Chemistry Reference
In-Depth Information
anchor pathway is promising, since cultured cells grow normally and suffer no
adverse consequences, even when they are unable to express GPI anchored pro-
teins on their surface. Such a defect in unicellular organisms however is lethal.
Ethyl methanesulfonate (EMS) has been extensively used to generate such mutants.
EMS generates primarily GC-to-AT transitions, most likely a result of unrepaired
O
6
-ethyl guanine adducts that mispair with thymine during replication. Subse-
quently, selection of mutants defi cient in GPI-anchored proteins is usually achieved
through the use of aerolysin, a secreted bacterial toxin from
Aeromonas hydrophila
,
which binds to GPI-anchored protein and kills normal cells by forming pores. Both
GPI and
N
- glycan moieties of GPI -anchored proteins are known to be involved in
effi cient binding of aerolysin. The complementation strategy used in this case
depends mainly on the use of mammalian cDNA libraries. Similarly, heterologous
complementation of conditional yeast lethal mutants has been successfully used
to isolate functional homologs from various species. Based on the high conserva-
tion of gene function in cells from different eukaryotic species, many of the known
or unknown essential genes from yeast are potential targets for heterologous
complementation screens, which depend on the availability of conditional lethal
mutants of the gene of interest [10, 11]. So far these mutants have been mainly
temperature sensitive mutants, which often revert with certain, sometimes high
frequency. The generation of yeast conditional lethal mutants depends mainly on
the use of the stringently regulated, glucose-repressed GAL-1 promoter. GAL-1
has been shown to be induced in the presence of galactose and be tightly repressed
in the presence of glucose. The expression of the endogenous gene of the yeast
mutants can be turned off by shifting the yeast cells from galactose-containing
medium, in which the promoter is not repressed, to glucose- containing medium.
In the coimmunprecipitation approach, a generic tag like glutathione S-transferase
or FLAG (N-DYKDDDDK-C) is usually fused to the protein of interest, stably
transfected into the cells and subsequently used to isolate protein-protein com-
plexes based on affi nity purifi cation.
9.3.2
Defects in GPI Anchor Biosynthesis
No inherited defects of GPI anchor biosynthesis have been identifi ed up to now.
As Chapter 22 on congenital diseases of glycosylation attests, reasons may be the
lack of a convenient assay or the labeling or the lethality caused. However, over
100 acquired somatic mutations occur in the X-linked gene PIG-A, which encodes
a GlcNAc transferase that initiates this pathway (see Table 9.2). Impaired GPI
anchor synthesis causes a defect called paroxysmal nocturnal hemoglobinuria
(PNH), a descriptive term for the clinical manifestation of red blood cells break-
down with release of hemoglobin into the urine that is manifested most promi-
nently by dark-colored urine in the morning (please see also Chapter 22.5). Lack
of GPI-anchored complement regulatory proteins, such as decay-accelerating
factor (CD55) and complement regulatory protein (CD59), results in complement-
mediated hemolysis and hemoglobinuria. Paroxysmal nocturnal hemoglobinuria
