Chemistry Reference
In-Depth Information
Info Box
The discovery of GPIs and their functions, their structural elucidation and the
unraveling of their complex biosynthesis is a fascinating chapter of glycobiol-
ogy. As early as 1963, the existence of protein-lipid anchors was suggested.
However, the efforts of many leading laboratories and scientists were needed
to obtain a clear picture. It was found that bacterial PI-PLC releases alkaline
phosphatase from mammalian cells. This was a quite surprising observation.
In detail, inositol-containing phospholipid protein anchors were assumed inde-
pendently by Hiro Ikezawa (Nagoya, Japan) and by Martin Low (New York,
USA). Later, Alan Williams (Oxford, UK) found that a cell-surface antigen (Thy-
1) showed both the properties of a glycolipid and the behavior of a glycoprotein.
The C-terminus of the Thy-1 glycoprotein was then found to contain both fatty
acids and ethanolamine. In 1981, Tony Holder and George Cross (Beckenham,
UK) showed that the soluble form of the variant surface glycoprotein ( VSG ) of
the African trypanosome
Trypanosoma brucei
contains carbohydrate attached to
its C-terminus via an amide linkage involving ethanolamine (since the carbo-
hydrate moiety is recognized by sera against different VSGs it is referred to as
immune cross-reactive determinant). Melvin Turner (Cambridge, UK) then
found that a membrane-bound form of VSG also exists. In 1985, the groups of
Hart and Englund at Johns Hopkins University (Baltimore, Maryland, USA)
showed that the lipid anchor on VSG is added within 1 min of the polypeptide's
synthesis in the endoplasmic reticulum (ER). They postulated that a preas-
sembled membrane anchor is attached
en bloc
before transfer to the nascent
polypeptide chain (in a process now called a transamidation reaction). In 1985
Michael Ferguson and colleagues (Oxford, UK) published a seminal paper on
the structural analysis of the glycolipid attached to the membrane-bound form
VSG of trypanosomes. These studies defi ned the term ' GPI ' .
9.1.1
Detection and Isolation of GPI - Anchored Proteins
Protocols used for the identifi cation and characterization of GPIs depend mainly
upon suffi cient amounts of material or metabolic labeling techniques using radio-
active GPI precursor molecules, organic solvent extraction procedures, the pres-
ence of nonacetylated glucosamine (GlcN; otherwise found in glycosaminoglycan
chains,please see Chapter 11), the use of GPI-specifi c phospholipases and the use
of thin-layer chromatography (TLC) analysis. One of the fi rst and the most power-
ful methods to identify a GPI anchor on a given protein remains the use of the
bacterial PI-PLC or GPI-PLC (see Info Box). After treatment with these enzymes,
the protein should loose its amphipathic character, which is refl ected in a change
of solubility. However, GPI anchors carrying a modifi ed inositol ring (for example
in the malaria parasite
Plasmodium falciparum
and erythrocytes) are insensitive to
