Analysis of the substrate binding sites of human galactosyltransferase by protein engineering

Daisuke Aoki, Hubert E. Appert, Dennis Johnson, Shan S. Wong, Michiko N. Fukuda

Research output: Contribution to journalArticle

92 Citations (Scopus)

Abstract

An expression vector, pIN-GT, encoding the soluble form of β1,4-galactosyltransferase (GT) has been constructed from human GT cDNAs and the pIN-III-ompA2 expression vector. Escherichia coli strain SB221 harboring the pIN-GT plasmid produces and secretes a fusion protein consisting of the ompA signal and GT. The expression of GT was detected by assaying enzymatic activity as well as by Western blotting using anti-GT antibodies. The recombinant GT was purified to homogeneity by N-acetylglucosamine-Sepharose affinity chromatography. The NH2-terminal peptide sequence of purified GT confirmed the cleavage site of the fusion protein by bacterial signal peptidase. This expression system was utilized to produce mutant forms of GT in order to identify specific amino acids involved in substrate binding sites. Photoaffinity labeling of GT with UDP-galactose analog, 4-azido-2-nitrophenyluridylylpyrophosphate (ANUP), followed by cyanogen bromide (CNBr) cleavage revealed that ANUP bound to a fragment of GT composed of amino acid residues from Asp276 to Met328. Within this peptide segment, Tyr284, Tyr287, Tyr309, Trp310 and Trp312 were separately substituted into Gly and Tyr287 into Phe by site-directed mutagenesis. Enzymatic activity assay showed drastic reduction of the activity in all of the mutants except that Tyr287 → Phe remained as active as wild-type GT. Kinetic studies of the mutated GT showed that Tyr284, Tyr309 and Trp310 are critically involved in the N-acetyglucosamine binding and Tyr309 is involved in UDP-galactose binding as well. These results indicate that these tyrosines and tryptophans in GT are essential for the binding of the acceptor N-acetylglucosamine, and that UDP-galactose also binds to residue(s) nearby where N-acetylglucosamine binds.

Original languageEnglish
Pages (from-to)3171-3179
Number of pages9
JournalEMBO Journal
Volume9
Issue number10
Publication statusPublished - 1990
Externally publishedYes

Fingerprint

Galactosyltransferases
Protein Engineering
Binding Sites
Substrates
Proteins
Uridine Diphosphate Galactose
Acetylglucosamine
Fusion reactions
Affinity chromatography
Amino Acids
Cyanogen Bromide
Agarose Chromatography
Mutagenesis
Peptides
Bacterial Proteins
Enzyme Assays
Site-Directed Mutagenesis

Keywords

  • Affinity labeling
  • Enzyme kinetics
  • Galactosyltransferase
  • Glycoprotein
  • Mutations

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

Cite this

Aoki, D., Appert, H. E., Johnson, D., Wong, S. S., & Fukuda, M. N. (1990). Analysis of the substrate binding sites of human galactosyltransferase by protein engineering. EMBO Journal, 9(10), 3171-3179.

Analysis of the substrate binding sites of human galactosyltransferase by protein engineering. / Aoki, Daisuke; Appert, Hubert E.; Johnson, Dennis; Wong, Shan S.; Fukuda, Michiko N.

In: EMBO Journal, Vol. 9, No. 10, 1990, p. 3171-3179.

Research output: Contribution to journalArticle

Aoki, D, Appert, HE, Johnson, D, Wong, SS & Fukuda, MN 1990, 'Analysis of the substrate binding sites of human galactosyltransferase by protein engineering', EMBO Journal, vol. 9, no. 10, pp. 3171-3179.
Aoki, Daisuke ; Appert, Hubert E. ; Johnson, Dennis ; Wong, Shan S. ; Fukuda, Michiko N. / Analysis of the substrate binding sites of human galactosyltransferase by protein engineering. In: EMBO Journal. 1990 ; Vol. 9, No. 10. pp. 3171-3179.
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AB - An expression vector, pIN-GT, encoding the soluble form of β1,4-galactosyltransferase (GT) has been constructed from human GT cDNAs and the pIN-III-ompA2 expression vector. Escherichia coli strain SB221 harboring the pIN-GT plasmid produces and secretes a fusion protein consisting of the ompA signal and GT. The expression of GT was detected by assaying enzymatic activity as well as by Western blotting using anti-GT antibodies. The recombinant GT was purified to homogeneity by N-acetylglucosamine-Sepharose affinity chromatography. The NH2-terminal peptide sequence of purified GT confirmed the cleavage site of the fusion protein by bacterial signal peptidase. This expression system was utilized to produce mutant forms of GT in order to identify specific amino acids involved in substrate binding sites. Photoaffinity labeling of GT with UDP-galactose analog, 4-azido-2-nitrophenyluridylylpyrophosphate (ANUP), followed by cyanogen bromide (CNBr) cleavage revealed that ANUP bound to a fragment of GT composed of amino acid residues from Asp276 to Met328. Within this peptide segment, Tyr284, Tyr287, Tyr309, Trp310 and Trp312 were separately substituted into Gly and Tyr287 into Phe by site-directed mutagenesis. Enzymatic activity assay showed drastic reduction of the activity in all of the mutants except that Tyr287 → Phe remained as active as wild-type GT. Kinetic studies of the mutated GT showed that Tyr284, Tyr309 and Trp310 are critically involved in the N-acetyglucosamine binding and Tyr309 is involved in UDP-galactose binding as well. These results indicate that these tyrosines and tryptophans in GT are essential for the binding of the acceptor N-acetylglucosamine, and that UDP-galactose also binds to residue(s) nearby where N-acetylglucosamine binds.

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