Carbohydrate Structures of HVJ (Sendai Virus) Glycoproteins"

The carbohydrate structures of two membrane gly- coproteins (HANA protein and F protein) of HVJ have been determined on materials purified from virions grown in the allantoic sac of embryonated chicken eggs. Both glycoproteins contain fhcose, mannose, galactose, and glucosamine but not galactosamine, indi- cating that their sugar chains are exclusively of the asparagine-linked type. The radioactive oligosaccha- ride fractions obtained from the two glycoproteins by hydrazinolysis followed by NaB[3H]4 reduction gave quite distinct fractionation patterns after paper electrophoresis. More than 75% of the oligosaccharides from F protein were acidic and separated into at least four components by paper electrophoresis. Only 18% of the oligosaccharide from HANA protein was an acidic single component. These acidic oligosaccharides could not be converted to neutral oligosaccharide by sialidase digestion. Structural studies of the neutral oligosaccharide fractions from HANA and F proteins revealed that both of them are mixtures of a series of high mannose type oligosaccharides and of complex type oligosaccharides with Gala1 -+ 4 (Fucal 4 3) GIcNAc group in their outer chain moieties.

The carbohydrate structures of two membrane glycoproteins (HANA protein and F protein) of HVJ have been determined on materials purified from virions grown in the allantoic sac of embryonated chicken eggs. Both glycoproteins contain fhcose, mannose, galactose, and glucosamine but not galactosamine, indicating that their sugar chains are exclusively of the asparagine-linked type. The radioactive oligosaccharide fractions obtained from the two glycoproteins by hydrazinolysis followed by NaB[3H]4 reduction gave quite distinct fractionation patterns after paper electrophoresis. More than 75% of the oligosaccharides from F protein were acidic and separated into at least four components by paper electrophoresis. Only 18% of the oligosaccharide from HANA protein was an acidic single component. These acidic oligosaccharides could not be converted to neutral oligosaccharide by sialidase digestion.
Structural studies of the neutral oligosaccharide fractions from HANA and F proteins revealed that both of them are mixtures of a series of high mannose type oligosaccharides and of complex type oligosaccharides with Gala1 -+ 4 (Fucal 4 3) GIcNAc group in their outer chain moieties.
It has been known that HVJ has an activity of inducing high frequency of cell to cell fusion. HVJ i s a negative strand RNA virus with sizes ranging from 150-250 nm. Like other paramyxoviruses, it consists of an inner nucleocapsid surrounded by a membranous envelope covered with spikes of approximately 130-150 a in length. The envelope of HVJ virions grown in the allantoic sac of embryonated hen's eggs contain six structural proteins, which are readily separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of thiol derivatives (1, 2). Three of them are glycoproteins. HANA protein with molecular size of 67,000 daltons has both hemagglutinating and sialidase activities and constructs HANA spikes of viral envelope (3). F1 protein (Mr = 51,000) and Fz protein (M, = 15,000) show neither hemagglutinating nor sialidase activities. In the viral envelope, these glycoproteins occur as F protein which is composed of 1 mol each of FI and F B proteins connected by a disulfide bond. F protein constructs F spikes (3) and plays essential roles in hemolysis, cell fusion, and infectivity of virons (2-5). In view of the recent development of the techniques to use HVJ as a means to fuse living cells, the mechanism of cell fusion induced 8 To whom correspondence should be addressed. by the envelope glycoproteins of HVJ is an important subject to elucidate. Structural information of these glycoproteins is essential for elucidating the biochemical basis of functions of these glycoproteins. This paper deals with the structural study of the carbohydrate moieties of HANA and F proteins. R e a c t i o i s were t e r m i n a t e d by h e a t i n g the r e a c t i o n m i x t u r e In a boiling v a t s ; b a t h f o r 2 min and the productll were a n a l y z e d by 810-Gel P-4 mlchromatography.

Fractionation of Oligosaccharides by Anionic Charges
When the radioactive oligosaccharide fractions obtained from HANA and F proteins are subjected to paper electrophoresis at pH 5.4, they gave quite distinct fractionation patterns (Fig. 2). More than 75% of the oligosaccharides from F protein were acidic, which could be separated into several components with different mobilities. In contrast, only 18% of the oligosaccharides from HANA protein were acidic components. Each peak in the electrophoretograms was eluted from paper with water as indicated by bars in Fig. 2. The yields of radioactive oligosaccharides FN, FA-1, FA-2, FA-3, FA-4, HN, and HA were 4.84, 7.52, 1.23, 2.83, 3.57, 5.71, and 1.29 X lo6 cpm, respectively. Each component from the deuterium-labeled oligosaccharide fraction was also recovered from electrophoresis paper. An aliquot (2 X lo4 cpm) of each radioactive oligosaccharide was hydrolyzed in 0.4 ml of 4 N HCl at 100 "C for 2 h, and the reaction mixture was freed from acid by repeated evaporation with water. The hydrolysate was Nacetylated and analyzed by paper electrophoresis using borate buffer as reported previously (24). N-Acetylglucosaminitol was the only radioactive component detected from all samples. Therefore, N-acetylglucosamine is located at the reducing termini of all oligosaccharides as expected from the hydrazinolysates of asparagine-linked sugar chains.
All five acidic oligosaccharides were completely resistant to sialidase digestion, indicating that the acidic nature of these oligosaccharides cannot be ascribed to sialic acid like most other asparagine-linked acidic sugar chains. After reduction with NaB[3H]4, the oligosaccharide mixture was subjected to paper electrophoresis at pH 5.4. BPB, bromphenol blue.

Fractionation of Neutral Oligosaccharides
When radioactive HN was subjected to Bio-Gel P-4 column chromatography, it was separated into five peaks (Fig. 3A). Radioactive FN was separated into one major and four minor peaks by the same treatment (Fig. 3B). Each component was pooled as indicated by bars in Fig. 3. The elution ranges of both HN-V and FN-V were wide, indicating that they were mixtures of several oligosaccharides. Therefore, these fractions were subjected to paper chromatography using Solvent 11. As shown in XylNAc, N-acetylxylosamine; subscript OT, NaBr3HIr-reduced oligosaccharides; subscript OH, NaBL-reduced oligosaccharides. All sugars mentioned in this paper were of D-COnfiguratiOn except for fucose, which has an L-configuration. The yields of the six oligosaccharides (HN-I, -11, -111, -IV, -V1, and -V2) from FIN fraction were 8.9 X lo5, 2.31 X lo6, 9.5 X lo5, 5.0 X lo5, 4.B X lo5, and 5.8 X lo5 cpm, respectively.
Complex-type Sugar Chains-The mobilities of FN-I11 and -1V in Bio-Gel P-4 column were 11.3 and 12.1 glucose units, respectively (Fig. 6, A and H). The radioactive FN-IV was not hydrolyzed by P-galactosidase, P-N-acetylhexosaminidase, Bacillus a-fucosidase, or jack bean or Aspergillus a-mannosidases. However, a-fucosidase I from Almond emulsin released 1 fucosyl residue from FN-IV and converted it to a radioactive component with the same mobility as FN-I11 (data not shown). The radioactive component gave exactly the same results as FN-111 in the following structural analyses.

Molar ratio of alditol acetates from hydrolysates ofpermethylated oligosaccharides
Both FN-V2 and FN-V3 were also separated into a major and a minor components (Fig. 8, A and B, respectively). They were not hydrolyzed by Bacillus a-fucosidase but were converted by almond emulsin a-fucosidase I digestion to two components with the same mobilities as those in FN-V1 (Fig.  8 0 . That the two components in Fig. 8C have the same structures as FN-V1 shown in Fig. 10 was c o n f i i e d by sequential exoglycosidase digestion (Fig. 8, D and E ) and by methylation analysis as described above. Therefore, FN-V2 and FN-V3 should be monofucosyl and difucosyl derivative of FN-V1, respectively. Methylation analysis of intact FN-V3 (Table 11) indicated that the 2 fucosyl residues are linked at the C-3 position of N-acetylglucosamine residues in the outer chain moieties (Fig. 10). This structure was also supported by the evidence that FN-V3 was completely resistant to P-galactosidase digestion (data not shown). Methylation analysis of intact FN-V2 (Table 11) indicated that it has 1 each of Gal@l+ 4(Fucal+ 3)GlcNAc and Gawl ".* 4GlcNAc residues in its outer chain moiety. When the radioactive FN-V2 was incubated with a mixture of P-galactosidase and P-N-acetylhexosaminidase, the two components decreased their sizes by approximately 3 glucose units (Fig.  8 0 , indicating that a GalPl"+ 4GlcNAc residue was removed from both oligosaccharides. Approximately 40% of the exposed a-mannosyl residues in both oligosaccharides were removed by jack bean a-mannosidase digestion. (Fig. 8G). Therefore, 40% of FN-V2 should have Galpl -+ 4(Fucal -+ 3)GlcNAc residue on the Manal + 6 side and 60% on the Manal + 3 side (Fig. 10).
Structural analyses on HN-V1 gave exactly the same results as FN-V1. The only difference was that the ratio of oligosac- charide with fucose to that without fucose was 3:2.
Structural analyses of HN-V2 gave the same results as FN-V2 except that the product corresponding to Fig. 8F was completely resistant to jack bean a-mannosidase digestion.
Therefore, G a l P l j 4(Fucal+ 3)GlcNAc outer chain of the two components in HN-V2 should exclusively be linked to Manal "+ 3 side as shown in Fig. 9.

DISCUSSION
Analyses of the carbohydrate composition of HANA and F proteins confirmed the report of Kohama et al. (26) that both glycoproteins contain fucose, mannose, galactose, and glucosamine but no galactosamine and sialic acids (data not shown). Therefore, all sugar chains in these glycoproteins should exclusively occur as asparagine-linked sugar chains, which must be completely released as oligosaccharides by hydrazinolysis described in this paper. Structural study of the sugar chains of vesicular stomatitis virus glycoprotein grown in different lectin-resistant Chinese hamster ovary cell lines indicated that the sugar chains of the viral glycoprotein reflect those produced in the plasma membrane glycoproteins of host cells (27). However, the distinct difference in the oligosaccharide patterns found in the two different glycoproteins of HVJ indicates that the sugar chains of viral envelope glycoproteins are not always a simple replica of host membrane glycoproteins. A possible explanation for the detection of different oligosaccharide patterns in the two glycoproteins is that more than a single biosynthetic machinery of sugar chains exist in endoplasmic reticulum and in Golgi apparatus of host cells, and production of the two viral glycoproteins is segregated from each other until they reach the plasma membrane of host cells. Another possibility is that the processing of asparagine-linked sugar chains is affected by the structure of polypeptide moiety to which they are linked and consequently results in the formation of different oligosaccharide patterns on different protein molecules.
Since the acidic oligosaccharides liberated from HANA and F proteins were not converted to neutral oligosaccharides by sialidase digestion, their structures have not been elucidated yet. However, these oligosaccharides must also be linked to the polypeptide moieties of HANA and F proteins through GlcNAc + Asn linkage, because they have N-acetylglucosamine at their reducing ends. That the two viral glycoproteins contain anionic residues other than sialic acid in their sugar chains is interesting. Since the viral envelope is enriched with sialidase, the viral glycoproteins may have acquired in compensation another anionic residue resistant to sialidase digestion. Whatever the anionic residue may be, it might be specific to viral glycoproteins or may occur in very minor glycoproteins of host cells because sialic acids are the common acidic residues of most animal glycoproteins (28, 29).
The complex type sugar chains of both HANA and F proteins contain Galpl + 4(Fucal + 3)GlcNAc residues in their outer chain moieties. This trisaccharide residue is rarely found in animal glycoproteins. So far, three glycoproteins, human al-acid glycoprotein (30), human parotid a-amylase (8), and human lactotransferrin (31), are shown to have asparagine-linked sugar chains with the particular trisaccharide residue. Consequently the functional role of these unusual outer chains is an interesting subject for future study. Comparative study of the distribution of the sugar chains shown in Fig. 10 in F, and Fz glycoproteins must also be performed before the functional role of sugar chains is considered.
It is well established that concanavalin A inhibits HVJinduced cell fusion. From the structures of the sugar chains in HVJ glycoproteins elucidated in this paper, the mechanism of the effect of concanavalin A can be estimated. Since HANA protein contains mainly high mannose type sugar chains, which binds strongly with concanavalin A, the lectin may cover HANA protein inhibiting viral attachment to cell surface and succeeding cell fusion.