Back to Search
Start Over
Structural and Biophysical Characterization of Human EXTL3: Domain Organization, Glycosylation, and Solution Structure
- Source :
- Biochemistry. 57:1166-1177
- Publication Year :
- 2018
- Publisher :
- American Chemical Society (ACS), 2018.
-
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.
- Subjects :
- Models, Molecular
0301 basic medicine
Protein Folding
Glycosylation
Protein Conformation
Protein domain
N-Acetylglucosaminyltransferases
Biochemistry
03 medical and health sciences
chemistry.chemical_compound
symbols.namesake
Protein structure
Protein Domains
X-Ray Diffraction
Polysaccharides
Scattering, Small Angle
Humans
Denaturation (biochemistry)
Protein secondary structure
Protein Stability
Heparan sulfate
Golgi apparatus
Dynamic Light Scattering
HEK293 Cells
030104 developmental biology
chemistry
Biophysics
symbols
Protein folding
Subjects
Details
- ISSN :
- 15204995 and 00062960
- Volume :
- 57
- Database :
- OpenAIRE
- Journal :
- Biochemistry
- Accession number :
- edsair.doi.dedup.....c869f79f85a0512a4a51974c35f0ed69