Glycobiology Advance Access originally published online on February 5, 2007
Glycobiology 2007 17(5):7G-9G; doi:10.1093/glycob/cwm013
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Letter to the Glyco-Forum: Catalytic domains of glycosyltransferases with ‘add-on’ domains
2 Structural Glycobiology Section, CCR Nanobiology Program
3 Basic Science Program, SAIC-Frederick, Inc., Center for Cancer Research, National Cancer Institute at Frederick, Building 469, Room 221, Frederick, MD, 21702
1 Tel: +1 301 846 1934; fax: +1 301 846 7149; e-mail : qasba{at}helix.nih.gov
Glycosyltransferases (GTs) belong to a large family of enzymes that are involved in the synthesis of oligosaccharide moieties of glycoproteins, glycolipids, and proteoglycans and have been assembled in a CAZy database (http://www.cazy.org). In the eukaryotic cells, most of these enzymes are Golgi-resident type II membrane proteins with a cytoplasmic domain, a transmembrane domain, and a stem region with a catalytic domain facing the Golgi-lumen, which transfers a sugar moiety from an activated sugar nucleotide donor to an acceptor molecule with either retention or inversion of the stereochemistry at the C1 position of the donor sugar.
Crystal structures of the catalytic domain of several GT enzymes have been recently reported. Most of these structures fall into a GT-A fold which has an N-terminal Rossmann-like nucleotide-binding domain and a C-terminal acceptor-binding domain, whereas a few fall into a GT-B fold that has two similar Rossmann-like folds. In this issue, the structure of the catalytic domain of 
1,6-fucosyltransferase, FUT8, is described (Ihara et al. 2007
), which transfers fucose (Fuc) in 1,6 linkage from GDP-Fuc to the Asn-linked GlcNAc in the N-glycan core of a glycoprotein. Although there is only one nucleotide-binding domain located toward the C-terminus of the catalytic domain, the FUT8 structure is better represented as a GT-B fold. On the basis of the nucleotide fold, the GDP-Fuc-binding site has only been tentatively identified.
To date, the detailed structure–function studies on GTs (Qasba et al. 2005), particularly on ß-1,4-galactosyltransferase (Ramakrishnan et al. 2004), have shown that in the vicinity of their catalytic pocket are one or two flexible loops that, upon binding of the nucleotide sugar donor substrate and the metal ion (when required as a cofactor), change conformation often from open to closed, creating an acceptor-binding site. The loop acts as a lid covering the bound donor substrate. After the transfer of the glycosyl unit to the acceptor, the product is ejected, and the loop reverts to its native conformation to release the remaining nucleotide moiety. The closed conformation of the loop is generally observed in the X-ray crystal structures when the enzyme is cocrystallized with the donor substrate. On the other hand, when the enzyme is crystallized in the absence of donor substrate, the loop, being flexible, remains in the open conformation, and often, the region cannot be observed in the crystal structures. In the metal-ion-dependent enzymes, the metal-ion-binding site is generally at the amino terminal hinge region of the flexible loop. In FUT8, which does not require metal ion for its enzymatic activity, the metal-ion-binding motif is absent in the structure. Interestingly, in the FUT8 structure, a flexible loop is also observed in the vicinity of the GDP-Fuc-binding site, and the authors report that the untraced residues in this flexible loop are essential for the catalytic activity (Ihara et al. 2007) and are conserved among many family members. The specificity of the sugar donor in many GTs is determined by a few residues in the sugar–nucleotide-binding pocket of the enzyme, which are conserved among the family members from different species (Ramakrishnan and Qasba 2007).
On the basis of the protein sequence comparison, nearly a 50-residue-long sequence at the C-terminus of the FUT8 catalytic region was predicted to be an SH3 (Src homology 3)-like domain (Javaud et al. 2000). In the FUT8 structure (Ihara et al. 2007), an SH3-like folded protein linked at the C-terminus of the catalytic domain has indeed been identified. The SH3 domains, which are small ß-barrel proteins that have five antiparallel ß-strands forming two orthogonal ß-sheets (Radha Kishan and Agrawal 2005), are present in many signal transduction enzymes and have been shown to be involved in the intermolecular and intramolecular interactions via the proline-rich motifs of the proteins and play important role in the activation–inactivation mechanism of the Src family tyrosine kinases (Huse and Kuriyan 2002; Li 2005). In the FUT8 enzyme (Ihara et al. 2007), upon binding GDP-Fuc, it is likely, although not yet proved, that the flexible loops located near the catalytic site also change the conformation that traps the donor substrate, whereas the SH3 domain may adjust its orientation to interact with the proline-rich portion of the protein that carries the N-linked glycan structure. This mechanism may explain the selectivity of fucosylation of the Asn-linked GlcNAc in the N-glycan core of a specific glycoprotein. Thus, the SH3 domain is an ‘add-on’ domain to the catalytic domain of FUT8, which accounts for the specificity of fucosylation of a particular N-linked glycoprotein acceptor, thereby imparting a unique function to the glycoprotein. For example, the core fucosylation of N-glycans of many glycoproteins in a cell, if altered, has severe pathological consequences: the core fucosylation of the TGF-ß1 or EGF receptors is essential for the receptor-mediated biological functions (Wang et al. 2006). On the other hand, the deficient core fucosylation of IgG1 improves the binding to the Fc fragment and thus enhances antibody-dependent cellular toxicity. Overexpression of the FUT8 gene causes steatosis in the liver and kidney.
To diversify the catalytic activity toward less preferred substrates, such as sugar acceptors or proteins or lipids or aglycons, the catalytic domains of GTs either interact (1) with an additional protein, (2) have acquired add-on domains at the C-terminus, or (3) acquired add-on domains at the N-terminus. For example, in the lactose synthase enzyme (Figure 1A), ß-1,4-galactosyltransferase in a closed conformation interacts with a mammary gland-specific protein, -lactalbumin (-LA), at its carboxyl terminal end, changing the acceptor specificity of the enzyme toward less preferred acceptor glucose (Ramakrishnan and Qasba 2001). The -LA protein, although not linked to ß-1,4-galactosyltransferase, acts as an add-on domain. Several other GTs have been shown (Lu et al. 2005) or suggested (Ramakrishnan et al. 2002) to require an activating protein which may interact in a manner similar to the lactose synthase system, but no structural studies are as yet available on these cases. In contrast to two interacting proteins, the catalytic domains of polypeptide--N-acetylgalactosaminyltransferases have a lectin domain that is linked to the catalytic domain via a linker region. The lectin domain is located at the C-terminus of the catalytic domain that determines the specificity toward a peptide or a glycopeptide (Fritz et al. 2006; Kubota et al. 2006) (Figure 1B). The loops in the catalytic domain of these enzymes also undergo a conformational change upon binding of the metal ion and the sugar donor, whereas the lectin domain moves, bringing in the bound glycopeptide acceptor in the catalytic pocket, in order to synthesize an O--GalNAc moiety on the glycopeptide. The catalytic domains of most ppGalNAc-Ts without their lectin domain by themselves exhibit catalytic activity, similar to ß-1,4-galactosyltransferase without -LA in the lactose synthase complex. However, together with their lectin domain and the linker region, their specificity is enhanced toward a specific glycopeptide acceptor (Kubota et al. 2006). Now, a second interesting example in this category is the FUT8 enzyme (Ihara et al. 2007), where an SH3 domain has been identified, which is linked at the C-terminus of the catalytic domain (Figure 1C). An example of the last type in which there is an add-on domain at the N-terminus of the catalytic domain is expected for the ß-1,4-N-acetylgalactosaminyltransferase IV which synthesizes N, N'-diacetyllactosediamine (Gotoh et al. 2004), even though its crystal structure is yet to be determined. Defining and determining the structure of these add-on domains will provide structure-based understanding of the specificities of GTs toward a specific acceptor substrate.
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Acknowledgments
We thank our colleagues at the Structural Glycobiology Section, NPR, CCR, NCI, for critically reading the manuscript and for helpful discussions. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. The content of this publication does not necessarily reflect the view or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. This project has been funded [in part] with Federal funds from the NCI, NIH, under contract no. N01-C0-12400.
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