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Biosynthesisin vitro of neolactotetraosylceramide by a galactosyltransferase from mouse T-lymphoma: purification and kinetic studies; synthesis of neolacto and polylactosamine core

  • Papers Dedicated To Dr Sen-Itiroh Hakomori
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Abstract

The galactosyltransferase, GalT-4, which catalyses the biosynthesisin vitro of neolactotetraosylceramide, nLcOse4Cer (Galβ1-4GleNAcβ1-3Galβ1-4Glc-Cer) from lactotriaosylceramide, LcOse3Cer (GlcNAcβ1-3Galβ1-4Glc-Cer), and UDP-galactose has been purified 107 500-fold from a mineral oil induced mouse T-lyphoma P-1798, using affinity columns. The purified enzyme is partially stabilized in the presence of phospholipid liposomes. Two closely migrating protein bands of apparent molecular weights 56 kDa and 63 kDa were observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis of highly purified mouse GalT-4. These two protein bands, when subjected to limited proteolysis, resulted in three peptides with identical mobilities indicating amino acid sequence identity between the proteins. Both protein bands from P-1798 gave a positive immunostain when tested with polyclonal antibody against bovine lactose synthetase (UDP-Gal:Glc β4-galactosyltransferase) following Western blot analysis on nitrocellulose paper. The enzyme has a pH optimum between 6.5 and 7.0 and like all other galactosyltransferases, GalT-4 has absolute requirements for divalent cation (Mn2+). TheK m values for the substrate LcOse3Cer and donor UDP-galactose are 110 and 250 µm, respectively. Substrate competition studies with LcOse3Cer and either asialo-agalacto-α1-acid glycoprotein orN-acetylglucosamine revealed that these reactions might be catalysed by the same protein. The only other glycolipid which showed acceptor activity toward the purified GalT-4 was iLcOse5Cer (GlcNAcβ1-1-3Galβ1-4Lc3), the precursor for polylactosamine antigens. However, competition studies with these two active substrates using the most purified enzyme fraction, revealed that these two reactions might be catalysed by two different proteins since the experimental values were closer to the theoretical values calculated for two enzymes. Interestingly however, it seems that the GalT-4 from P-1798 has an absolute requirement for anN-acetylglucosamine residue in the substrate since the lyso-derivative (GlcNH2β1-3Galβ1-4Glc-sphingosine) of the acceptor glycolipid LcOse3Cer is completely inactive as substrate while theK m andV max of the reacetylated substrate (GlcNacβ1-3Galβ1-4Glc-acetylsphingosine) was comparable with LcOse3Cer. Autoradiography of the radioactive product formed by purified P-1798 GalT-4 confirmed the presence of nLcOse4Cer, as the product cochromatographed with authentic glycolipid. The monoclonal antibody IB-2, specific for nLcOse4Cer, also produced a positive immunostained band on TLC as well as giving a positive ELISA when tested with radioactive product obtained using a highly purified enzyme from mouse P-1798 T-lymphoma.

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Abbreviations

EDTA:

ethylenediamine tetraacetate

ME:

β-mercaptoethanol

PEG:

polyethylene glycol

PBS:

phosphate buffered saline

Suc:

sucrose

Mn2+ :

manganese

Gal:

galactose

GlcNAc:

N-acetylglucosamine

UDP-Gal:

Uridine diphosphate galactose

Ab:

antibody

SDS:

sodium dodecyl sulphate

PAGE:

polyacrylamide gel electrophoresis

ECB:

embryonic chicken brain

Cer:

ceramide

nLc4 or NlcOse4Cer:

Galβ1-4GleNacβ1-3Galβ1-4Glc-Cer, neoLactotetraosylceramide

Lc3 or LcOse3Cer:

GlcNacβ1-3Galβ1-4Glc-Cer, lactotriaosylceramide

iLc5:

iLcOse5Cer, GlcNAcβ1-3nLcOse4Cer

nLc6:

nLcOse6Cer, Galβ1-4iLcOse5Cer

SA−Gal−α1AGP:

asialo-agalactoα1-acid glycoprotein

TLC:

thin layer chromatography

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Author notes

  1. Farhat Khan

    Present address: Department of Psychiatrics, The Johns Hopkins Medical chool, 720 Rutland Avenue, 21205, Baltimore, MD, USA

  2. Federica Rossi

    Present address: Faculty of Pharmacy, Institute of Physiology and Biochemistry of Milan, Milan, Italy

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Notre Dame, 46556, Notre Dame, IN, USA

    Manju Basu, Shu-Ai Weng, Hongyu Tang, Farhat Khan, Federica Rossi & Subhash Basu

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  1. Manju Basu
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  2. Shu-Ai Weng
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  3. Hongyu Tang
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Basu, M., Weng, SA., Tang, H. et al. Biosynthesisin vitro of neolactotetraosylceramide by a galactosyltransferase from mouse T-lymphoma: purification and kinetic studies; synthesis of neolacto and polylactosamine core. Glycoconjugate J 13, 423–432 (1996). https://doi.org/10.1007/BF00731475

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  • DOI: https://doi.org/10.1007/BF00731475

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