LARGE1 Processively Polymerizes Matriglycan Using Active Sites on Alternate Protomers

Joseph S, Schnicker N, Xu Z, Yang T, Hopkins J, Watkins M, Chakravarthy S, Davulcu O, Anderson M, Venzke D, Campbell K, SSRN Electronic Journal () DOI

SASDNG8 – LARGE xylosyl- and glucuronyltransferase 1 (LARGE1dTM) dimer treated with PNGase F enzyme

Xylosyl- and glucuronyltransferase LARGE1
MWexperimental 207 kDa
MWexpected 179 kDa
log I(s) 1.58×100 1.58×10-1 1.58×10-2 1.58×10-3
Xylosyl- and glucuronyltransferase LARGE1 small angle scattering data  s, nm-1
ln I(s)
Xylosyl- and glucuronyltransferase LARGE1 Guinier plot ln 1.58×100 Rg: 4.3 nm 0 (4.3 nm)-2 s2
(sRg)2I(s)/I(0)
Xylosyl- and glucuronyltransferase LARGE1 Kratky plot 1.104 0 √3 sRg
p(r)
Xylosyl- and glucuronyltransferase LARGE1 pair distance distribution function Rg: 4.4 nm 0 Dmax: 18 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Xylosyl- and glucuronyltransferase LARGE1 DAMMIF model
Synchrotron SAXS data from solutions of PNGase F treated LARGE1dTM in buffer (20 mM HEPES pH 7.4, 150 mM NaCl) were collected on the BioCAT 18-ID-D beamline at the Advanced Photon Source (APS) (Chicago, IL, USA) using a Eiger2 XE 9M detector at a sample-detector distance of 3.67 m and at a wavelength of λ = 0.1033 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC)-MALS-SAXS was employed. The SEC parameters were as follows: A 500 μl sample at 1 mg/ml was injected at a 0.6 ml/min flow rate onto a Superdex 200 increase 10/300 GL column (GE healthcare) at 23°C. 2500 successive 1 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

Xylosyl- and glucuronyltransferase LARGE1 (LARGE1)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Dimer
Mon. MW   89.7 kDa
 
UniProt   O95461 (34-756)
Sequence   FASTA