Analysis of the substrate binding sites of human galactosyltransferase by protein engineering.
An expression vector, pIN-GT, encoding the soluble form of beta 1,4-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Aoki
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La Jolla Cancer Research Foundation, CA 92037.
Appert
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Johnson
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Wong
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Fukuda
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005700:Galactosyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D009844:Oligosaccharides / Q000032:analysis; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D015345:Oligonucleotide Probes; D009844:Oligosaccharides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D005700:Galactosyltransferases