190 U. Theopold et al. Insect Biochemistry and Molecular Biology 31 2001 189–197
similarities to enzymes involved in carbohydrate metab- olism Yuan et al., 1997; Haecker et al., 1997; Sen et
al., 1998. The substrates for these enzymes have not been identified in all cases. Analysis of developmental
changes in glycosylation has so far mostly relied on the use of lectins with specificity for specific sugar con-
figurations Fredieu and Mahowald, 1994; Callaerts et al., 1995. The carbohydrate containing moieties, which
include proteins and lipids remained mostly unidentified. Given the underlying complexities, changes in the glyco-
sylation patterns have not been studied extensively in whole animals, whereas Drosophila cell-lines were suc-
cessfully used for this purpose Kramerov et al., 1983. One developmental aspect in particular has been
addressed in cell lines; namely the influence of ecdysone on the regulation of glycosylation Berger et al., 1978;
Kramerov et al., 1983; Cherbas et al., 1989. In most cell lines, ecdysone has an anti-proliferative effect
Kramerov et al., 1983. In vivo, this effect is only observed in tissues, which are replaced during metamor-
phosis Talbot et al., 1993. In developing tissues, ecdy- sone orchestrates the differentiation. The differential
effects of ecdysone are mediated by different isoforms of the ecdysone receptor Talbot et al., 1993.
Here we describe the differential glycosylation of hemomucin, a cell surface mucin, which we used as a
model to study glycosylation in insects Theopold et al., 1996. One rationale for using this protein is the highly
glycosylated nature of mucins which facilitated our analysis. Another rationale is the fact that hemomucin is
involved in insect immune reactions and that ecdysone has been shown to enhance immune reactivity in Droso-
phila
by inducing a number of proteins involved in insect immunity Dimarcq et al., 1997. Hemomucin an inducer
of insect immunity was originally isolated from Droso- phila
hemocyte-like cell lines using Helix pomatia agglutinin HPL, a lectin with specificity for GalNAc
Theopold et al., 1996. Here we present evidence for the stage- and tissue-specific appearance of two different
hemomucin glycoforms in Drosophila.
2. Experimental procedures
2.1. Flies Flies carrying the w
1118
mutation were kept on cornmealyeast food at 25
° C with a 1014 h lightdark
cycle. 2.2. RNA slot blots
RNA blots were hybridized according to conventional protocols Sambrook et al., 1989.
2.3. Preparation of antisera The hemomucin specific antiserum was prepared as
described Theopold et al., 1996. For the production of an antiserum against recombinant hemomucin a PCR
amplified fragment covering amino acids 178–299 Theopold et al., 1996 was expressed in the expression
vector pQE32 Quiagen. The resulting fusion-protein was purified according to the instructions of the manu-
facturer and excised from a preparative polyacrylamide gel. Rabbits were immunized according to Harlow and
Lane 1988 with approximately 10
µ g protein used for
each immunization. 2.4. Purification of glycoproteins using lectin columns
Purification on lectin columns was performed essen- tially as described Theopold et al., 1996. The sugar
used to elute mucin from a PNA column was 125 mM lactose. For smaller scale precipitations of mucin from
cell- or tissue-lysates, 50 µ
l of column material contain- ing 50 beads was incubated with the lysate for 10 min
at 4 °
C, washed six times in the buffer used for binding and eluted by boiling with SDS–PAGE loading buffer.
2.5. Electrophoretic techniques SDS–polyacrylamide gel electrophoresis on a Mini-
Protean II electrophoresis unit Bio Rad was essentially performed according to Laemmli 1970. Molecular
weights were determined using prestained SeeBlue
TM
or Mark12
TM
molecular weight markers Novex. The pro- teins were blotted onto a nitrocellulose membrane
Amersham as described by Theopold and Schmidt 1997. The amount of protein loaded was ca. 5
µ glane
in Western blots or as indicated in the figure legends. The blotting efficiency was determined by staining the
blot with Ponceau S. In Western blots, peroxidase-conju- gated HPL Sigma, Bandeira Griffonia simplicifolia
lectin BSL and Vicia villosa B
4
lectin VVL were used at a concentration of 0.1
µ gml. Peroxidase-conjugated
PNA was used at a concentration of 1 µ
gml. For detec- tion of signals by enhanced chemiluminescence, the BM
Chemiluminescence reagents
from Boehringer
Mannheim were used according to the instructions of the supplier. Detection by antibody on the immnoblot
and by lectin-mediated chemiluminescence on an auto- radiograph allowed us to superimpose the two patterns
and to identify signals that stained with both the specific antibody and the lectin.
2.6. Treatment of cells prior to Western blot analysis Schneider cells SL2 cells, a cell line which has been
used as a model system to study ecdysone induction and immune reactions Kramerov et al., 1983; Theopold and
191 U. Theopold et al. Insect Biochemistry and Molecular Biology 31 2001 189–197
Schmidt, 1997 were diluted 10-fold and grown for another day to obtain an exponentially growing cell sam-
ple. For induction, 20-hydroxyecdysone Sigma was added to a concentration of 5
µ gml for the duration indi-
cated in the figure legends. Taxol Sigma was added at the concentrations indicated and the cells were left to
grow for another five days.
2.7. Treatment of hemomucin with O-glycosidase Approximately 1
µ g of hemomucin eluted from a
PNA column was incubated for 20 h after addition of 5 mU of O-glycosidase Boehringer in 50
µ l reaction
buffer 50 mM TrisHCl, pH 7.5, 0.5 Nonidet P40. After that time, an aliquot of 20
µ l was taken for analy-
sis. The remainder was heat-denatured for 10 min at 65
° C and incubated with the same amount of freshly
added O-glycosidase for another two days. Only the 105 form of hemomucin was digested by O-glycosidase
when incubated alone or in a mixture with the 100 kDa form, which remained undigested data not shown.
2.8. Staining of ovaries and cells Freshly emerged Drosophila females were transferred
into a new culture vessel and incubated for another two to four days. Ovaries were dissected and fixed in 4
paraformaldehyde in PBS overnight. After four washes 5 min each in PBS, FITC-conjugated PNA was added
to a concentration of 50
µ gml in PBS containing 0.5
NP40. After 1 h, the sample was washed twice to remove excess
PNA and
analyzed using
fluorescence microscopy. Other ovaries were analyzed in Western
blots using the hemomucin specific antiserum.
3. Results
