Structural Basis for the Initiation of Glycosaminoglycan Biosynthesis by Human Xylosyltransferase 1

Abstract

SummaryProteoglycans (PGs) are essential components of the animal extracellular matrix and are required for cell adhesion, migration, signaling, and immune function. PGs are composed of a core protein and long glycosaminoglycan (GAG) chains, which often specify PG function. GAG biosynthesis is initiated by peptide O-xylosyltransferases, which transfer xylose onto selected serine residues in the core proteins. We have determined crystal structures of human xylosyltransferase 1 (XT1) in complex with the sugar donor, UDP-xylose, and various acceptor peptides. The structures reveal unique active-site features that, in conjunction with functional experiments, explain the substrate specificity of XT1. A constriction within the peptide binding cleft requires the acceptor serine to be followed by glycine or alanine. The remainder of the cleft can accommodate a wide variety of sequences, but with a general preference for acidic residues. These findings provide a framework for understanding the selectivity of GAG attachment.

Deep evolutionary analysis reveals the design principles of fold A glycosyltransferases

“…2). Out of these, only GT14 has representative crystal structures where a glutamate serves as the catalytic base (26). For other inverting families with a non-conserved xED-Asp, residues from other structural regions may serve as a catalytic base.…”

“…3A). For example, GT14 and prokaryotic members of GT6 that fall on different clades, have independently lost the DxD motif and no longer require a metal ion for activity (26, 29).…”

Identification of Putative Non-Substrate-Based XT-I Inhibitors by Natural Product Library Screening

“…Both XTs catalyze the transfer of d -xylose from uridine diphosphate (UDP)- d -xylose onto defined serine residues on similar proteoglycan (PG) core proteins and, thus initiating a series of posttranslational modifications necessary for the transport and secretion of PGs [ 2 ], which are essential components of the extracellular matrix (ECM) [ 3 ]. Both XTs consist of a short amino-terminal region facing the cytosolic side, a single transmembrane helix, a stem region necessary for Golgi retention [ 1 ], a catalytic glycosyltransferase (GT)-A domain facing the Golgi lumen [ 4 , 5 ] and a C-terminal domain named Xylo_C according to the Pfam database [ 5 , 6 ]. XT-I and XT-II are differentially expressed in tissues and cell types due to variations in the promotor region and cellular transcriptional regulations [ 7 , 8 , 9 , 10 ], but the reason for the existence of these two isoforms in all higher organisms remains unknown.…”

Xylosyltransferase 1 and the GAG Attachment Site

“…In this issue of Structure, Briggs and Hohenester (2018) describe the structure of XT1 involved in the first step of PG biosynthesis and its specificity for UDPxylose donor and for the peptide sequence in the core protein acceptor. How XT1 selects the serine residue of the GAG attachment site in the core protein acceptor is of great interest to researchers in the PG field.…”

“…Although enzymes can recognize subdomains and organize these in the GAG chain, our current understanding of biosynthetic control of GAG chain structure is still insufficient to understand how GAGs have a defined or limited number of sequences. Briggs and Hohenester (2018) report the structures of ternary complexes of XT1, polypeptide, and UDP-xylose. XT1 shows a stringent specificity for UDP-xylose donor but a relatively lax specificity for the polypeptide acceptor, which begins to explain the observed diversity of PGs (Figure 1).…”