Chemistry Reference
In-Depth Information
cleavage by (G)PI-PLC. Alternative methods to identify a GPI anchor and/or to
confi rm results obtained by (G)PI-PLC treatment are the use of a mammalian
GPI - specifi c phospholipase D or deamination by nitrous acid (HNO
2
can be pre-
pared
in situ
in a test tube by adding any weak acid to sodium nitrite) (Figure 9.2 ).
Both treatments cleave GPI anchors from solubilized proteins, but marginally
from intact cells. The sensitivity of proteins to these GPI-specifi c treatments can
be determined by a shift in mobility of the protein in sodium dodecylsulfate- poly-
acrylamide gels. The deamination of GPIs (extracted mainly by organic solvent)
using nitrous acid (HNO
2
) leads to the cleavage of the linkage between the inositol
moiety and the non-acetylated glucosamine present in the GPI structure (Figure
9.2). Usually in standard protocols, the anhydromannose generated at the reducing
terminus of the GPI glycan is consequently converted to anhydromannitol using
sodium borohydride (NaBH
4
) , which resists unspecifi c destruction as it does not
contain a reducing-end sugar moiety. After purifi cation, the hydrophilic fragments
can be analyzed using size-exclusion chromatography. Neutral core glycans are
prepared by dephosphorylation, deamination and reduction. The released core
glycans are completely desalted prior analysis. They are analyzed by high pH
anion-exchange chromatography along with an internal standard of
- glucan oligo-
mers. Exoglycosidase (for example mannosidases) treatments are used to confi rm
the predicted structures of the GPI glycans. In addition to this strategy, alternative
methods are also available. For instance, GPI-anchored proteins can also be identi-
fi ed by temperature-induced phase separation in 1- 2% Triton X - 114. This deter-
gent is normally used to solubilize membranes and whole cells, and the soluble
material is submitted to phase separation. This technique distinguishes between
GPI-anchored proteins and integral membrane proteins versus cytosolic proteins.
It is based on the ability of the nonionic detergent Triton X-114 to partition into
two distinct phases at 30 ° C - a detergent - rich phase, with the major part of the
membrane proteins, and an aqueous phase, containing predominantly nonmem-
brane proteins. Having thus explained structural aspects including characteriza-
tion, we now turn our attention to their biosynthesis.
β
Figure 9.2
Procedures for the identifi cation and characterization of GPIs.
