses relaxed donor substrate specificity in N-glycan synthesis

Structure–function analysis of silkworm sucrose hydrolase uncovers the mechanism of substrate specificity in GH13 subfamily 17 exo-α-glucosidases

B4GALNT1 enhances cell proliferation and growth in oral squamous cell carcinoma via p38 and JNK MAPK pathway

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