Recent advances on glycosyltransferases involved in the biosynthesis of the proteoglycan linkage region

Abstract

Proteoglycans (PGs) are an essential family of glycoproteins, which can play roles in many important biological events including cell proliferation, cancer development, and pathogen infections. Proteoglycans consist of a core protein with one or multiple glycosaminoglycan (GAG) chains, which are covalently attached to serine residues of serine-glycine dipeptide within the core protein through a common tetrasaccharide linkage. In the past three decades, four key glycosyl transferases involved in the biosynthesis of PG linkage have been discovered and investigated. This review aims to provide an overview on progress made on these four enzymes, with foci on enzyme expression/purification, substrate specificity, activity determination, product characterization, and structure-activity relationship analysis.

Keywords: Biosynthesis; Chemoenzymatic synthesis; Enzymes; Linkage region; Proteoglycan.

Fig. 1

Schematic demonstration of the structure of proteoglycans. The tetrasaccharide linkage is highlighted in the blue box.

Fig. 2

Biosynthetic assembly of the PG linkage region.

Fig. 3

XT-I acceptor specificity. Eight peptides complexed with XT-I are superimposed.

Fig. 4

UDP-xylose binding pocket of XT-I. Residue W392 is found in close proximity to the C-5 of xylose providing a potential rationale for the donor specificity.

Fig. 5

Active site of XT-I in complex with UDP-xylose donor and a peptide acceptor.

Fig. 6

Structures of representative peptide acceptors (1–4) transformed by human XT-I to glycopeptide products (5–8) with the serine xylosylation sites highlighted in red.

Fig. 7

Molecular docking of glucose into the binding pocket of Drosophila β4GalT7.O-2, O-3 and O-4 hydroxy groups of the docked glucose molecule are in close proximity to catalytic residues D211/D212. Residue Y177 imposes steric hindrance on the C-6/O-6 atom of the glucose molecule, implying only xylose would be accommodated by the enzyme.

Fig. 8

Stereoview of the molecular modeling of human β4GalT7 in complex with UDP-Gal. (A) Predicted complex formed with UDP-Gal, Mn²⁺, and ¹⁶³DVD¹⁶⁵/²⁵⁷HLH²⁵⁹; (B) predicted interaction between β-phosphate of UDP-Gal and residue W224. The protein α-carbon backbone is colored green. Key residues in the active sites, UDP-Gal, and Mn²⁺, are highlighted.

Fig. 9

(A) Xylobiose binding to Drosophila β4GalT7 in a closed conformation. The active site is colored green. (B) Overview of proposed key interactions of xylosides and UDP-Gal in the β4GalT7 binding pocket (reprinted with copyright permission from Elsevier).

Fig. 10

D211N β4GalT7 in complex with UDP-Gal, Mn²⁺ and a xyloside analog. The protein is colored blue. UDP-Gal and the xyloside analog are highlighted gray.

Fig. 11

The active site of human β4GalT7 in complex with UDP-Gal, Mn²⁺ and 4-MUX. The protein α-carbon backbone is colored gray. Key residues in the active site and substrates are highlighted.

Fig. 12

(A) Structures of representative peptide acceptors (6–11) transformed by β4GalT7 to glycopeptide products (12–17) with the serine glycosylation sites highlighted in red. (B) One-pot, two-enzyme reactions with XT-I and β4GalT7 could convert the peptide to a glycopeptide bearing a Gal-Xyl disaccharide in higher yields than those of the stepwise reactions.