The Structures of the Carbohydrate Moieties of Bovine Blood Coagulation Factor IX (Christmas Factor) OCCURRENCE OF PENTA- AND TETRASIALYL TRIANTENNARY SUGAR CHAINS IN THE ASPARAGINE-LINKED SUGAR CHAINS*

Bovine blood coagulation factor M (Christmas factor) contains four asparagine-linked sugar chains in one molecule. The sugar chains were quantitatively liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The structures of these sugar chains were determined by sequential exoglycosidase digestion in combination with methylation analysis

Bovine factor IX contained two unique penta-and tetrasialyl triantennary sugar chains with the structures shown below in addition to tetra-, tri-, and disialyl biantennary sugar chains of Siaa2 4 3 G a l f l l j 3(Siaa2 + G)GlcNAcfll+ 2 M a n a l 4 6[Siaa2 "-* 3Gal/31"-* 3(Siaa2 "-* 6)GlcNAcfll + 2Manal"-* %]Manjt1+ 4GlcNAcfll-4GlcNAc, Siaa2 + 6Galftl+ 4GlcNAcfll-2Mancul-6[Siaa2 + 3Galftl+ 3(Siaa2 -+ G)GlcNAcfll+ 2Manal+ 3]Manfl1+ 4GlcNAcfll+ 4GlcNAc, and Siaa2 + 6 G W l + 4GlcNAcfll + 2Manal -G(Siaa2 + 6Galfll + 4GlcNAcfll + 2Manal + 3)Ma@1+ 4GlcNAcfl1+ 4GlcNAc and their partially desialized forms. Blood coagulation factor IX (Christmas factor) is one of the plasma glycoproteins that requires vitamin K for its biosynthesis. It plays an important role in the intrinsic pathway of the blood coagulation process (1). Individuals with factor IX deficiency (Christmas disease or hemophilia B) show bleeding symptoms essentially identical with those of classic hemophilia or hemophilia A (2,3). In the blood coagulation process, factor IX is converted to the active form factor IXa by the action of factor XIa in the presence of calcium ions, releasing an activation glycopeptide of 10, OOO Da (4). Factor IXa then acts as the enzyme responsible for the proteolytic conversion of factor X to factor Xa in the presence of factor VIII, phospholipid, and calcium ions (1). Studies of the activation mechanism and amino acid sequence of bovine factor IX have shown that the protein contains four asparagine-linked sugar chains of which three contain up to as much as 75% of the total carbohydrate and are localized in the activation peptide of only 35 amino acid residues (4, 5). To elucidate the struc-tures and physiological roles of the sugar moieties of blood coagulation factors, tlie structures of all sugar chains in bovine factor IX were investigated.

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Portions of this paper (including "Experimental Procedures," "Results," Table I OT used $ this paper to indicate NaB3H4-reduced oligosaccharides.
In the same way, subscript OD is used to indicate NaB2H4-reduced oligosaccharide. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 82M-3075, cite authors, and include a check or money order for $6.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

DISCUSSION
Bovine factor IX is composed of a single polypeptide chain of 416 amino acid residues with M, -55,400. The protein contains as much as 15% carbohydrate which is linked to four asparagine residues (residues 158,168, 173, and 261) (5). Upon activation by factor XIa in the pesence of calcium ions, a highly glycosylated peptide of 35 amino acid residues (residues 147-182), activation peptide, is released from the middle of the molecule to form the active factor IXa (4). Although the physiological role of sugar chains of factor IX has not been elucidated yet, it is obvious that this activation peptide contains three sialic acid-rich sugar chains at Asn-158, Asn-168, and Asn-173, as much as 75% of the total carbohydrate.
Abundance of sialic acid residues in this region, as shown in the present study, may contribute to maintaining the conformation of the factor IX molecule by their negative charge, and their release with the activation peptide upon activation may cause a drastic change in the conformation to generate the active form factor IXa. Alternatively, it is conceivable that three sialic acid-rich sugar chains in a small region of only 35 amino acid residues protect this region of the factor IX molecule, which is likely to be exposed on the surface of the molecule, from undesirable proteolytic attack until its activation by factor XIa takes place in the coagulation process. In fact, the activation peptide region of bovine factor IX is not cleaved in vitro by proteolytic enzymes such as pronase and subtilisin without removal of sialic acid residues (5).
In the present study, approximately 4 mol of sugar chains were liberated by the hydrazinolysis from 1 mol of bovine factor IX on the basis of its M, = 55,400, indicating that the carbohydrate moiety of the protein is almost quantitatively released under the conditions employed if it is linked to four asparagine residues as previously shown by the amino acid sequence analysis (5).
The sugar chain structures of factor IX are unique among those of the vitamin K-dependent bovine blood coagulation factors so far studied, because they are composed of novel tetra-and pentasialyl triantennary complex-type oligosaccharides (Fig. 5, A and B ) as well as di-, tri-, and tetrasialyl biantennary complex-type oligosaccharides (Fig. 5, C-E) already found in bovine prothrombin (7) and bovine factor X (Stuart factor) (15). Interesting evidence is that the triantennary oligosaccharides contain the oligosaccharide D in Fig. 5 as their partial structures. Enzymatic background of this structural rule is an interesting subject for future study.
Finding both N-acetyl-and N-glycolylneuraminic acid in the sugar chains of factor IX casts doubt on our previous speculation that the asparagine-linked sugar chain contains only N-acetyheuraminic acid (15). At the initial stage of developing the hydrazinolysis technique, we noticed that the N-glycolyl group is removed more slowly than the N-acetyl group. However, we later found that the glycolyl group can mostly be removed by 10-h hydrazinolysis. In our recent experiment, we also found that the glycopeptide which contains amino acid residues 34 to 41 of the heavy chain of factor X contains both N-acetyl-and N-glycolylneuraminic acid.' Therefore, our previous speculation must be withdrawn. The d e t a i l s Of '"Experimental Procedures", "Results", The radioactive authentic oligosaccharides and the enzymes described i n this paper were Obtained as described i n the ptevious paper respectively (Table   11: Therefore. it was concluded t h a t t h e s i a l i c acid residues in the sugar chains Of f a c t o r IX e a s t as Slam2-3Gs1, Siaio2+6Gall and Gal81*3ISiao2-6)GlcNA1lc.
IAl-%A91 are s-rired i n Table 1 i n d i c a t e d t h a t A8 has 2 mol each of Siac2+3Gal and Galgl*3lSiac2*61Gl~Ac A8 was concluded t o be tetrasglalyl NZ. wethylation data ~n Table I groups i n i t s Outer chain moiety. Therefore, the Structure of A8 was proposed as Oligosaccharide C in Fig