Structures of the Asparagine-linked Oligosaccharide Chains of Human von Willebrand Factor OCCURRENCE OF BLOOD GROUP A, B, AND H(0) STRUCTURES*

The asparagine-linked oligosaccharide chains of human von Willebrand factor (vWF) purified from pooled plasma were quantitatively liberated from the poly-peptide moiety by hydrazinolysis. After N-acetylation, these were fractionated by paper electrophoresis and sequential chromatography on lectin-affinity columns of concanavalin A, Phaseolus vulgaris erythrophyto-hemagglutinin, Datura stramonium agglutinin, Ricinus communis agglutinin 120, and Ulex europaeus agglutinin I and on a Bio-Gel P-4 column. Their structures were investigated by sequential exoglycosidase digestion in conjunction with methylation analysis. The glycoprotein was shown to be unique in its great diversity of oligosaccharide structures. Another note-worthy finding which had not been reported previously was the occurrence of asparagine-linked oligosaccharide chains with blood group A, B, and H(0) structures. present study, this glycoprotein to mono- (0.4% the total oligosaccharides), bi-(78.2%), tri- tetraantennary

The asparagine-linked oligosaccharide chains of human von Willebrand factor (vWF) purified from pooled plasma were quantitatively liberated from the polypeptide moiety by hydrazinolysis. After N-acetylation, these were fractionated by paper electrophoresis and sequential chromatography on lectin-affinity columns of concanavalin A, Phaseolus vulgaris erythrophytohemagglutinin, Datura stramonium agglutinin, Ricinus communis agglutinin 120, and Ulex europaeus agglutinin I and on a Bio-Gel P-4 column. Their structures were investigated by sequential exoglycosidase digestion in conjunction with methylation analysis. The glycoprotein was shown to be unique in its great diversity of oligosaccharide structures. Another noteworthy finding which had not been reported previously was the occurrence of asparagine-linked oligosaccharide chains with blood group A, B, and H(0) structures. In the present study, this glycoprotein was shown to contain mono-(0.4% of the total oligosaccharides), bi-(78.2%), tri-(12.3%), and tetraantennary (2.3%) complex type oligosaccharides in addition to a series of high mannose type oligosaccharides, Man6-9GlcNAc2 (0.8%). Biantennary complex type oligosaccharide chains were those with (8.2%) and without (70.0%) a bisecting GlcNAc residue and approximately 13.2%, 2.2%, and 0.4% of these contained blood group H(O), A, and B structures, respectively. The tri-and tetraantennary complex type chains were those with and without N-acetyllactosamine repeats, and about 13.0% of the triantennary chains without the N-acetyllactosamine repeat contained the blood group H ( 0 ) structure. Occurrence of these asparagine-linked oligosaccharides with blood group A and B structures suggest that the repeated use of factor VIII/vWF pooled concentrate for the treatment of hemophiliacs could result in the production of antibodies against vWF with a different blood group from that of the patient, and this development may be pathogenic. * This study was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan and a grant-in-aid from Fujita Health University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $To whom correspondence and reprint requests should be addressed.
von Willebrand factor (vWF)' is a plasma glycoprotein which is known to play a key role in platelet adhesion to the subendothelium and is a carrier of factor VI11 (1, 2). vWF circulates as a disulfide-linked multimer with a mass of 0.5-20 x lo6 Da and is composed of 270-kDa subunits (1,3). The complete amino acid sequence of the mature subunit (2050 residues) has been elucidated (4), and several functional domains such as binding domains for heparin (5), platelet glycoproteins (6, 7), collagen (8), and factor VI11 (9) were identified using fragments obtained by limited proteolysis of the glycoprotein and antibodies specific for the domains.
Human vWF contains 12 N-linked and 10 0-linked oligosaccharide chains in a subunit, accounting for about 15% of the total weight (4). Although the functional significance of the carbohydrate moiety of vWF is still unclear, it is suggested to play a role in protection from proteolytic degradation (IO), multimerization (ll), and interaction with platelets (12-14) or collagen (15). The carbohydrate portion appears to be influential in the conformation of glycoprotein since asialo vWF induces platelet aggregation in the absence of a cofactor such as ristocetin (16). Carbohydrate-deficient vWF which is devoid of activity was reported in a certain variant of von Willebrand disease (17). Until now, only the structures of two N-linked oligosaccharides of the bi-and tetraantennary complex types and one 0-linked oligosaccharide have been determined for the human factor VIII/vWF complex of which 90% is vWF (18)(19)(20). In a previous study using a series of horseradish peroxidase-conjugated lectins, we demonstrated the presence of N-linked oligosaccharides reactive with Ulex europaeus agglutinin I (UEA-I) which has a strong affinity for oligosaccharides with the Fuccu1+2Gal~l+4GlcNAc~l+ group, blood group H ( 0 ) structure (21).
In the present study, as a step toward elucidation of the functional role of the carbohydrate moiety of human vWF and in order to determine the structures of all N-linked oligosaccharides, the structures of the N-linked oligosaccharide chains of human vWF highly purified from commercial factor VI11 concentrate were investigated by enzymatic microsequencing of the chains released by hydrazinolysis and radiolabeled with NaB3H, and by methylation analysis. We describe here the great diversity in oligosaccharide studied. Among these, biantennary complex type chains with or without bisecting N-acetylglucosamine residues and triantennary The abbreviations used are: vWF, von Willebrand factor; ConA, concanavalin A; HPLC, high performance liquid chromatography; E-PHA, Phuseolus vulgaris erythropbytohemagglutinin; DSA, Datura stramonium agglutinin; RCA120, Ricinus communis agglutinin 120; UEA-I, Ulex europaeus agglutinin I; Fuc, fucose; GalNAc, N-acetylgalactosamine; subscript OT, NaB3H,-reduced oligosaccharides. All sugars mentioned in this paper were of the D-configuration except for fucose with the L-configuration.

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Total asialo oligosaccharides complex type chains, both of which contain blood group H structures, were found. In addition, oligosaccharides with blood group A and B structures were detected in biantennary complex type chains, suggesting that the repeated use of commercial factor VI11 concentrate may be pathogenic.

RESULTS'
Radioactive N-linked oligosaccharide chains obtained from human vWF were subjected to high voltage paper electrophoresis at pH 5.4. As shown in Fig. 1 in the Miniprint, two acidic (A1 and A2) and one neutral (N) oligosaccharide fractions were obtained. The ratio of N, Al, and A2 calculated from incorporated radioactivity was 145630. Upon incubation with sialidase, acidic fractions A1 and A2 were both completely converted to neutral components, indicating that their acidic nature can be attributed to sialic acid residues. Results obtained after partial desialylation by mild acid hydrolysis of each acidic fraction revealed that A1 and A2 contain 1 and 2 sialic acid residues, respectively (data not shown).
For further fractionation, the total radioactive oligosaccharides were incubated with sialidase, and the total asialo oligosaccharides thus obtained were subjected to serial lectin affinity chromatography (Fig. 2). ConA-Sepharose, E-PHA- agarose, and DSA-agarose columns and an RCA12O-HPLC column were used in series as shown in Fig. 2, and fractions designated A to L were obtained (Fig. 2). The percent molar ratio of AB:C:D:E:F:G:H:I:J:K:L calculated from their radioactivities was about 2.8:6.9:59.9:0.8:0.81.1:5.2:0.8:1.5:2.9: 6.3:ll.O. When these were subjected to size separation using a Bio-Gel P-4 column, it was shown that all fractions from A to L were a mixture of several oligosaccharides (Fig. 3 in the Miniprint).
Structures of oligosaccharides in fractions A to L were determined as shown in Fig. 6 by enzymatic microsequencing in conjunction with methylation analysis, as described in detail in the Miniprint.

DISCUSSION
In the present study, structures of about 94% of the Nlinked oligosaccharides released by hydrazinolysis from human vWF were identified after desialylation, as shown in Fig.  6. The great structural diversity among the N-linked oligosaccharides of human vWF was of particular interest, as was the finding that these oligosaccharides have blood group A, B, and H(0) structures. Until now, only the structures of two N-linked oligosaccharides corresponding to the biantennary chain with a Fuc residue in fraction C and the tetraantennary chain with a Fuc residue in fraction L2 shown in Fig. 6 have been reported for human factor VIII/vWF, a complex of which 90% is vWF (18,19).
The N-linked oligosaccharides released from human vWF by hydrazinolysis and labeled with NaB3H4 were neutral (14%), monosialylated (56%), and disialylated (30%) in type, and the sialic acids were shown to be linked mainly to the C-6 position of the Gal residue. Sequential chromatography on lectin-affinity columns in which ConA, E-PHA, DSA, and RCAl2O had been immobilized and on a Bio-Gel P-4 column revesled that the total asialo oligosaccharide fraction obtained by sialidase treatment of the total radioactive oligosaccharides derived from this factor contains a great diversity of oligosaccharide chains. Almost all were of the complex type, although 0.8% were made up of a series of high mannose type, Man6-gGlcNAc2. Complex type oligosaccharide chains had mono-(0.4%), bi-(78.2%), tri-(12.3%), and tetraantennary (2.3%) structures, some with and others without a Fuc residue linked to the reducing terminal GlcNAc. Biantennary complex type oligosaccharide chains were the major component (78.2%) of N-linked oligosaccharides of human vWF of which 8.2% had and 70.0% lacked a bisecting GlcNAc. In addition, about 32% of the chains with this GlcNAc residue contained one or two blood group H ( 0 ) structures Fucal+2Galpl--* 4GlcNAcPl+ in the outer chain moieties, whereas 11%, 2.4%, and 0.4% of the chains without a bisecting GlcNAc residue contained the blood group H(O), A, and B structures Fucal-2Galpl+4GlcNAcpl+, GalNAcal+3(Fucal~2)Gal/31-4GlcNAc(31+, and Gala1~3(Fucal-+2)Gal~l+4GlcNAc~l-+, respectively. About 12% of the triantennary oligosaccharide chains contained one or two N-acetyllactosamine repeats, Galpl4GlcNAcfil+. In addition, about 25% of the chains with a Man residue branched at C-2,4 positions and no Nacetyllactosamine repeat contained the blood group H(0) structure in the outer chain moiety. About 9% of the tetraantennary oligosaccharide chains contained the N-acetyllactosamine repeat. Finally, after desialylation, about 94% of the N-linked oligosaccharides of human vWF were identified. The remaining 6% were thought to have complex type chains which could not be characterized further because of the limited amounts available. Some of these oligosaccharides were thought to have the blood group H(0) structure in the outer  chain moiety. There have been no previous reports giving complete descriptions of the biantennary complex type chains with blood group A and B structures, the biantennary chains with both the bisecting GlcNAc residue and the blood group H(0) structure, or the triantennary chains with the blood group H(0) structure of the N-linked oligosaccharides of glycoproteins.
In the preliminary experiment in which the immunoprecipitates of vWF from plasma of individuals of blood groups A, B, and H(0) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting, antisera against blood groups A and B reacted only with vWF derived from an individual whose blood group was A or B, respectively (data not shown). Human vWF used as the source of the N-linked oligosaccharides investigated in this paper was purified from commercial factor VI11 concentrate prepared from pooled plasma. Therefore, oligosaccharides with blood group A and B structures are thought to exist on vWF molecules in plasma of individuals whose blood group is A or B, respectively. These findings suggest that the repeated use of factor VI11 pooled concentrate for treatment of hemophiliacs could result in production of antibodies against vWF with a different blood group, and this development may be pathogenic.
The occurrence of N-linked oligosaccharides with blood group A, B, and H(0) structures in human vWF revealed in the present study can explain the observation by Sodetz et al.
(45) that human factor VIII/vWF purified from pooled plasma inhibited hemagglutination induced by antisera against blood groups A and B and exhibited the acceptor activity for Nacetylgalactosaminyltransferase involved in formation of blood group A structure. Oligosaccharides with the blood group H(0) structure, which was reactive with a lectin, UEA-I, accounted for about 12% of the total N-linked oligosaccharides of vWF (Fig. 6). vWF is known to be synthesized in both endothelial cells and megakaryocytes (1,2). Factor VIIIrelated antigen (vWF) and UEA-I receptors have been used effectively as markers of endothelial cells in immunohistochemical and pathological studies (46, 47). The present study has clearly demonstrated the occurrence of N-linked oligosaccharides reactive with UEA-I on the vWF molecule. Hormia et al. (48) reported that 140-kDa, 120-kDa, and 80-kDa surface glycoproteins from human endothelial cells bind to UEA-Iagarose. It would be interesting to see if these glycoproteins are derived from human vWF since their molecular masses are the same as derivatives from this glycoprotein (3, 21).

Although blood group antigens have been found on various epithelial and endothelial cells (49, 50), the biological significance of oligosaccharides with blood group structures is unclear.
A relationship between the concentration of vWF in plasma and AB0 blood group has been reported (51,52). It was shown that a relatively low concentration of vWF is present in individuals with blood group H(0) and that the ratio of blood group H(0) in patients with type I von Willebrand disease is significantly higher than in the normal population (51). It remains to be shown how the occurrence of blood group A, B, and H(0) structures in the N-linked oligosaccharides of vWF is related to these clinical observations. It is also important to investigate the structures of these oligosaccharides in patients with various types of von Willebrand disease. Furthermore, elucidation of the function of the carbohydrate moiety of this factor awaits further study. 1.