A C T A U N I V E R S 1 T A T J S L O D Z I E N S I S
F O L IA B IO C H I M IC A E T B IO P H Y S IC A 14, 1999
Anna K rześlak, A nna L ipińska
GLYCOPROTEINS ASSOCIATED WITH NUCLEAR MATRICES FROM HAMSTER, CHICKEN AND FROG LIVER CELLS:
DETECTION AND CHARACTERIZATION
N u clear m atrices glyco p ro tein s from ham ster, chicken an d fro g liver cells were stu d ied . O u r results show the sim ilarity in the profiles o f liver n u c le ar m atrices g lycoproteins recognized by co n can av alin A (C onA ) o f the exam ined an im al species in c o n tra ry to the ones o b tain ed after stain in g w ith G alanthus nivalis a g glutinin (G N A ). It m ay reflect the differences in the stru c tu re o f olig o sacch arid e ch ain s o f som e liver n u c le ar m atrice s g lycoproteins fro m ham ster, chicken and frog.
IN T R O D U C T IO N
G lycosylation is one o f the m ajo r natu rally occurring m od ificatio ns o f nu clear proteins covalent stru ctu re [17, 18, 20, 22, 31, 39], A n u m b er of n u clear p ro teins such as nuclear pore com plex proteins (designated nuc- leoporins) [31], R N A polym erase II [25] and som e tran sc rip tio n facto rs [7, 23] w ere glycosylated in an unconventional way. T hey con tained single A -acetylglucosam ine (G lcA A c) residues attached by 0 -lin k a g e directly to serine and th reo n in e residues at m ultiple sites. T h e specific functio ns o f O -G lcA A cylation have no t been elucidated. O -G lcA A cy lation has been p o stu lated to play a role in m ediating assem bly o f m ultim eric pro tein com plexes, in targ etin g proteins from the cytoplasm into the nucleus, in reversibly blocking sites o f p h o sp h o ry latio n on serine o r th reo n in e residues o f protein, in nucleus-cytoplasm ic exchanges and in tran scrip tio n al regulation [16, 19, 20]. O -G lcA A cylation ap p ears to be highly dynam ic in a m an n er sim ilar to p ro tein p h o sp h o ry latio n [15, 20], T h e GlcTVAc levels are regulated by the interplay o f G lcA A c transferase(s) and specific iV-acetyl-//-L)-gluco- sam inidase(s) [8, 13, 14]. T h e G lcA A c transferase from ra t liver cytosol capable o f ad d in g G lcA A c to serine and th reo n in e residues o f pro tein a n d a n e u tra l so lu b le A,-a c e ty l-/i-D -g lu c o sa m in id a se fro m r a t spleen
cytosol, which was highly efficient a t rem oving GlciVAc have been purified and characterized [8, 13, 14].
jV-glycosylated p ro tein s, in which o ligo saccharides are a tta c h e d to asp arag in e residues, have been identified as nuclear co m p o n en ts n o t only in the nuclear envelope [3, 12, 40, 41], eu chrom atin fractio n [24] but also in the nuclear m atrix [4, 5, 10, 11, 29, 34], T h e im po rtan ce o f A/-bound oligosaccharide chains o f nuclear m atrix proteins for this stru ctu re was indicated by the results o f the enzym atic deglycosylation experim ents which show ed solubilization o f a significant fraction o f m atrix p rotein s (25% ) on tre a tm e n t o f the m atrix with N -glycosidase F [11]. T h u s, it can be suggested th a t ca rb o h y d ra te -p ro tein in teractio ns are ad d itio n al factors, a p a rt from disulfide cross-linkages, responsible for stabilization o f the native nuclear m a trix structure. T h e glycosylated IIM G 1 4 and 17 proteins belonging to high m o b ility g ro u p o f n o n h isto n e protein s bind preferen tially to th e nuclear p rotein m atrix of m am m alian cells [32], F u rth erm o re , this association appears to be m ediated by the glycosyl chains since enzym atic rem oval o f these m o difications from the lIM G s greatly reduced their binding to the nu clear m atrix. It is tem pting to suggest th a t this ca rb o h y d ra te m od ificatio n o f H M G proteins m ay have functional significance for the arch itectu ral o rg a n iz atio n o f the active dom ains o f ch ro m a tin in cells.
T he m ain aim o f the present study was the detection and ch aracterization o f glycoproteins originating from liver nuclear m atrices o f the th ree anim al species, i.e., ham ster, chicken an d frog.
M A T E R IA L S A N D M E T H O D S
Tissues
Liver o f three anim al species: h am ster (M esocricetus auratus), chicken
(Gallus sp.) and frog (R ana esculenta) were used for experim ents.
Preparation o f liver nuclei
All prep ared solutions were used at 4°C. Liver nuclei from exam ined anim al species were isolated by a m odified sucrose m eth o d [6]. T h e tissue was hom ogenized in the p ro p o rtio n s o f 1 g to 10 m l o f 0.25 M sucrose, 5 m M M g C l2, and 0.8 m M K H 2P 0 4 at pH 6.7, filtered th ro u g h fo u r layers o f gauze and spun dow n at 800xg fo r 7 m in. T h e pellet w as resuspended
in the above solution and T rito n X-100 was added to a final co n c en tratio n o f 0.5% and th en the suspension was hom ogenized again and centrifuged a t 800xg fo r 7 m in. T h e crude nuclear pellet was suspended in 10 vol 2.2 M sucrose, 5 m M M g C l2 and centrifuged a t 40,000xg for 45 m in. T h e purity and integrity o f the nuclei were checked by light m icroscopy.
Nuclear matrix isolation
C arefully purified nuclei from the above m entioned species (2-2.5 m g D N A /m l) were suspended in 0.25 M sucrose, 5 m M M g C l2, 1 m M P M S F , 5 m M T ris-H C l buffer (pH 7.4) and digested by en do genou s nucleases for 45 m in at 37°C. T h e suspension was spun dow n at 7700xg for 15 m in to sep a rate nuclease-released p ro d u cts (N R P ). T h e pellet was ex tracted three tim es w ith LS buffer: 0.2 m M M g C l2, 1 m M P M S F , 10 m M T ris-H C l (pH 7.4) and three times with IIS buffer: 2 M N aC l, 0.2 m M M g C l2, 1 m M P M S F , 10 m M T ris-H C l (pH 7.4) at 4°C. T h e residual pellet was w ashed twice w ith LS buffer yielding nuclear m atrices. C en trifu g atio n s were a t 2000xg fo r 15 m in d u rin g initial LS buffer extractio ns an d a t 7700xg fo r 15 m in for all subsequent extractions [33, 37],
SD S-PA G E o f proteins
F o r sodium dodecyl sulphate-polyacrylam ide gel electrop ho resis (SDS- -P A G E ), nuclear m atrices sam ples were m ixed with 0.9 vol o f solubilizing buffer (20% glycerol, 4 % sodium dodecyl sulp hate /S D S /, 25 fig pyron i- neY /m l, 0.125 M T ris-H C l buffer, pH 6.8) and 0.1 vol o f 2 -m ercaptoeth an ol, an d th en heated in a boiling w ater b ath fo r 5 m in. O ne-dim ensional electrophoresis was perform ed in slab polyacrylam ide gels co n tain in g 0.1% SD S and 8% acrylam ide (pH 8.8) with 3% stacking gel (pH 6.8) according to L a e m m 1 i [28] at 25 m A /slab gel until the py ronine Y m a rk e r reached th e end o f the stacking gel, and then at 35 m A /slab in th e resolving gel until th e m a rk e r dye reached the b o tto m o f the gel. T h e gel slabs were stained w ith C oom assie brilliant blue R-250 according to F a i r b a n k s et al. [9], Excess o f stain was rem oved w ith 10% acetic acid co n tain in g 5% m eth an o l. M olecular m asses o f protein bands were calculated by com pariso n w ith the stan d ard proteins: m yosin (205 kD a), //-galactosidase (116 kD a), p h o sp h o ry lase b (97 kD a), bovine serum album in (66 k D a ), ovalbu m in (45 k D a ) and ca rb o n ic anhydrase (29 k D a ) (Sigm a, St Louis, M issouri, U .S.A .).
Transfer o f proteins from SDS gels
P roteins separated in S D S -polyacrylam ide slab gels were tran sferred o n to Im m obilon-P tran sfer m em b ran e (pore size 0.45 /¿m) by electro p h o retic b lo ttin g in 20% m eth an o l, 192 m M glycine, 25 m M T ris (pH 8.3) for 15 h a t 60V and 4°C [36]. P roteins im m obilized o n tran sfer m em b ra n es were stained by a 5 m in incub atio n at room tem p eratu re w ith 0.2% P on ccau S in 3% acetic acid, followed by destaining in H 20 until th e p ro tein b an ds were visible, in o rd e r to co n tro l the efficiency o f b lottin g an d to m ark the location o f stan d ard proteins. T he Ponceau S staining o f the p ro tein b an ds disap p eared d u rin g subsequent incu b atio n o f the m em b ran e in the blocking solution.
Detection o f lectin-binding glycoproteins
T he detection o f lectin-binding glycoproteins im mobilized on Im m obilon-P sheets was accom plished by th e m eth o d o f H a s e l b e c k et al. [21]. T h e m em branes were treated for 30 m in at room tem p eratu re w ith 20 m l 0.5% blocking reagent (w/v) in T ris buffered saline (TBS: 50 m M T ris-H C l, 1 5 0 m M N aC l, pH 7.5), and were w ashed twice w ith TBS and once w ith buffer one consisting o f TBS, 1 m M M n C l2, 1 m M M g C l2, 1 m M C a C l2, a t pH 7.5 (50 ml each). T h e m em branes were incubated fo r 1 h at ro o m te m p e ra tu re w ith the lectin-digoxigenin (D IG ) con ju gate (G N A , Galanthus
nivalis agglutinin, 1 pg/m l; C onA , C oncan avalin A, 10 /¿g/ml) in 20 m l o f
buffer one.
T h en , the m em branes were w ashed three tim es w ith 50 m l TB S, and polyclonal sheep anti-digoxigenin F a b fragm ents conjugated w ith alkaline p h o sp h atase (a n ti-D IG A P , 750 U /m l) were added in a 1:1,000 dilu tio n in 20 m l TB S and incubated for 1 h at room tem p eratu re. T h e m em branes were again washed three times with 50 m l TBS and th e alkaline p h o sp h atase re actio n was carried ou t by incub atin g the m em b ran e w ith o u t shak in g in 20 ml o f the follow ing freshly prep ared solution: 75 ¡A 5-brom o-4-chloro-3- -indolyl p h o sp h ate 4-toluidine salt (B C IP 50 m g/m l, in dim ethylfo rm am id e) an d 100 /¿I 4-nitro blue tetrazolium chloride (N B T 75 m g/m l, in 70% dim ethylform am ide) in 20 ml o f buffer tw o (100 m M T ris-H C l, 50 m M M g C l2, 100 m M N aC l at pH 9.5). T he reactio n was com pleted w ithin a few m inutes. T h e m em branes were raised w ith H 20 to stop the re actio n and
Analytical procedures
P rotein was estim ated using bovine serum album in as a stan d ard by the m eth o d o f L o w r y et al. [30]. D N A c o n c en tratio n was determ ined from the ab so rb an ce at 260 nm (1 m g D N A /m l co rresp o n d s to 20 O .D . units) o f sam ples dissolved in 5 M u ltra-p u re urea.
Reagents
D igoxigenin-labelled lectins, blocking reagent and polyclonal sheep an ti- -digoxigenin F a b fragm ents conjugated w ith alkaline p h o sp h atase , used for the detection and characterization o f glycoproteins, were o b tained from B oehringer M an n h eim , G erm any. F o r b lottin g the Im m o b ilo n -P tran sfer m e m b ra n e (p o re size 0.45 /im ) o b ta in e d from M illip o re C o rp o ra tio n , B edford, M assachusetts, U .S.A ., was used. O th er chem icals were o f the highest available purity and were from Sigm a, St Louis, M issouri, U .S.A .
R E S U L T S A N D D IS C U S S IO N
T h e nuclear m atrix is o p eration ally defined as th e residual nuclear stru ctu re th a t is yielded by sequential trea tm e n t o f isolated nuclei with d eterg ents, nucleases and buffers o f high ionic stren gth (for reviews see [1, 2, 26, 27]). T h e isolated nuclear m atrix is com posed o f th ree m a jo r electron m icroscopically identifiable stru ctu ral dom ains, nam ely: th e su rro u n d in g p o re com plex-lam ina, residual nucleoli and an internal fib ro g ra n u la r m atrix. T h e nuclear m atrix represents the three-dim ensional fibrillar p ro tein stru ctu re c o n stitu tin g the fram ew ork o f the in terphase nucleus. In a d d itio n to its role in m ain tain in g th e nuclear architectu re and the higher o rd e r stru ctu re o f ch ro m a tin , the nuclear m atrix has been rep o rted as being involved in vario u s n uclear activities such as D N A replication, D N A tran sc rip tio n , R N A processing and stero id -h o rm o n e action [1, 26, 38], It was stated th a t som e n uclear m a trix proteins are cell-, tissue-, d ifferen tiatio n- and tu m o r- specific [2, 35], M oreover, evidence was d em o n strated fo r the presence o f a com m o n set o f polypeptides in the nuclear m atrices o f various cell types [1, 2]. A lth o u g h the presence o f glycoproteins associated w ith the nuclear m a trix was described by m an y au th o rs [4, 5, 10, 11, 29, 34], until now th eir ch aracterizatio n from nuclear m atrices o f different anim al species h as been very lim ited.
T h e present study concerns the JV-glycosylated pro teins recognized by C o n A and G N A from liver nuclear m atrices o f three anim al species, i.e., ham ster, chicken and frog. Nuclei from liver cells o f these v erteb rates were carefully isolated and checked fo r integrity and purity by o b serv atio n with light m icroscopy. Liver nuclear m atrices from the purified nuclei o f the exam ined species were obtained by the technique developed in B crezney’s laboratory [33, 37], N uclear m atrix proteins were separated by one-dim ensional electrophoresis perform ed in .slab polyacrylam ide gels co n tain in g 0.1% SDS and 8% acrylam ide (pH 8.8) with 3% stacking gel (pH 6.8) accordin g to L a e m m l i [28], T ypical polypeptide profiles o f liver n u clear m atrices o f the exam ined anim al species o btained by staining w ith C oom assie brilliant blue R-250 are show n in Fig. 1. T he proteins o f w hole nu clear m atrices separated by S D S -polyacrylam ide slab gel electrophoresis (Fig. 1) were tran sferred o n to Im m obilon-P m em b ran e and tested for D IG -C o n A and D IG -G N A binding (see M aterials and M ethods). T h e effects o f these experim ents are d em onstrated in Fig. 2. T he resulting p attern s show n in Fig. 2A allow to com pare the nuclear m atrices glycoproteins recognized by C on A from liver o f ham ster, chicken and frog. T h e detailed analysis o f all sep aratio n s indicated the sim ilarity in the profiles o f the m ain glycoprotcins. H ow ever, the lim itation which was given by used system o f electrophoresis m akes im possible the com parison o f glycoproteins existing as m in o r co m ponents.
It is rem arkable, th a t the p attern s o f liver nuclear m atrices glycoproteins recognized by C onA o f exam ined species differed significantly from those o b tain ed after staining w ith G N A - a lectin specific fo r term in al b ound m an n o ses (a l-2 , 1-3, 1-6 to m an n o se) (Fig. 2B). A t least six glycoproteins o f nuclear m atrix o f h am ster liver stained by C onA were recognized also by G N A . It was observed th a t G N A reacts strongly with glycoproteins w ith m o lecu lar m ass to a b o u t 180 and 46 k D a. In th e case on nu clear m atrix glycoproteins o f chicken liver seven o f them occurring betw een 46 and 158 k D a were stained w ith G N A as well as w ith C onA . F ro m six glycoproteins o f nuclear m atrix from frog liver the m ajo r G N A bin ding p ro tein h as an ap p ro x im ate m olecular m ass o f 60/62 k D a .
T h e results presented in this study indicate th a t the differences betw een glycoproteins associated with nuclear m atrices o f the three anim al species m ay be attrib u ted to a different level o f their glycosylation. It is possible th a t the differences in the stru ctu re o f oligosaccharide chains o f nuclear m atrices glycoproteins from different anim al species m ay affect diversity of th eir function.
M M
1 2 3
Fig. 1. S D S -P A G E o f iiver n u c le ar m atrice s p ro te in s fro m h a m ste (1), chicken (2) an d fro g (3) o n 8% acry lam id e slab gel. A rro w s in d icate th e p o sitio n s o f m a rk e r p ro te in s o f 205, 116, 97, 66, 45 an d 29 k D a fro m to p to b o tto m . G el w as stain ed w ith C o o m assie b rillia n t blue
R -250. A b o u t 50 ¿ig p ro te in s w ere applied p e r gel
1 2 3 1 2 3
F ig. 2. L iver n u c le a r m atrices p ro te in s fro m h a m s te r (1), chicken (2) a n d fro g (3) e le ctro p h o re se d on 8 % a cry lam id e slab gel were tran sfe rre d to Im m o b ilo n -P m em b ran e s a n d tested fo r D IG -C o n A
ACKNOWLEDGEMENTS
T h e au th o rs wish to express th eir th an k s to J. G ierak , M .Sc. fo r typing the m a n u scrip t, and M . R ad w an fo r p h o to g rap h y . T his w ork w as sup p o rted by g ra n t 505/254 from the U niversity o f Łódź.
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W płynęło d o R ed ak cji D e p a rtm e n t o f C y to b io c h e m istry
F o lia b iochim ica et biop h y sica U niv ersity o f Ł ó d ź
24.04.1998
Anna Krześlak, Anna Lipińska
G L IK O P R O T E IN Y Z A S O C JO W A N E Z M A T R IK S JĄ D R O W Ą K O M Ó R E K W Ą T R O B Y C H O M IK A , K U R Y I ŻA B Y :
ID E N T Y F IK A C JA I C H A R A K T E R Y S T Y K A
B a d an o g lik o p ro tein y m atrik s jąd ro w ej w ątro b y ch o m ik a, k u ry i żaby. N asze w yniki w ykazują p o d o b ień stw o w ykresów g likoprotein rozp o zn aw an y ch przez k o n k a n a w a lin ę A (C o n A ) m a trik s ją d ro w e j w ą tro b y b a d a n y c h g a tu n k ó w zw ierząt w p rzeciw ień stw ie d o w y k resó w g lik o p ro te in o trzy m an y ch po zasto so w an iu lektyny z Galanthus nivalis (G N A ). M o że to o d zw iercied lać ró ż n ic e w s tru k tu rz e ła ń c u ch a o lig o sac h ary d o w eg o p e w n y ch g lik o p ro te in m a trik s jąd ro w ej w ą tro b y c h o m ik a, k u ry i żaby.
