Recent structural and mechanistic insights into protein O-GalNAc glycosylation

Abstract

Protein O-GalNAcylation is an abundant post-translational modification and predicted to occur in over 80% of the proteins passing through the Golgi apparatus. This modification is driven by 20 polypeptide GaINAc (N-acetylgalactosamine)-transferases (GalNAc-Ts), which are unique in that they possess both catalytic and lectin domains. The peptide substrate specificities of GalNAc-Ts are still poorly defined and our understanding of the sequence and structural features that direct O-glycosylation of proteins is limited. Part of this may be attributed to the complex regulation by coordinated action of multiple GalNAc-T isoforms, and part of this may also be attributed to the two functional domains of GalNAc-Ts that both seems to be involved in directing the substrate specificities. Recent studies have resulted in 3D structures of GalNAc-Ts and determination of the reaction mechanism of this family of enzymes. Key advances include the trapping of binary/ternary complexes in combination with computational simulations and AFM/small-SAXS experiments, which have allowed for the dissection of the reaction coordinates and the mechanism by which the lectin domains modulate the glycosylation. These studies not only broaden our knowledge of the modes-of-action of this family of enzymes but also open up potential avenues for the rational design of effective and selective inhibitors of O-glycosylation.

The Multiplicity of Polypeptide GalNAc‐Transferase: Assays, Inhibitors, and Structures

“…[1a] GalNAc-T8, -T9, -T18, and -T19 have been proven to lack activity in the in vitro enzyme assays; [7,8] however,t hese four isoformsc an be classified as subfamily Ie by sequence alignment.A ll GalNAc-Tsr equireamanganese ion (Mn 2 + )t o exert their enzymaticf unction,t houghthe catalytic mechanism remains largely unknown. [16,24] The catalytic domain of GalNAc-Ts has two conserved regions, namely,t he GT1 and Gal/ GalNAc-T motifs ( Figure 1A). The GT1 motif features aD XH sequencethat is involved in the binding to UDP-GalNAc, whereas the Gal/GalNAc-Tm otif has ah ighly conserved WGGE motif that recognizes not only the donor but also the peptide substrate.…”

“…[8] GTsc an be classified into retaining enzymes and inverting enzymes,w hich form a-glycosides and b-glycosides, respectively,b yt ransferring the GalNAc moiety to the acceptor. [16,24] All the isoformso fG alNAc-Tsb elong to the retainingi soenzyme. [26] In light of two currently proposed mechanisms for the glycosylation reaction, [15,24] that is, "double displacement"a nd "front-side single displacement", the plausible glycosylation reaction catalyzed by GalNAc-T can be described as follows (Figure 1B).…”

“…[15b] However,t hese proposed mechanisms are still under debate. [16] It appearst hat different GTs even within the same subfamily,f or example, the GT6 family of retaining enzymes,c an employ either of the two mechanisms to form the glycosidic bond. [24] 3.…”

UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase- 6 (pp-GalNAc-T6): Role in Cancer and Prospects as a Drug Target

“…This group of enzymes catalyses the addition of N-acetyl-D-galactosamine (GalNAc) to threonine or serine residues in proteins using UDP-N-acetyl-D-galactosamine (UDP-GalNAc) as the donor, generating an oxygen-linked GalNAc (O-GalNAc) with the release of UDP [4,5]. The addition of GalNAc to a peptide is often the first step in the process of glycosylation [6]. The second step involves GalNAc: glycosyltransferases adding further monosaccharides to the O-GalNAc, in a step-wise process, creating an O-glycan chain [7].…”

Synthesis of Amino‐Acid–Nucleoside Conjugates

“…[2a, 5] The peptide to which the carbohydrate is transferred interacts with the receptor in the active site of the enzyme. The GalNAcT [6] familya nd OGT [7] are representative protein glycosyltransferases that have been the object of extensive studies during the last few years, [8] owing to their implications in severald iseases,i ncluding cancer [9] and Alzheimer'sd isease. [10] As an example, an X-ray structure of enzymeG alNAc-T2 is shown in Figure 1, which highlights the substrates at the active site.…”