We report a new chemoenzymatic strategy for the rapid and sensitive detection of O-GlcNAc posttranslational modifications. The approach exploits the ability of an engineered mutant of beta-1,4-galactosyltransferase to selectively transfer an unnatural ketone functionality onto O-GlcNAc glycosylated proteins. Once transferred, the ketone moiety serves as a versatile handle for the attachment of biotin, thereby enabling chemiluminescent detection of the modified protein. Importantly, this approach permits the rapid visualization of proteins that are at the limits of detection using traditional methods. Moreover, it bypasses the need for radioactive precursors and captures the glycosylated species without perturbing metabolic pathways. We anticipate that this general chemoenzymatic strategy will have broad application to the study of posttranslational modifications.
1,4-Galactosyltransferase I (Gal-T1) normally transfers Gal from UDP-Gal to GlcNAc in the presence of Mn 2؉ ion. In the presence of ␣-lactalbumin (LA), the Gal acceptor specificity is altered from GlcNAc to Glc. Gal-T1 also transfers GalNAc from UDP-GalNAc to GlcNAc, but with only ϳ0.1% of Gal-T activity. To understand this low GalNAc-transferase activity, we have carried out the crystal structure analysis of the Gal-T1⅐LA complex with UDP-GalNAc at 2.1-Å resolution. The crystal structure reveals that the UDP-GalNAc binding to Gal-T1 is similar to the binding of UDP-Gal to Gal-T1, except for an additional hydrogen bond formed between the N-acetyl group of GalNAc moiety with the Tyr-289 side chain hydroxyl group. Elimination of this additional hydrogen bond by mutating Tyr-289 residue to Leu, Ile, or Asn enhances the GalNAc-transferase activity. Although all three mutants exhibit enhanced GalNAc-transferase activity, the mutant Y289L exhibits GalNAc-transferase activity that is nearly 100% of its Gal-T activity, even while completely retaining its Gal-T activity. The steady state kinetic analyses on the Leu-289 mutant indicate that the K m for GlcNAc has increased compared to the wild type. On the other hand, the catalytic constant (k cat ) in the Gal-T reaction is comparable with the wild type, whereas it is 3-5-fold higher in the GalNAc-T reaction. Interestingly, in the presence of LA, these mutants also transfer GalNAc to Glc instead of to GlcNAc. The present study demonstrates that, in the Gal-T family, the Tyr-289/Phe-289 residue largely determines the sugar donor specificity.
Beta-1,4-galactosylransferase (beta4Gal-T1) participates in the synthesis of Galbeta1-4-GlcNAc-disaccharide unit of glycoconjugates. It is a trans-Golgi glycosyltransferase (Glyco-T) with a type II membrane protein topology, a short N-terminal cytoplasmic domain, a membrane-spanning region, as well as a stem and a C-terminal catalytic domain facing the trans-Golgi-lumen. Its hydrophobic membrane-spanning region, like that of other Glyco-T, has a shorter length compared to plasma membrane proteins, an important feature for its retention in the trans-Golgi. The catalytic domain has two flexible loops, a long and a small one. The primary metal binding site is located at the N-terminal hinge region of the long flexible loop. Upon binding of metal ion and sugar-nucleotide, the flexible loops undergo a marked conformational change, from an open to a closed conformation. Conformational change simultaneously creates at the C-terminal region of the flexible loop an oligosaccharide acceptor binding site that did not exist before. The loop acts as a lid covering the bound donor substrate. After completion of the transfer of the glycosyl unit to the acceptor, the saccharide product is ejected; the loop reverts to its native conformation to release the remaining nucleotide moiety. The conformational change in beta4Gal-T1 also creates the binding site for a mammary gland-specific protein, alpha-lactalbumin (LA), which changes the acceptor specificity of the enzyme toward glucose to synthesize lactose during lactation. The specificity of the sugar donor is generally determined by a few residues in the sugar-nucleotide binding pocket of Glyco-T, conserved among the family members from different species. Mutation of these residues has allowed us to design new and novel glycosyltransferases, with broader or requisite donor and acceptor specificities, and to synthesize specific complex carbohydrates as well as specific inhibitors for these enzymes.
-1,4-Galactosyltransferase 1 (Gal-T1) transfers galactose (Gal) from UDP-Gal to N-acetylglucosamine (GlcNAc), which constitutes its normal galactosyltransferase (Gal-T) activity. In the presence of ␣-lactalbumin (LA), it transfers Gal to Glc, which is its lactose synthase (LS) activity. It also transfers glucose (Glc) from UDPGlc to GlcNAc, constituting the glucosyltransferase (Glc-T) activity, albeit at an efficiency of only 0.3-0.4% of Gal-T activity. In the present study, we show that LA increases this activity almost 30-fold. It also enhances the Glc-T activity toward various N-acyl substituted glucosamine acceptors. Steady state kinetic studies of Glc-T reaction show that the K m for the donor and acceptor substrates are high in the absence of LA. In the presence of LA, the K m for the acceptor substrate is reduced 30-fold, whereas for UDP-Glc it is reduced only 5-fold. In order to understand this property, we have determined the crystal structures of the Gal-T1⅐LA complex with UDP-Glc⅐Mn 2؉ and with N-butanoyl-glucosamine (N-butanoyl-GlcN), a preferred sugar acceptor in the Glc-T activity. The crystal structures reveal that although the binding of UDP-Glc is quite similar to UDPGal, there are few significant differences observed in the hydrogen bonding interactions between UDP-Glc and Gal-T1. Based on the present kinetic and crystal structural studies, a possible explanation for the role of LA in the Glc-T activity has been proposed.
The vast number of proteins that sustain the currently living organisms have been generated from a relatively small number of ancestral genes that has involved a variety of processes. Lysozyme is an ancient protein whose origin goes back an estimated 400 to 600 million years. This protein was originally a bacteriolytic defensive agent and has been adapted to serve a digestive function on at least two occasions, separated by nearly 40 million years. The origins of the related goose type and T4 phage lysozyme that are distinct from the more common C type are obscure. They share no discernable amino acid sequence identity and yet they possess common secondary and tertiary structures. Lysozyme C gene also gave rise, after gene duplication 300 to 400 million years ago, to a gene that currently codes for alpha-lactalbumin, a protein expressed only in the lactating mammary gland of all but a few species of mammals. It is required for the synthesis of lactose, the sugar secreted in milk. alpha-Lactalbumin shares only 40% identity in amino acid sequence with lysozyme C, but it has a closer spatial structure and gene organization. Although structurally similar, functionally they are quite distinct. Specific amino acid substitutions in alpha-lactalbumin account for the loss of the enzyme activity of lysozyme and the acquisition of the features necessary for its role in lactose synthesis. Evolutionary implications are as yet unclear but are being unraveled in many laboratories.
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