Jessica Gilliland
Biochemical Characterization of C. elegans ESP Glycoproteins
April 22, 2010
Abstract:
C. elegans has been utilized as a model for the interactions between host and parasitic
nematodes. It has been shown that several different pathogens infect C. elegans via interaction
with the exterior nematode cuticle. However, few studies have encompassed surface
glycosylation or glycoproteins in C. elegans. This nematode contains an intricate range of
enzymes dedicated to the synthesis of an array of complex carbohydrates, wherein said
carbohydrates are involved in several biological processes including protein folding,
oligomerization, stability, immune response, and host pathogen interactions. This study aims to
explore, via lectin binding, whether the excreted secreted products (ESP) of wild type N2 and
srf-3 C. elegans contain differing surface glycoproteins and corresponding sugar moieties. Srf-3
is a nucleotide sugar transporter of the Golgi apparatus membrane that is exclusively expressed
in secretory cells whose phenotype is expressed on the surface of the nematode, thus it is a good
candidate for exploration of surface glycoproteins. This study was intended to characterize and
catalog the various lectins that bind to the surface of wild type N2, and mutant srf-3 and their
accompanying sugar moieties.
Introduction:
Caenorhabditis elegans is a well characterized nematode that is frequently used as a
model organism for many other different organisms (1). More specifically, C. elegans is
therefore used as a model for studying parasitic infection (2). The exterior of the nematode is
known to be surrounded by a collagenous cuticle (3). Furthermore, it has been shown that several
different pathogens infect C. elegans via interaction with the exterior nematode cuticle (1).
However, few studies have encompassed surface glycosylation or glycoproteins in C. elegans.
This nematode contains an intricate range of enzymes dedicated to the synthesis of an array of
complex carbohydrates, wherein said carbohydrates are involved in several biological processes
including protein folding, oligomerization, stability, immune response, and host pathogen
interactions (4). Moreover, glycosylation is the most complex form of posttranslational
modification wherein the biological functions of the glycans are independent of the
accompanying protein (5). Although the biosynthesis of C.elegans N- and O- linked glycans are
similar to vertebrate glycans, the terminal structures are notably different (4). It is these
differences that have not been characterized.
It has been shown that the lectins, wheat germ agglutinin (WGA) and soybean agglutinin
(SBA) bind to the surface of several mutant varieties of C. elegans, including the mutant srf-3.
However, it is not known how or why the surface restricts lectin-binding to specific regions of
the cuticle (3). This study is intended to characterize and catalog the various other lectins that
bind to the surface of wild type N2, and mutant srf-3.
SRF-3 is a nucleotide sugar transporter of the Golgi apparatus membrane that is
exclusively expressed in secretory cells (2). Furthermore, SRF-3 is involved in modification of
the cuticle and surface of C. elegans (2). UDP-galactose and UDP-N-acetylglucosamine are
substrates for SRF-3 (2). It has been shown that srf-3 mutants have an altered glycocongugate
surface composition made apparent by the lack of attachment of Yersinia pseudotuberculosis and
Microbacterium nematophilum that normally infect wild type (2). Moreover, it has been shown
that in srf-3 a subset of glycoproteins are altered when compared to that of wildtype (1).
Parasitic nematode infections are one of the most frequent diseases of humans and
animals (6). These infections have high morbidity but low mortality since the parasite can
establish a long term infection via escaping immediate elimination by the host’s immune system
(7). The surface coat is involved in immune evasion by the parasitic nematode enabling the
parasite to survive the immune response and infect the host for long periods of time in otherwise
immune competent hosts (8). The nematode is able to evade said host immune response by
shedding their surface cuticle thereby shedding attached antibodies (8). The evasion of
antibodies disallows the host to efficiently combat a parasitic infection. Cuticle structure and
composition is conserved among nematodes, parasitic or not (8). Therefore C. elegans is a good
model for study of parasitic nematodes.
Subsequent studies indicate that ESP is involved in such processes as nutrition, digestion,
blood coagulation, host immune evasion, and tissue penetration (9). ESP contributes to parasitic
invasion and evasion. In parasitic nematodes, ESP has been shown to contain glycans as
excretory/secretory (ES) products and surface coat components (10). Therefore, collection and
analysis of ESP glycoproteins in C. elegans may lead to an understanding of parasitic and nonparasitic nematode surface glycan composition.
Methods:
Gravid C. elegans were bleached using a 75% distilled water, 20% sodium hypochlorite,
5% 10M NaOH solution. The pellet was re-suspended in solution and distributed across agar
plates for rehatching. The hatched C. elegans were collected using sterile media and the pellet
was centrifuged at 1200 rpm for 3 minutes. The larvae were re-suspended and washed twice
more using sterile media. The sample was then incubated in M9 for 1 hour. Media was then
collected and centrifuged. The sample was then washed three times with M9 media. The C.
elegans were then transferred to clean M9 media for ESP collection for 3.5 hours.
The sample was centrifuged and the supernatant was collected. The supernatant was
transferred to 0.22𝜇m sterile filters with a vacuum pump and filtrated. Protease inhibitors were
added to N2 and Srf-3 samples; 10mM EDTA, 100ug/ml PMSF, 1ug/ml leupeptin, and pepstatin
A. The sample was then placed in an Amicon-15 filter to concentrate the ESP protein. EZQ was
performed.
The sample was loaded on a 10% agarose gel and SDS-PAGE was performed. The
proteins were transferred to a pvdf membrane. SPYRO RUBY was performed as well. The
Western blot was then blocked for 1 hour in a solution of TBS with 10% Tween. The ESP
proteins were probed with 2.0μg/ml of the selected biotinylated lectin in TTBS and incubated at
room temperature for 1 hour, covered, with gentle shaking. The blots were washed three times
for 10 minutes each with 10ml TTBS. The blots were then probed with 0.01μg/ml avadin-HRP
in TTBS and incubated at room temperature for 1 hour with gentle shaking. The blots were
washed three times for 10 minutes each in TTBS. The bound lectins were then imaged using an
ECL kit.
Results:
Optimization of Biotinylated Lectins
Seven biotinylated lectins were screened against a C. elegans N2 soluable fraction in
order to optimize and thus allow for screening of ESP. Erythrina Cristagalli Lectin (ECL, ECA),
Vicia Villosa Lectin (VVL), Jacalin, Datura Stramonium Lectin (DSL), Griffonia (Bandeiraea)
Simplicifolia Lectin (GSL) II, Lycopersicon Esculentum Lectin (LEL), and Solanum Tuberosum
(Potato) Lectin (STL, PL) were screened. Three of the seven were optimized at 2μg/ml; Jacalin,
VVL, and LEL. All three lectins bind with high molecular weight proteins suggesting significant
amounts of the corresponding glycoprotein moieties. The biotinylated lectins were visualized
using avadin-HRP. Figure 1 shows the affinity of each lectin for the N2 sample. The
optimization of VVL, Jacalin, and LEL, allowed for each of these lectins to be probed against the
ESP samples in order to test for the presence of corresponding glycoproteins.
ECL
VVL
LEL
Jacalin
STL
DSL
2°
GSL II
RUBY
Figure 1. Biotinylated Lectin Optimization. N2 C. elegans soluble fractions Western Blots
were probed using various biotinylated lectins at 2μg/ml to determine specificity. Lanes were
loaded with 4.33mg/ml of protein. Avadin-HRP was used as a secondary and as a control at
0.01μg/ml. Each lectin was probed against their competing sugars. In each blot, from left to
right, the lanes read; lectin and lectin plus competing sugar. Competing sugars from left to right;
lactose, GalNAc, galactose, Chitin Hydrolysate, Chitin Hydroylsate, Chitin Hydrolysate, and
Chitin Hydrolysate. All were used at concentrations dictated by vector labs. Blots were imaged
using Avadin-HRP and ECL.
Western Blot Analysis of C. elegans ESP Fraction Using Biotinylated Jacalin
ESP fraction collected from C. elegans was probed with the biotinylated lectin, Jacalin, at
2.0μg/ml. Jacalin was imaged using avadin-HRP. Controls of Jacalin, competing sugar and
avadin-HRP, and avadin-HRP alone, were successful. In Figure 2 there are two differences
between N2 wild type and the Srf-3 mutant. Probing showed that there is more binding of Jacalin
to one glycoprotein in the Srf-3 sample than that of the N2 sample. There is also more binding to
one oligosaccraride in the N2 sample than that of the Srf-3 sample. Both are interpreted as high
molecular weight proteins, but a molecular weight marker was mistakenly omitted and thus it is
difficult to tell the molecular weight of any proteins in Figure 2.
RUBY
N2
Srf-3 E. coli
Jacalin
N2 Srf-3 E. coli
Jacalin + Galactose
N2 Srf-3 E. coli
avadin-HRP
N2 Srf-3 E. coli
Figure 2. ESP Western Blot Analysis With Jacalin. C. elegans ESP from both strains, N2 and
Srf-3, were probed with biotinylated Jacalin at 2.0μg/ml and imaged using avadin-HRP and
ECL. E. coli is used as a control, as well as Jacalin with competing galactose and avadin-HRP,
and avadin-HRP alone. Jacalin is competed with 800mM galactose as suggested by vector labs.
The lanes were loaded with 8μg of N2, 22μg of E. coli, and 11.2μg of srf-3.
Discussion:
Several attempts were made to optimize a lectin to the sought after ESP fraction. FITClectins were originally used with concentrations and conditions as suggested by Nakayama et al.
However, the resulting FITC-lectin experiments had immense amounts of unspecific binding.
When the conditions suggested could not be replicated or adjusted, an Anti-FITC IgG and an
IgG-HRP were utilized in fractionated amounts in attempts to combat the unspecific binding.
However, these were also abandoned after several failed and repeated attempts over a period of
several months as these conditions were not able to be adjusted. Ultimately, biotinylated lectins
were able to be optimized to a N2 soluble sample, thus they were optimized to a wild type
glycoprotein sample. Optimizing the biotinylated lectin to a wild type glycoprotein allowed for
the subsequent probing of the ESP glycoproteins. Three lectins were able to be optimized to the
N2 soluble sample; Jacalin, LEL, and VVL.
The binding of the lectin, Jacalin, indicates the presence of O-glycosodically linked
oligosaccrarides. The subsequent probing of ESP glycoproteins with Jacalin resulted in a higher
percentage of O-glycosodically linked oligosaccrarides in both of the Srf-3 and N2 samples, thus
suggesting a surface modification in the srf-3 ESP fraction to that of O-glycosodically linked
oligosaccrarides when compared to wild type N2. Figure 2 shows an increase of Oglycosodically linked oligosaccrarides in srf-3 in the second ESP protein indicated and a
decrease in the amount of O-glycosodically linked oligosaccrarides in the first protein indicated
when compared to that of wild type N2. Although there is a clear difference between the two
samples of ESP, this experiment should be repeated in that probing with Jacalin was only able to
be performed once in that several months of failed attempts preceded the experiment. All
preceding results were inconclusive. Probing the ESP with Jacalin should be repeated with the
same conditions, in addition to a molecular weight ladder for sake of duplication as well as for
determining an approximate molecular weight of the modified glycoprotein moieties in the srf-3
sample.
Furthermore, VVL was probed in parallel with Jacalin. The experimental conditions were
run in parallel; however, the results were inconclusive. Again the experiment was only run in
singlet. Therefore, both experiments should be repeated. No attempts were made to probe with
LEL. Future research should also probe with LEL and should seek to optimize the rest of the
lectins in the initial screening.
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