Structural and Biophysical Characterization of Human EXTL3: Domain Organization, Glycosylation, and Solution Structure

Abstract

Heparan sulfate proteoglycans are proteins substituted with one or more heparan sulfate (HS) polysaccharides, found in abundance at cell surfaces. HS chains influence the activity of many biologically important molecules involved in cellular communication and signaling. The exostosin (EXT) proteins are glycosyltransferases in the Golgi apparatus that assemble HS chains on HSPGs. The EXTL3 enzyme mainly works as an initiator in HS biosynthesis. In this work, human lumenal N-glycosylated EXTL3 (EXTL3ΔN) was cloned, expressed in human embryonic kidney cells, and purified. Various biophysical and biochemical approaches were then employed to elucidate the N-glycosylation sites and the function of their attached N-glycans. Furthermore, the stability and conformation of the purified EXTL3ΔN protein in solution have been analyzed. Our data show that EXTL3ΔN has N-glycans at least at two positions, Asn290 and Asn592, which seem to be critical for proper protein folding and/or release. EXTL3ΔN is quite stable, as high temperature (∼59 °C) was required for denaturation. Deconvolution of the EXTL3ΔN far-UV CD spectrum revealed a substantial fraction of β sheets (25%) with a minor proportion of α-helices (14%) in the secondary structure. Solution small-angle X-ray scattering and dynamic light scattering revealed an extended structure suggestive of a dimeric arrangement and consisting of two distinct regions, narrow and broad, respectively. This is consistent with bioinformatics analyses suggesting a 3-domain structure with two glycosyltransferase domains and a coiled-coil domain.

Specific functions of Exostosin-like 3 (EXTL3) gene products

“…It has been shown to exhibit GlcNAc transferase I and II activities. Since the glycosyltransferase domain on the N-terminal side (GT47) of EXT1/EXT2 was predicted to be GlcA transferase II, based on a bioinformatics analysis [32], the GT47 domain of EXTL3 may exhibit no enzyme activity. The GT64 domain in EXTL3 appears to have both GlcNAc transferase I and II activities.…”

“…The conformation of the EXTL3 protein was recently characterized [32]. Awad et al prepared the human EXTL3 protein without the sequence for the N-terminal cytosolic and transmembrane regions (EXTL3ΔN) and examined its stability and conformation.…”

Potential role for Ext1-dependent heparan sulfate in regulating P311 gene expression in A549 carcinoma cells

“…5) (Kellokumpu, Hassinen, & Glumoff, 2016). The physical characteristics of EXTL proteins have been demonstrated based on the crystallographic structures of EXTL2 (Pedersen et al, 2003) and EXTL3 (Awad, Kjellstrom, Svensson Birkedal, Mani, & Logan, 2018) that exhibit similarly conserved cysteine amino acid residues in their catalytic domains (Zak, Crawford, & Esko, 2002). EXTL2 is the smallest member with 330 amino acid (a.a) residues followed by EXTL1 (676 a.a.), EXT2 (718 a.a.), EXT1 (746 a.a.) and EXTL3 is the largest with about 919 a.a. residues (Busse-Wicher et al, 2014).…”

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

“…The crystal structure revealed that EXTL2 exists as a symmetric homodimer, and that the GT64 domain adopts a GT-A fold, binding UDP-GlcNAc or UDP-GalNAc with the aid of a manganese cofactor. While the GT47 domain of the larger exostosins has been tentatively predicted to adopt a GT-B fold 30 , the overall bi-domain structure remains to be determined, and the GT47 fold is yet to be described. This missing information could provide insight into the mechanism of HS chain extension and the diverse activities of GT47 family members in plants 17 , 31 .…”