Advanced

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

Awad, Wael LU ; Kjellström, Sven LU ; Svensson Birkedal, Gabriel LU ; Mani, Katrin LU and Logan, Derek T. LU (2018) In Biochemistry 57(7). p.1166-1177
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... (More)

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.

(Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biochemistry
volume
57
issue
7
pages
12 pages
publisher
The American Chemical Society
external identifiers
  • scopus:85042285849
ISSN
0006-2960
DOI
10.1021/acs.biochem.7b00557
language
English
LU publication?
yes
id
b80090c1-d51c-454e-8001-9531c33738d1
date added to LUP
2018-03-06 08:16:46
date last changed
2018-06-06 03:00:26
@article{b80090c1-d51c-454e-8001-9531c33738d1,
  abstract     = {<p>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.</p>},
  author       = {Awad, Wael and Kjellström, Sven and Svensson Birkedal, Gabriel and Mani, Katrin and Logan, Derek T.},
  issn         = {0006-2960},
  language     = {eng},
  month        = {02},
  number       = {7},
  pages        = {1166--1177},
  publisher    = {The American Chemical Society},
  series       = {Biochemistry},
  title        = {Structural and Biophysical Characterization of Human EXTL3 : Domain Organization, Glycosylation, and Solution Structure},
  url          = {http://dx.doi.org/10.1021/acs.biochem.7b00557},
  volume       = {57},
  year         = {2018},
}