Participation of a Trisaccharide-Lipid in Glycosylation of Oviduct Membrane Glycoproteins*

Preincubation of a hen oviduct membrane preparation with UDP-N-acetyl[~‘lClglucosamine and bacitracin, followed by incubation with GDP-mannose, leads to formation of a chloroform/methanol (Z/l)-extractable glycolipid. Treatment of the lipid with mild acid results in release of a trisaccharide shown to have the structure ~-mannosyl-N-acety1glucosaminyl-N-acetylg1ucosamine. Incubation of purified trisaccharide-lipid with oviduct membranes in the presence of sodium deoxycholate, Mn2+, and GDP-mannose leads to formation of a labeled glycoprotein with an apparent molecular weight of 25,000. Under these conditions no oligosaccharide-lipid is formed from the trisaccharide-lipid. Structural studies revealed that the labeled glycoprotein contained trisaccharide side chain. When incubation conditions were altered by omission of sodium deoxycholate and Mn*+, trisaccharide-lipid was converted containing to chains to form labeled glycoprotein(s). These results provide direct evidence for the enzymatic formation of a lipid-linked trisaccharide containing a P-mannosyl unit, and its subsequent participation in the glycosylation of membrane glycoprotein(s).

Preincubation of a hen oviduct membrane preparation with UDP-N-acetyl[~'lClglucosamine and bacitracin, followed by incubation with GDP-mannose, leads to formation of a chloroform/methanol (Z/l)-extractable glycolipid. Treatment of the lipid with mild acid results in release of a trisaccharide shown to have the structure ~-mannosyl-N-acety1glucosaminyl-N-acetylg1ucosamine.
Incubation of purified trisaccharide-lipid with oviduct membranes in the presence of sodium deoxycholate, Mn2+, and GDP-mannose leads to formation of a labeled glycoprotein with an apparent molecular weight of 25,000. Under these conditions no oligosaccharide-lipid is formed from the trisaccharide-lipid. Structural studies revealed that the labeled glycoprotein contained a trisaccharide side chain. When incubation conditions were altered by omission of sodium deoxycholate and Mn*+, trisaccharidelipid was converted to an oligosaccharide-lipid containing 7 to 9 glycose units. Under these conditions both the trisaccharide-lipid, and the oligosaccharide-lipid formed from it, served as a donor of their carbohydrate chains to form labeled glycoprotein(s). These results provide direct evidence for the enzymatic formation of a lipid-linked trisaccharide containing a P-mannosyl unit, and its subsequent participation in the glycosylation of membrane glycoprotein(s).
Although the participation of polyisoprenol-linked sugars in synthesis of the oligosaccharide chains of certain glycoproteins is now well established (for reviews see Refs. 1 and 2), a number of key questions about this process remain unanswered. One of these questions relates to the intermediates involved in the assembly of the oligosaccharide chain that is ultimately transferred to protein. The enzymatic synthesis of a disaccharide-lipid, shown by Herscovics et al. to be identical with synthetic N,N'-diacetylchibiosyl-a-P-P-dolichol (3), has been demonstrated in a number of systems (4)(5)(6)(7)(8). Moreover it has been shown that this disaccharide-lipid is converted to an oligosaccharide-lipid containing multiple n-mannosyl units (5,7,9). However, little is known about possible intermediates between the disaccharide-lipid and the oligosaccharide-lipid. Levy et al. (7) have presented preliminary evidence suggesting the formation of a trisaccharide-lipid.
Recently Heifitz et al. (8) reported the synthesis of a family of oligosaccharide-lipids of differing carbohydrate chain lengths, one of which has been shown to contain a tetrasaccharide unit. Smith degradation of this tetrasaccharide yielded a trisaccharide with the structure ~-mannosyl-N-acety1glucosaminyl-N-acetylglucosamine (P-Man-GlcNAc-GlcNAc).
In previous GlcNAc-(l-4)-GlcNAc (10). Similar findings have been reported in a mouse myeloma system by Hsu et al. (12). In this study we have investigated the possible involvement of a trisaccharide-lipid in synthesis of this oligosaccharide-lipid and glycoprotein (Fig. 1)  (l-*4)-N-acetylglucosaminitol were prepared as previously described (10 by the multiple extraction procedure previously described (16). The pellet, containing the labeled glycoprotein was incubated with 0.5 ml of 5% Triton X-100 at 100" for 20 min. The assay tube was then immersed in an ice bath and the contents were transferred to a counting vial by rinsing the tube three times with 5 ml of Hydromix scintillation fluid (Yorktown Research Co.). An aliquot (200 to 400 pg) of the labeled protein fraction was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea as previously described (11). Proteolytic Digestion of [GlcNAc-'%JGlycoprotein -The labeled protein fraction (27,000 cpm, 18 mg) was dispersed by sonication in 2 ml of 10 mM CaCl,, 50 mM Tris/HCl (pH 7.9). After 1 drop of toluene was added the suspension was digested with 2.5 mg of pronase for 24 h at 37", followed by another addition of 2.5 mg of the enzyme for 24 h. The digest was then centrifuged in a clinical centrifuge, and the pellet was washed twice with 2 ml of H,O. The combined supernatant solution (26,600 cpm) was concentrated to 1 ml and purified on a Sephadex G-25 column (1.2 x 94 cm) eluted with 0.1 M acetic acid. The fractions comprising the single peak of radioactivity were pooled and lyophilized.
Alkaline Degradation of LGlcNAc-" CIGlycopeptide -The labeled glycopeptide (8,900 cpm) was heated with 1 M NaOH, 1 M NaBH, for 18 h at 100" in a tightly capped tube. The reaction mixture was immersed in an ice bath and treated with a few drops of glacial acetic acid to destroy the excess NaBH,. N-Acetylation and desalting of the reduced product was carried out as previously reported (10). The final product was examined by paper chromatography as described under "Results." Digestion with Mannosidases-One aliquot (5,000 cpm) of the reduced [GlcNAc-14Cltrisaccharide prepared from the [G~cNAc'~CItrisaccharide-lipid or glycopeptide as described above was incubated in 500 ~1 (final volume of 50 mM sodium acetate (pH 4.75), containing 6.4 units of purified oc-n-mannosidase for 24 h at 37". The reaction mixture was deproteinized by passage over a column of phosphoethylcellulose (0.9 x 2 cm) in water and then concentrated under reduced pressure. A second aliquot (3,000 cpm) of the reduced [GlcNAc-WItrisaccharide was incubated in 500 ~1 of 50 rnM sodium acetate (pH 2.8) containing 0.12 unit of purified P-n-mannosidase for 24 h at 37". The reaction mixture was deproteinized and concentrated as described above. The products of a-or P-mannosidase digestion were examined by paper chromatography oligosaccharide-lipid, and glycoprotein were separated as described above. The lGlcNAc-'-lCloligosaccharide-lipid was subjected to mild acid hydrolysis (5), and the water-soluble oligosaccharide product was analyzed by paper chromatography in Solvent System A. The labeled glycoprotein (2,500 cpm) was subjected to pronase digestion as described above, and the resulting glycopeptide fraction was analyzed by gel filtration on a Bio-Gel P-4 column (2.0 X 75 cm).

DISCUSSION
This study has established that a hen oviduct membrane preparation catalyzes incorporation of GlcNAc from UDP-GlcNAc into three CHCl,/CH,,OH-extractable lipids. Two of these are mono-and disaccharides of GlcNAc, presumably linked to dolichol via a pyrophosphate bridge. The third lipid, which contains a trisaccharide unit with the structure p-Mann-GlcNAc-GlcNAc, is present at an extremely low level. It represents only 2% of the CHCl&H:,OH-extractable labeled products and less than 0.9% of the total labeled GlcNAc incorporated into the CHCl,/CH:IOH, CHCl,,/CH,,OH/H,O, and protein fractions. When 1 mM bacitracin is present during the incubation, incorporation of ["ClGlcNAc into the CHCl,/ CH,,OH-extractable fraction is increased l.&fold, and formation of labeled trisaccharide-lipid is increased 12.5-fold.
Further studies involving variation in the incubation conditions revealed that in the absence of Mn2', the oviduct membrane preparation catalyzed transfer to protein at a decreased rate. Furthermore, when both Mn" and deoxycholate were omitted from the incubation mixture, both oligosaccharidelipid and glycoprotein synthesis from trisaccharide-lipid was observed. Because of the relatively low level of labeling of the glycoprotein fraction under these conditions it was not possible to analyze the product by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
However, it was possible to establish by analysis of the pronase digestion products of the labeled glycoprotein fraction that two glycopeptides, corresponding in size to authentic trisaccharide-peptide and oligosaccharidepeptide, were present.
In bacteria, bacitracin blocks the action of a phosphatase that converts undecaprenol pyrophosphate to undecaprenol phosphate, thus preventing it from recycling as a sugar carrier in peptidoglycan synthesis (18). Bacitracin has also been shown to inhibit sterol and squalene synthesis in cell-free preparations of rat liver and, although its mode of action in eukaryotic systems has not been studied in detail, it has been suggested that it interferes with all reactions involving polyisoprenol pyrophosphates (19). Possibly, it functions in a similar fashion in the oviduct membrane system and binds to polyisoprenol pyrophosphates, thus causing an accumulation of certain intermediates, such as the trisaccharide-lipid. However, further studies will be necessary to test this speculation.
These findings, coupled with earlier studies of the transfer of exogenous oligosaccharide-lipid to a membrane protein acceptor (5,111, indicate that the oviduct membranes can catalyze transfer of both (a-Man),,-P-Man-GlcNAc-GlcNAc and p-Man-GlcNAc-GlcNAc from their lipid carrier to an endogenous protein of identical apparent molecular weight.
In any case, because bacitracin causes a preferential accumulation of the trisaccharide-lipid it was practicable to isolate substrate quantities of it in radiochemically pure form. On the basis of chromatographic and enzymatic studies it has been established that the trisaccharide moiety contains a terminal p-mannosyl unit linked to N, N'-diacetylchitobiose.
The origin of the mannosyl unit has not been investigated in detail, but it is clear that only trace levels of trisaccharide-lipid are formed from UDP-['"C]GlcNAc if GDP-mannose is not present in the incubation mixture. Based on the preliminary studies of Levy et al. (71 it seems likely that GDP-mannose, rather than mannosylphosphoryldolichol, serves as the donor of the pmannosyl unit of the trisaccharide-lipid. It is of interest to consider these results in relation to recently summarized information (20) on the structure of the oligosaccharide chains of glycoproteins containing an oligosaccharide linked to the polypeptide via an N-glycosidic bond between GlcNAc and an asparaginyl residue. Seventeen glycoproteins of this category are now known to contain an identical core unit with the structure p-Man-p-GlcNAc-p-GlcNAc-Asn. Excluding consideration of distal sugars (sialic acid, galactose, and N-acetylglucosamine) the number of cr-mannosyl units in these different glycoproteins varies considerably. Thus, the carbohydrate chain in human IgG, IgE, and IgA, contains only 2 a-mannosyl units whereas that in the linkage region of yeast proteo-mannan contains 11 a-mannosyl residues. Clearly, the present studies do not explain this diversity in structure of the oligosaccharide chains to different glycoproteins in terms of possible specificity of the enzyme(s) catalyzing transfer of carbohydrates from their lipid carriers to proteins. However, it is hoped that extension of such studies, using saccharide-lipids of known carbohydrate chain length, to a recently developed system utilizing specific exogenous protein acceptors (21) will cast light on this important question.
Addition of purified [GlcNAc-'Cltrisaccharide-lipid to oviduct membranes in the presence of Mn'+ and sodium deoxycholate did not lead to formation of oligosaccharide-lipid, although relatively efficient transfer of the trisaccharide moiety into the protein fraction was observed. Examination of the labeled protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that virtually all of the label was localized in a glycoprotein with an apparent molecular weight of 25,000. As reported earlier (111, a protein of this molecular weight is the principal product formed when exogenous oligosaccharide-lipid (or GDP-mannose) is used as substrate.