J. Biol. Chem., Vol. 262, Issue 7, 2968-2972, Mar, 1987
The use of avidin-biotinylglycan as the model for in vitro glycoprotein processing
MC Shao and F Wold
In an attempt to evaluate the effects of the protein matrix on the
specificity of glycoprotein processing in Golgi membranes, we have
developed a model neoglycoprotein consisting of biotinylated glycans bound
noncovalently to avidin (Chen, V. J., and Wold, F. (1986) Biochemistry 25,
939-444) with which the protein effect on processing can be evaluated as
the difference in substrate efficiency between a free biotinylated glycan
and the same biotinylated glycan bound to avidin. The avidin
(streptavidin)-glycan complex stability was found to be proper for the
experimental design; the complex remains intact for extended periods of
incubation at the concentrations used, but the glycan can be completely
liberated and recovered by heating the complex at 95 degrees C for 10 min
in the presence of a 10-fold molar excess of biotin. By measuring the
relative rates of [14C]sugar incorporation into the free and bound
substrates it was demonstrated that the protein indeed influences the
processing reactions; under conditions where free glycans such as
biotinyl-Asn-Glc-NAc2-Man5 and 6-(biotinamido)hexanoyl- Asn-Glc-NAc2-Man5
could be converted to the biantennary products R-Asn-
GlcNAc2-Man3-GlcNAc2-Gal2-sialyl2 in the presence of UDP-GlcNAc, UDP- Gal
and CMP-sialic acid and Golgi enzymes, the avidin-bound derivative without
the extension arm gave only low levels of product and the
streptavidin-bound one remained unaltered. The presence of the extension
arm in the substrates significantly improved the yield of some products in
the complex, apparently by reducing or eliminating the avidin inhibition of
the early steps, but not of the late ones. There are consequently two types
of effect of the protein matrix on processing efficiency. One is expressed
only when the glycan is close to the protein surface and affecting
primarily early steps (mannosidases and GlcNAc transferases). The other is
apparently independent of the proximity of the glycan core and the protein,
and affects primarily late steps, in particular the incorporation of the
second sialic acid residue into a biantennary complex glycan.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
