Biomedical Engineering Reference
In-Depth Information
Golgi UDP-GlcNAc transporter (SLC35A3) reduces the availabil-
ity of UDP-GlcNAc substrate necessary for the addition of GlcNAc
to extend both
N
- and
O
-glycans; knockdown of MGAT1 or
C1GaLT1 inhibits the extension of
N
-glycans or
O
-glycans, respec-
tively, and thus Gal-
ʲ
1,4-GlcNAc structures. Thus knockdown of
each of the three genes all leads to a reduction in ECA staining.
Phaseolus vulgaris
leukoagglutinin (PHA-L) has specifi city for
tri- and tetra-antennary
N
-glycans, binding preferentially to
GlcNAc in a
ʲ
1,6 linkage with the trimannosyl core [
18
,
19
].
Datura stramonium
lectin (DSL) has a relatively broad specifi city
for poly-LacNAc extended
N
- and
O
-glycans as well as tri- and
tetra-antennary
N
-glycans [
20
-
22
]. To impair
N
-glycan synthesis,
it is possible to co-knockdown STT3A and STT3B, subunits of the
oligosaccharyltransferase complex, which initiates
N
-glycosylation
by catalyzing the transfer of a lipid-linked high-mannose oligosac-
charide to an asparagine residue on nascent polypeptide chains.
This depletion inhibits both PHA-L and DSL binding.
Maackia amurensis
lectin II (MAL-II) binds sialic acid resi-
dues, preferentially
O
-linked glycans containing the trisaccharide
Sia-
ʱ
2,3-Gal-
ʲ
1,3-GalNAc [
23
]. Sialic acid (Or N-acetyl-
neuraminic acid, Neu5Ac) is the terminal sugar added to glycan
chains and cannot be further elongated; thus a decrease in sialylated
structures may indicate incomplete glycosylation. Knockdown of
synthesizing enzyme ST3 beta-galactoside alpha-2,3-sialyltransfer-
ase 1 (ST3GAL1) reduces MAL-II staining, as does depletion of
the CMP-sialic acid transporter (SLC35A1), which reduces the
availability of CMP-sialic acid substrate necessary for the capping.
Alternatively, in vitro sialidase treatment, which cleaves off the
sialic acid residues, also reduces MAL-II staining. These lectins,
their binding specifi cities, and control genes to verify their speci-
fi cities are summarized in Table
1
.
Limitations to the technique, of course, exist. While the glycan
structure recognized by a lectin may be well characterized, the full
glycan on which the particular structure is expressed may not be
known. What the binding can reveal is a change in synthesis of the
particular structure, which could refl ect such factors as enzyme
expression/activity, substrate availability, enzyme localization, or
protein cargo traffi cking. Also, due to potential steric hindrance
between glycans, which can be relatively large macromolecules, as
well as the diversity of glycan structures bearing a lectin-binding
motif, lectin binding cannot be considered fully quantitative. It
should also be noted that some lectins exhibit cell density-
dependent staining [
24
]. It is thus good practice to test a range of
cell numbers to determine this dependency or to use the whole
screen dataset to correct for it accordingly (Fig.
3
).
Finally, while lectin specifi city can be tested as in the above
examples given, the researcher should be prepared to carefully
