The structure of EXTL3 helps to explain the different roles of bi-domain exostosins in heparan sulfate synthesis

Abstract

Heparan sulfate is a highly modified O-linked glycan that performs diverse physiological roles in animal tissues. Though quickly modified, it is initially synthesised as a polysaccharide of alternating β-d-glucuronosyl and N-acetyl-α-d-glucosaminyl residues by exostosins. These enzymes generally possess two glycosyltransferase domains (GT47 and GT64)—each thought to add one type of monosaccharide unit to the backbone. Although previous structures of murine exostosin-like 2 (EXTL2) provide insight into the GT64 domain, the rest of the bi-domain architecture is yet to be characterised; hence, how the two domains co-operate is unknown. Here, we report the structure of human exostosin-like 3 (EXTL3) in apo and UDP-bound forms. We explain the ineffectiveness of EXTL3’s GT47 domain to transfer β-d-glucuronosyl units, and we observe that, in general, the bi-domain architecture would preclude a processive mechanism of backbone extension. We therefore propose that heparan sulfate backbone polymerisation occurs by a simple dissociative mechanism.

DALI shines a light on remote homologs: One hundred discoveries

“…For half of the pairs in this subset, a function has been previously assigned on both sides, indicating that these proteins are potentially involved in similar biological processes. Obvious cases share the same or synonymous terms in their descriptions as experimentally determined structures (PF12062 deacetylases, PF13898 deubiquitinases, PF14130 endonucleases, PF18168 DNA primase subunits, PF10307 RNA repair proteins, and PF12222 peptide‐N‐glycosidase). PF08889 are putative glycine transferases with shared conserved residues and structural similarity to cysteine‐adding enzymes in the family PF10079 (CL0039). The exostosin glucosyltransferases (PF03016) join the GT‐B clan (CL0113), which contains diverse glucosyltransferases with the Rossmann fold. Function loss has been implicated in some exostosin subfamilies (Wilson et al, 2022). Cell wall proteins pertain to the family PF11765, whose members are involved in sialidase activity and are structurally similar to pectate lyases. The spin‐doc zinc‐finger (PF18658) matches the zinc finger domain of PF19088, a member of a diverse nucleotidyltransferase superfamily (CL0260). Carbonic anhydrase is involved in CO 2 transport, a common denominator of probable inorganic carbon transporters (PF10070) and carboxysome shell carbonic anhydrase (PF08936). There are no structures associated to clan CL0370 of membrane transporters in Pfam 35.0.…”

The biosynthesis, degradation, and function of cell wall β-xylosylated xyloglucan mirrors that of arabinoxyloglucan

“…2b–e). In each case, the aligned nucleotide was placed below the side chain of Asp407—a highly conserved residue that has since been shown to bind sugar nucleotides in other GT47 family members (Leisico et al ., 2022; Wilson et al ., 2022; Li et al ., 2023); hence, this is the likely binding pocket for UDP-Gal in At XLT2.…”

“…Where only nucleotide sequences were available, we searched for GT47-A sequences with TBLASTN (Altschul et al ., 1990), using all the Arabidopsis GT47-A protein sequences as individual queries; redundant hits were removed by CD-HIT clustering (Li & Godzik, 2006; Fu et al ., 2012) with a 95% similarity threshold. TBLASTN hits were named Xx GT47-A_1, Xx GT47-A_2 etc.. All sequences were then truncated to the GT47 domain (residues 123–512 in At XLT2) as previously described (Wilson et al ., 2022). Following this, sequences shorter than that of At XLT2 by more than 33% were removed.…”

“…Dried XyGOs (without further desalting), or reductively aminated oligosaccharides (passed through a GlycoClean S column) were analysed by mass spectrometry (MS) and tandem MS–MS, respectively, as previously described (Tryfona & Stephens, 2010; Yu et al ., 2022; Wilson et al ., 2022). Data were acquired on reflector positive ion mode (mass range 300– 2,000 Da).…”

β-1,4-Xylan backbone synthesis in higher plants: How complex can it be?

“…(B) AlphaFold-Multimer model of a heterotrimer of the IRX9L (blue), IRX10L (violet), and IRX14 (yellow) globular domain. A putative nucleotide-binding residue, Asp296 ( Wilson et al., 2022 ), is shown in IRX10L. Note the predicted α-helix of IRX14 at the interaction surfaces of both IRX9L and IRX10L (asterisk).…”