Aug 23, 1979 - Bovine blood coagulation factor X contains both 8s- paragine-linked and threonine-linked oligosaccha- rides. The asparagine-linked chain is a ...
THE~ O U R N A I . O P BIOLOGICAL CHEMISTRY Vol 255. No. 8. Issue of Aprd 95, pp :3526-3531. 1980 Pnnted m I 1 . S A .
The Structures of the Carbohydrate Moietiesof Bovine Blood Coagulation FactorX* (Received for publication, August 23, 1979)
Tsuguo Mizuochi,S Katsuko Yamashita, Kazuo Fujikawa,@Koiti Titani,@land Akira Kobata From the Department of Biochemistry, Kobe University School of Medicine, Ikuta-ku, Kobe, Japan and the §Department of Biochemistry, University of Washington, Seattle, Washington 98195
Bovine blood coagulation factor X contains both 8sparagine-linked and threonine-linked oligosaccharides. The asparagine-linked chain is a mixture of a tridecasaccharide NeuAca2 + 3GalP1+ a(NeuAca2 + 6)GlcNAcPl + 2Manal -+ 6[NeuAca2 + 3GalPl -+ 3(NeuAca2 + 6)GlcNAcPl + 2Manal -+ 3IManPl + 4GlcNAcPl + 4GlcNAc and a dodecasaccharide NeuAca2 6 GalPl + 4GlcNAcP1 -+ 2Manal -+ 6peuAca2 + 3Galbl -+ S(NeuAca2 + 6)GlcNAcPl + 2Manal-+3JManfil 4GlcNAc/31+ 4GlcNAc and their partial desialylation products. The threonine-linked chain is a mixture of NeuAca2 + 3GalPl-+ 3(NeuAca2 G)GaMAc, NeuAca2 -+ 3GalPl + 3(NeuGlya2 -+ 6)GaMAc,NeuGlya2 -+ 3Ga1P1 + 3 (NeuAca2 -+ 6)GalNAc, and NeuGlya2 + 3GalP1-+ 3(NeuGlya2 -+ 6)GalNAc, and their partial desialized forms. The carbohydrate moieties of the factor X subgroups, factors XI and XZ,are identical. --f
--f
--f
DISCUSSION
Because the molecular weights and amino acid compositions of factors X, and X, are almost identical, a structural of these glycoproteins has difference in carbohydrate moieties been suspected. The present study eliminates this possibility. Analysis of both the intact molecules and the glycopeptides from theCOOH-terminalportions gaveexactly thesame patterns of structure in the asparagine-linked and threoninelinked oligosaccharides, although some structural heterogeneities are present within each group of sugar chains as shown in Figs. 1 and 2. Quite recently, Morita and Jackson reported that the aminoacid reside at position 18 in the heavy chainof factor X2 is tyrosine sulfate (4) while that of factor X , is tyrosine as reported by Titani et al. ( 5 ) .This difference may account for the differences apparent between factors X, and X2.
NeuAca 2 i
Blood coagulation factor X (Stuart factor) is one of the plasma glycoproteins produced by vitamin K-dependent pathway. It haswidely been noted that factorX can be separated chromatographically into two components(factors X, and Xz). Jackson reported that the difference of the two components canbeascribed totheircarbohydrate moieties (1). However, the analysis of the carbohydrate compositions of the two components by Fujikawa et al. ( 2 ) showed no significant difference between these glycoproteins. We have recently found that bovine prothrombin has a series of very unique complex type asparagine-linked sugar chains, in which Gal/?1 + 3GlcNAc grouping is included at the outer chainmoieties (3). In order to find out if these unique sugar chains commonly occur in vitamin K-dependent glycoproteins and also to find out any structural difference of the carbohydrate moieties factors XI and Xz, the structural study of the sugar chainsof the two glycoproteins was performed.’ * This work has been supported in part by research grants from the Scientific Research Fund of the Ministry of Education, Science and Culture of Japan, from the Mitsubishi Foundation, and from the National Institutes of Health (HL-16919,GM-15731, and AM-7902). The costs of publication of this article were defrayed in part by the be hereby payment of page charges. This articlemusttherefore marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. +This paper is a part of the dissertationsubmitted to Kobe University School of Medicine for the requirement of Doctor of Medical Sciences. 7 Investigator of the Howard Hughes Medical Institute. ’ Portions of this paper (including “ExperimentalProcedures,” “Results,” “References,” Figs. 4 to 11,and TablesI to V) are presented in miniprint at theend of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full-size photocopies are available
6 NeuAca2*3G~1%1*3GlcNAc61-2Man~l~~ Man%1-4GlcNAcE1-4GlcNAcOT NeuAca2*3Gal%1~3Gl~NAc%l*2Man~l~~ 6
R
t
NeuAca2
NeuAco2+6GalB1*4GlcNAcBl~2Manal., 6M~nB1-4GlcNAc01-4ClcNA~~~ NeuA~o2*3GalBl*3GlcNAcB1-r2Man~l~~
B -
6
t NeUAca2
FIG. 1. Fully sialylated asparagine-linked s u g a r chains of bovine factor X, and XZ.
Factor XI has themolecular structure depictedin Fig. 3 ( 5 , 6). The complex asparagine-linkedoligosaccharides with NeuAca2 -+ 3GaW1 ”-f 3(NeuAca2 -+ 6)GlcNAc components in their outer chains, which were originally found in bovine prothrombin (3),are also present in factor X (Fig. 1).However, oligosaccharide with only NeuAca2 + 6GalDl -+ 4GlcNAc groups in its outer chain moietywas absent in factor X. The occurrence of the unusualasparagine-linked oligosaccharides in both glycoproteins might be related to their similar polypeptide structures. In this context, the structure of the asparagine-linked sugar chains of other vitamin K-dependent glycoproteins, such as factor IX and protein C, which is similar in polypeptide structure to prothrombin and factorX, might be an interesting subject for study. The possibility that the unique asparagine-linked sugar chains of factor X may play key roles in their binding to tissues such as platelet( 7 , 8 ) also warrants future study. The different distribution of sialic acids in the two kinds of oligosaccharides is worthy of note. In contrast to the threo__~ . ____ ~. . from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Maryland 20014. Request Document 79111-1720,cite authors, and include a check or money order for $4.50 per set of photocopies.
3526
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~~~~
~
~
3527
Carbohydrate of Blood Coagulation FactorX N:
GalB1+3GalNAcOT
A-I-a:
NeuAca2+3GalB1+3GalNAcoT
A-I-b:
NeuGlya2+3Ga161+3GalNAcOT
rFJJj 1
FIG. 3. Schematic model of bovine factor x. 0, asparaginelinked sugar chain; 0,threonine-linked sugar chain; Y, y-carboxylglutamic acid residue.
NeuAca2
c
responsible for the formation of the mucin type sugar chains which use both CMP-NeuAc and CMP-NeuGly as substrates. When intact factor XI and factor XZwere subjected to alkaNeuGlya2 line-sodium borohydride treatment, the neutral oligosacchac ride N and the acidic oligosaccharide A-I-c and A-I-d shown 6 in Fig. 2 were notdetected a t all, while six other acidic Ga161+3GalNAcOT A-I-d: oligosaccharides were detected as in the case of the COOHterminal glycopeptides (data not shown). Therefore, oligosacNeuAca2 c charides N, A-I-c, and A-I-d are probably produced by desi6 alylation of other acidic sugar chains, during the preparation A-11-a: NeuAca2+3Ga161+3GalNAcOT of the glycopeptides. Fragment 1,a glycopeptide releasedfrom bovine plasma high molecular weight kininogen by the action NeuGlya2 of plasma kallikrein, also contains the same sugar chains as c 6 the threonine-linked sugar chain of factor X (9). In that case A-11-b: NeuAca2+3Ga161+3GalNAcOT also, only Siaa2 + 3GalP1- 3GalNAcoT was detected as the NeuAca 2 monosialyl sugar chain (9). Therefore, thebiosynthetic patht way for the formation of the disialyl oligosaccharides may 6 NeuGlya2+3Ga161+3GalNAcoT proceed by the sequence, Galb1- 3GalNAc ”+ Thr Siaa2 + 3Galp1+ 3GalNAc + Thr Siaa2 + 3GalP1- 3(Siaa2 NeuGlya2 + 6)GalNAc + Thr. c The possible occurrence of serine-linked sugar chainsin the 6 peptide (residues 3 to 32) of the heavy chain of factors X, and A-11-c: NeuGlya2+3Gal61+3Ga1NAcoT X2 has recently been suggested by Morita and Jackson (4). FIG. 2. Structures of threonine-linked oligosaccharides obSince the intact factor X gave a similaroligosaccharide pattern tained from the COOH-terminalglycopeptidesof bovine factor XI and Xz. The names of the oligosaccharides correspond to those in as COOH-terminal glycopeptide, the serine-linked sugar chain of factor X, if any, may have the same structures as that Fig. 11 in the miniprint. shown in Fig. 2. A-I-C:
6 Ga161+3GalNAcOT
-
-
nine-linked sugar chainswhich contain both N-acetyl and N- Acknowledgments-We would like to express our gratitude to Dr. glycolylneuraminic acids in random distribution, the aspara- E. W. Davie for his continuous interestin this study. Thanks arealso gine-linked sugarchainscontain onlyN-acetylneuraminic due to Miss J. Fujii for her expert secretarial assistance. acid. This evidence implies that the sialyl transferases which REFERENCES act on asparagine-linked sugar chains are specific for CMPNeuAc and aredifferent from the sialyl transferases which are References are found on p. 3529.
3528
Carbohydrate ofCoagulation Blood Factor
X
RESULIS ASPARAGINE-LINKED
SUGAR C H A I N S
X
Carbohydrate of Coagulation Blood Factor
3529
REFERENCES 1. 2. I. 4.
5. 6. 7. 8.
9.
6908-6916 E n d o . Y..
Yamashita.
X.,
W a n . Y. N.,
iwanaqa. S.,
a n d Kobata, A .
10. 11. 12.
13. 14.
15. 16. 17. 18. 19. 20. 21. 22. 23.
2.3.4,6-Tetra-i)-n?thyl 11.5-dl-c-acetylI 3.4.6-Trz-g-methyl (1.2.5-trl-O-acetyl) 2,4-DL-O-nethyl
0 2.06
11.3,5~6-retra-~-acecyIl
2.10 0
2.00 1'00
1.00
119")
Carbohydrate of Blood Coagulation FactorX
3530 Table IV.
Molar r a t i o of a l d l t o la c e t a t e so b t a l n e d
from t h e
h y d r o l y s a t e s of p e r m e t h y l a t e da c i d i co l l q o s a c c h a rides A - 1 .
A-2,
2' a
1
,
Q a
liberated from f a c t o r
and A - 4
A-3
3 a
@
X,.
A
> + .> .c.
A-1
N
n
s ge B
A
N .
A
A-1 -
0
r M t h y l a t r o n a n a l y s e ao f
Table V .
chhe
o l i g o s a c c h a r i d e sa n d
aslalo o l l g o s a c c h a r l d e bO b t a l n e d
from f a c t o r
i f
thelr
glym-
XI
10
20
30
3
peptide
N A-I-a
1.00
0
0.90
0
0
1.00
0.87
0
1.00
0
0.92
0
0
1.00
0.94
0
aslalo A-I-b
1.00
0
0.92
0
A-I-c
1.00
0
0
1.12
a 5 1 d l o A-I-'
1.00
0
0.91
0
A-I-d
1.00
0
0
1.03
1.00
0
0.93
0
0
1.00
0
1.15
1.00
0
1.00
0
0
1.00
0
1.03
1.00
0
0.92
0
0
1-00
4
1.10
1.00
0
0.98
0
a s l a l o A-I-a A-I-b
aslalo A-I-d A-11-a
aslalo A-11-a A-11-b a s l a 1 0 A-11-b A- I I - c
a s l a l o A-II-c
a1
Numhe~swere c a l c u l a t e db y
d e r 1 v a t l v e S as 1 . 0 0 .
rnaklng che v a l u e of g a l a c t l t o l
A
F
A
A-!!
e
0
D
N-1
N-2
10 20 30 distance from origin (cm)
X
Carbohydrate ofCoagulation Blood Factor 1098 7 6
V.Sl412
444444444 4 4 4
4
5
4
3
2
4
4
4
4
3531
'
i\i +n +m +w i c_ii.
Y
A
D E
400 elution
500 volume ( m l )
?VlB A 0
Fiq.
IO.
A
10 20 30 distance from origin (Cm)
P a ~ e relectroohoretoorams
Of alimosaccharldes a b t a l n e d
p e r f a m d a t 37-c
r
0
10
-
20 30 40 distance from origin (cm)
distance from origin ( c m )
from t h e
