Biomedical Engineering Reference
In-Depth Information
enzymes whose activities depend on Golgi compartmentation.
Most methods for glycan analysis such as mass spectrometry, nuclear
magnetic resonance, and chromatography-based techniques [
7
],
though potentially exhaustive, precise, and quantitative, are rather
labor and time intensive and are not easily amenable to high
throughput.
Lectins, proteins able to bind specifi c glycans, have long been
used to detect changes in glycan expression [
8
]. Numerous lectins
from various origins, from plants to invertebrates, have been char-
acterized [
9
]. In addition, glycan-specifi c antibodies have also been
developed. Using fl uorescence staining and quantitative intensity
analysis, these lectins and antibodies can be used to obtain a rapid
readout of multiple glycosylation pathways for a large number of
genes. In most cases, it is also possible to combine staining with
multiple lectins targeting different glycan structures to obtain a
simultaneous measurement of multiple glycans. Some lectins may
bind to different glycans in vitro and in vivo, potentially complicat-
ing the interpretation of results. However, it is possible to assess
the in vivo signal specifi city by depleting relevant glycosylation
enzymes or sugar transporters. We describe here some examples of
lectins that can be used to probe a variety of structures in the
mucin-type
O
-glycosylation and
N
-glycosylation pathways.
Helix pomatia
lectin (HPL),
Vicia villosa
lectin (VVL), and
peanut agglutinin (PNA) bind to the early structures in the biosyn-
thetic pathway of mucin-type (
O
-linked GalNAc)
O
-glycans. HPL
and
-linked
O
-GalNAc
(N-acetylgalactosamine), widely known as the Tn antigen [
10
,
11
]. PNA recognizes terminal core 1
O
-glycan, also widely known
as the Thomsen-Friedenreich antigen (TF or T), Gal-
VVL
recognize
terminal
ʱ
ʲ
1,3-GalNAc-
ʱ
1-Ser/Thr [
12
]. The Tn and TF antigens are both frequently
associated with malignant transformation and their levels correlate
with metastatic potential [
13
]. The TF structure is generated
mostly by one enzyme, the core 1 galactosyltransferase (C1GALT1),
and its knockdown almost completely abolishes PNA staining. In
contrast, multiple enzymes can be involved in generating a specifi c
glycan. The Tn antigen is generated by a family of enzymes called
polypeptide N-acetylgalactosaminyltransferase (GALNTs) with
overlapping substrate specifi cities [
14
]. While over 20 different
enzymes have been identifi ed, GALNT1 and 2 are the members
that are ubiquitously expressed. We found that concurrent deple-
tion of GALNT1 and 2 practically abolished HPL staining in HeLa
cells (Fig.
1a
).
For early
N
-glycan structures, concanavalin A (Con A) binds
mainly
ʱ
-
D
-mannosyl and
ʱ
-
D
-glucosyl groups, having a high
affi nity for the
N
-glycan trimannosyl core; thus it reveals high-
mannose
N
-glycans [
15
]. Mannosyl
ʱ
1,3-glycoprotein
ʲ
1,2-N-
acetylglucosaminyltransferase (MGAT1) transfers the fi rst
N-acetyl-D-glucosamine (GlcNAc) residue onto the mannosyl
core of
N
-glycans and is thus essential for the conversion of high-
mannose to hybrid and complex
N
-glycans. MGAT1 knockdown
