Cvetković, Ivana

Grigorov, Ilijana

Milosavljević, Tanja

Petrović, M.

English

 Digital Repository of Archived Publications - Institute for Biological Research Sinisa Stankovic (RADaR)

Arch. Bioi. Sci., Belgrade, 53 (3-4), 251'-261', 2001.
GLYCOSYLATION OF RAT LIVER NUCLEOPROTEINS INVOLVED IN THE REGULATION OF
HAPTOGLOBIN GENE EXPRESSION IS CHANGED DURING ACUTE PHASE RESPONSE. Ivana
Cvetkovic, IIijana Grigorov, Tanja Milosavljevic and M. Petrovic. Department for Molecular Biology,
Institute for Biological Research Sinisa Stankovic, 11060 Belgrade, Yugoslavia.
UDe 591.044:547.963.3
kD
Fig. I. Daffinity chromatography of nuclcoprotcins from the livers of con-
trol (lane I) and turpentine treated (lane 2) rats.
Haptoglobin (Hp) is an acute phase protein (API') syn-
thesis of which significantly increases during acute phase re-
sponse (APR) as a consequence of increased transcription of
its gene in liver (B a u man nand G a u I die 1994). It
has been established that transcriptional regulation of the rat
IIp gene is controlled by a mechanism relying on structural
modification of liver trans-acting nucleoprotcins (NPs) (M i -
los a v I j e vic c: al. 1997) which act viii hormone regula-
tory sequence termed ABC element. Numerous studies implied
that glycosylation is a prevalent form of structural modification
of many nuclear proteins including transcription factors such as
Spl, HNF-l and p53 (J a c k son and T j ian 1988;
S n a w ct ill. 1996; Com e I' and H a rt 1991). Qualita-
tive carbohydrate content analyses of these transcription fac-
tors have shown that N-acctylglucosamine (GlcNAc), mannose
(Man) and glucose (Glc) arc the most common types of sugar
moieties and their presence or absence determines their DNA
binding affinity and/or transactivation potential (Eufemi et, al,
1991). Based of these data, the aim of this study was to investi-
gate whether GlcNAc, Man and Glc sugar moieties exist in rat
liver trans-acting NPs involved in the I-Ip gene transcriptional
regulation during APR. In order to examine that, we isolated
rat liver trans-acting Nl's by DNA affinity chromatography.
In order to detect if GlcNAc, Glc and Man sugar moie-
ties are attached to the rat liver trans-acting proteins amino
acids side chains we performed lectin Western blot analyses.
This method enabled us to detect GlcNAc moieties with
wheat-germ agllutinin (WGA) and Glc and Man moieties with
concanavalin A (Con A). Equal amounts of traIlS-acting NPs
from the livers of control and turpentine-treated rats were
separated by II % SDS-PAGE and transferred on nitrocellu-
lose membrane (Hybond-C, Amersham) after the elution from
ABC affinity column. A membrane was firstly incubated in the
TBST buffer (100 mM Tris-HCl, pH 7.5, 0.15 M NaC!, 0.1%
Tween-20 with 3 mg/mL bovine serum albumin (BSA) for 30
min, and then with biotinylated WGA or Con A
(10 mg/mL of TBST buffer) for 45 min, with or without
D( +)GlcNAc and D( +)Glc as specific competitors. To remove
unbound lectin, the membranes were rinsed (3xlO min) with
TBST and then incubated with avidine-biotinylated alkaline
phosphatase complex (30 min) and rinsed (3xlO min). Binding
reaction was visualised with chromofore 5-bromo-4chloro-3-
indolyl phosphate and nitro blue tetrazolium (BCIP/NBT)
(INC kit SI'-2001, Vector Laboratories).
DNA affinity column was prepared with regulatory ABC
element (-170/-56 bp) linked to a CNBr-activated Sepharose
4B (Pharmacia) according to the method of K ado nag a
and T j ian (I986). Nuclear extracts (NEs) were isolated
from the livers of control and 12 b-turpentine-treated rats
(G 0 I' ski et ill. 1986) and then applied on to the ABC af-
finity column. ABC-binding NPs were eluted with the buffer
(25 mM Hcpes, pI-! 7.9, 12.5 M MgCI2, hnM DTT, 20% glyc-
erol) containing 1M KCI. Equal amounts of ABC-binding NPs
from the livers of both groups of rats were subjected to 11%
SDS-polyacrylamide electrophoresis (SDS-PAGE) as de-
scribed by L a e m m I i (1970). Silver staining of ABC-
binding NPs electrophoretical profiles (Fig. 1) showed that
DNA binding affinity of several NPs with molecular weights
(Mw) ranging from 25 to 70 kD is changed during the course of
APR. Namely, NPs with Mw from 26 to 45 kD displayed in-
creased binding affinity to ABC element (Fig. 1, lane 2) in
relation to the control sample (Fig. 1, lane 1), while NPs mo-
lecular mass of 25 (p25) and 51 kD (p51) expressed the binding
affinity only during APR. At the same time, binding affinity of
p62, p64, p68 and p70 was decreased.
oM
21
20-
66-
45-
36-
29-
251'
261'
Fig. 3. ConA Western blot .uialyscs 01 trans-acting nuclcoproteins from the
livers of control (lane 1) and turpentine treated rats (lane 2) in the absence
(A) or presence (B) of 0.3 M D (+ )Gle as a specific competitor.
Acknowledgments - This work was supported by the Ministry for
Science and Technology of Serbia.
References: Baumann, H. and Gauldie, J. (1994). Immunol.
Today 15, 74-80. - Choy, T-Y., Dang, C. V. and Hart, G. W.
(1995).l'mc. Natl, Acad. Sci. USA 92,4417-4421. - Comer, I. F
and Hart, W. G. (1999). Biochim. Biophys. Acta 1473, 161-171. -
Gorski K., Carneiro M. and Schibler U. (1986). Cell 47, 767-
776. - Eufemi, M., Altieri, F., Cervoni L., Spoto G. and Ferraro,
A. (1991). Biochem Int. 23, 35 -42. - Jackson, S. P. and Tjian,
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(1986). Proc. Nail. Acad. Sci. USA 83, 5889 -5893. - Laernmli,
U. K. (1970). Nature 227, 680-685. - Milosavljevic, T., Gri-
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17,423-426
For these reasons, our future investigations will be aimed
at mapping glycosylation sites of rat liver trans-acting NPs what
would enable us to learn the exact contribution of glycosylation
to the regulation of the rat Hp gene expression.
ConA Western blot profiles of rat liver trans-acting pro-
teins (Fig. 3A) showed a pronounced presence of D(+)Glc and
D( +)Man sugar moieties in the control trans-acting p42, p45,
pSI, p68 and p70, while APR was followed by their deglyco-
sylation and intensive glycosylation of p25, p62 and p64. Dis-
appearance of coloured bands after lectin Western blot analy-
ses in the presence of 0.3 M D( + )Glc proved the specifity of
trans-acting NPs-ConA interactions (Fig. 3B).
The observed changes at the level of D(+ )GlcNAc as
well as D( + [Glc, D( +)Man glycosylation of the mentioned
trans-acting Nl's following APR induction coincide with the
changes in their ability to bind ABC clement. Therefore we
assume that glycosylation could be one of the regulatory
mechanisms involved in the regulation of the rat Hp gene tran-
scription. This assumption is supported by the results of several
authors who demonstrated the influence of glycosylation on
DNA binding ability of several transcriptional factors
(J a c k son and T j ian 1988; S haw et ill. 1996;
T rei s m a n 1992). In the cases when glycosylation in-
creases the affinity of transcriptional factors to bind DNA gly-
cosylation sites are quite far from DNA binding domain and it
is most likely that glycosylation alters protein conformation in
such a way that its DNA binding ability is changed (S haw
ct ill. 1996). Besides, glycosylation can also appear within
transactivation domain (C hoy ct ill. 1995) or within PEST
sequences (R e c h s t e i n e r et nl. 1996) and thereby cause
changes of transactivating potential or stability of transcription
factor.
B
B
I. 1
•
45·
JO.
B·
24·
1
Fig. 2. WGA Western blot analyses of trans-acting nucleoproteins from the
livers of control (lane I) and turpentine treated rats (lane 2) in the absence
(A) or presence (B) of 0.5 M D( +) GlcNAe as a specific competitor
Results of WGA Western blot analyses of rat liver trans-
acting NPs (Fig. 2A) revealed that APR is accompanied by
intensified GlcNAc glycosylation of p2'), p32, p35 and pSI as
well as dcglycosylation of p62, pM, p68 and p70 (Fig 2A, lane
2) in relation to the corresponding controls (Fig.2A, lane I).
Specifity of rat liver trans-acting Nl's-WGA interactions was
confirmed by performing WGA Western blot analyses in the
presence of 0.5 M D( +) GlcNAc (Fig. 213, lanes I and 2).


Glycosylation of rat liver nucleoproteins involved in the regulation of haptoglobin gene expression is changed during acute phase response

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Glycosylation of rat liver nucleoproteins involved in the regulation of haptoglobin gene expression is changed during acute phase response

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