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Generation of novel chimeric LacdiNAcS by gene fusion of α-lactalbumin and β1,4-galactosyltransferase 1

Abstract

Novel chimeric lacdiNAc (GalNAc(β1-4)GlcNAc) synthase (c-LacdiNAcS) was generated by gene fusion of α-lactalbumin (α-LA) and β1,4-galactosyltransferase 1 (β1,4-GalT1). c-LacdiNAcS was expressed in Lec8 Chinese hamster ovary (Lec8 CHO) cells and exhibited N-acetylgalactosaminyltransferase (GalNAcT) activity in the absence of exogenous α-LA as well as other glycosyltransferase activities including lactose synthase (LacS), and β1,4-GalT. These glycosyltransferase activities of c-LacdiNAcS were compared to those activities induced in LacS system under the co-presence of bovine β1,4-GalT1 and α-LA, indicating that each domain of α-LA and β1,4-GalT1 on c-LacdiNAcS is not only folding correctly, but also interacting together. Furthermore, c-LacdiNAcS was found to be auto-lacdiNAcylated and can synthesize lacdiNAc structures on cellular glycoproteins, demonstrating that GalNAcT activity of c-LacdiNAcS is functional in Lec8 CHO cells.

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Abbreviations

β1,4-GalT1:

β1,4-galactosyltransferase 1

LacS:

lactose synthase

GalNAcT:

N-acetylgalactosaminyltransferase

c-LacdiNAcS:

chimeric lacdiNAc synthase

lacNAc:

Gal-β1,4-GlcNAc

lacdiNAc:

GalNAc-β1,4-GlcNAc

α-LA:

α-lactalbumin

GlcNAc:

N-acetylglucosamine

Lec8 CHO:

Lec8 Chinese hamster ovary

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Acknowledgments

This work was supported by Ajou Research Grant (ARG 2003–2004, Ajou University) and by Glycomics Research Project (GRP 2008–2012, KOSEF) from MOST. I thank Dr. Ki-Young Lee (GG pharmaceutical Institute) for technical assistance and critically reading the manuscript. I also thank Dr. Richard D. Cummings (Emory University) and Dr. Kwame Nyame for kindly giving the SMLDN1.1 monoclonal antibody.

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Correspondence to Su-Il Do.

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Do, SI. Generation of novel chimeric LacdiNAcS by gene fusion of α-lactalbumin and β1,4-galactosyltransferase 1. Glycoconj J 26, 567–575 (2009). https://doi.org/10.1007/s10719-008-9208-7

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  • DOI: https://doi.org/10.1007/s10719-008-9208-7

Keywords

  • β1,4-GalT1
  • α-LA
  • LacdiNAc
  • c-LacdiNAcS
  • Lec8 CHO cells