Substrate Recognition by Oligosaccharyltransferase STUDIES ON GLYCOSYLATION OF MODIFIED ASN-X-THR/SER 'fRIPEPTIDES*

The minimum primary structural requirement for N-glycosylation of proteins is the sequence -Asn-X-Thr/Ser-. In the present study, NHz-terminal deriva- tives of Asn-Leu-Thr-NHz and peptides with asparagine replacements have been tested as substrates or inhibitors of N-glycosylation. The glycosylation of a known acceptor, N"-[3H]Ac-Asn-Leu-Thr-NHCH3, was optimized in chicken oviduct microsomes. The reaction was shown to be dependent upon Mn2+ and linear for 10 min at 30 "C; the apparent K , for the peptide was found to be 10 NM. N"-Acyl derivatives of Asn-Leu-Thr-NHz (N-acetyl, or N-t-butoxycarbonyl) inhibited the glycosylation of N"-r3H] Ac-Asn-Leu-Thr-NHCH3 in a dose-dependent manner; additional experiments demonstrated that these com- pounds were alternative substrates rather than true inhibitors. The benzoyl and octanoyl derivatives were 10 times as effective as N"-Ac-Asn-Leu-Thr-NH, in inhibiting glycosylation. In contrast, peptides containing asparagine modifications or substitutions were neither substrates

The minimum primary structural requirement for N-glycosylation of proteins is the sequence -Asn-X-Thr/Ser-. In the present study, NHz-terminal derivatives of Asn-Leu-Thr-NHz and peptides with asparagine replacements have been tested as substrates or inhibitors of N-glycosylation. The glycosylation of a known acceptor, N"-[3H]Ac-Asn-Leu-Thr-NHCH3, was optimized in chicken oviduct microsomes. The reaction was shown to be dependent upon Mn2+ and linear for 10 min at 30 "C; the apparent K , for the peptide was found to be 10 NM. N"-Acyl derivatives of Asn-Leu- Thr-NHz (N-acetyl, N-benzoyl, N-octanoyl, or N-tbutoxycarbonyl) inhibited the glycosylation of N"-r3H] Ac-Asn-Leu-Thr-NHCH3 in a dose-dependent manner; additional experiments demonstrated that these compounds were alternative substrates rather than true inhibitors. The benzoyl and octanoyl derivatives were 10 times as effective as N"-Ac-Asn-Leu-Thr-NH, in inhibiting glycosylation. In contrast, peptides containing asparagine modifications or substitutions were neither substrates nor inhibitors of N-glycosylation. They did not compete for glycosylation of 3H-peptide at 100fold greater concentrations, and did not deplete endogenous pools of oligosaccharide-lipid. Thus, the asparagine side chain is an absolute requirement for recognition by the transferase. The majority of the glycosylated product (61%), but only l% of the unglycosylated peptide, remained associated with the microsomes after high speed centrifugation.
A large 41amino acid residue acceptor peptide, cu-lac17-ae, was a poor substitute for glycosylation unless detergent was added to the microsomes. In contrast, glycosylation of tripeptide acceptors was not stimulated by detergent. Both of these findings suggest that the tripeptides are freely permeable to the microsomal membrane and support the earlier conclusion that glycosylation of proteins occurs at the luminal face of the microsomes.
Several lines of evidence indicate that formation of Nglycosidically linked glycoproteins is a co-translational event * 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. involving transfer of oligosaccharide from oligosaccharylpyrophosphoryldolichol to nascent polypeptide acceptors attached to membrane-bound polysomes (1,2). The N-glycosylation reaction is catalyzed by an integral membrane protein of the endoplasmic reticulum, oligosaccharyltransferase (3), and the minimum primary structural determinant for the acceptor substrate is the amino acid sequence -Am-X-Thr/ Ser-(4). Transfer of oligosaccharide to peptide is thought to take place at the luminal face of the endoplasmic reticulum; i n vitro topology experiments have shown that the newly synthesized dolichol-linked oligosaccharide faces the lumen (5, 61, and that the glycosylated product formed by transfer of oligosaccharide to endogenous acceptors or translated polypeptide is sequestered within the endoplasmic reticulum (7).
Other features of the polypeptide backbone must also play a role in recognition of substrate by oligosaccharyltransferase because many potential glycosylation sites within proteins are not glycosylated in vivo (8). One factor involved in recognition of glycosylatable sequences by oligosaccharyltransferase appears to be protein folding. Intact proteins containing potentially glycosylatable -Am-X-Thr/Ser-sites are not glycosylated in vitro; however, these same proteins are acceptors after denaturation by reduction and alkylation (8). Small peptides containing proline in the X amino acid position are not acceptors, whereas replacement of proline with many different amino acids results in active acceptor substrates (9, 10). Furthermore, small cyclic peptides are poorer acceptors than their linear counterparts, presumably due to conformational limitations in their cyclic backbone (11). Some penta-, hexa-, and heptapeptides, but not tripeptide substrates, have circular dichroism profiles which are altered by the presence of dimethyl sulfoxide or phospholipid. Addition of dimethyl sulfoxide to the larger peptides makes them better substrates, whereas tripeptide acceptor activity is unaffected (12, 13). Other factors involved in recognition by enzyme, such as the role of polypeptide interaction with membrane or the effect of amino acid side chain analogs within -Am-X-Thr/Ser-, have not been studied in great detail. Substitution of asparagine with glutamine and serine or threonine with homoserine, 0-methylthreonine, or P-hydroxynorvaline appears to destroy acceptor activity in vitro (14-16).
In the present study, we have developed a sensitive competition assay to measure the ability of various NHz-terminal and asparagine derivatives of the tripeptide -Am-Leu-Thr-to act as substrates for or inhibitors of oligosaccharyltransferase in intact hen oviduct microsomes. The results of studies on the NH2-terminal derivatives indicate that a variety of chemical modifications are permitted in this region of the acceptor without loss of acceptor activity; in fact, activities increase as hydrophobicity of the acceptors increase. In contrast, the asparagine derivatives were found to be inactive as acceptors and inhibitors. Therefore, the asparagine side chain appears to be an absolute requirement for recognition by oligosaccharyltransferase.

Methods
Preparation of 3H-labeled Peptide-Five mg of Asn-Leu-Thr, dissolved in 200 p1 of sodium borate, pH 10.0, containing 2% (v/v) dimethyl sulfoxide was N-acetylated by treatment with 25 mCi of [3H]acetic anhydride (10 Ci/mmol) at room temperature. After 1 h, nonradioactive acetic anhydride was added in five equal aliquots, with each aliquot containing a 10-fold molar excess over primary amino groups. The pH of the reaction was maintained between 9 and 10 by addition of NaOH. Following acetylation, the reaction mixture was treated with 1 M hydroxylamine hydrochloride for 1 h at pH 10.0 to reverse any 0-acetylation. The reaction components were then separated by gel filtration chromatography on Sephadex (2-10 in 0.1 M NH4HC03, pH 7.8. The 3H-labeled component (representing approximately 25% of the starting radioactivity) that eluted immediately after Vo was recovered, lyophilized, and treated with 1 M methylamine HCI, pH 4.7, in the presence of 0.25 M l-ethyl-3-(3-dimethylamino-propy1)carbodiimide HCI for 8 h a t room temperature. The reaction products were then separated by gel filtration on a Sephadex G-10 column. Material in the 3H-labeled peak was recovered, lyophilized, dissolved in a small volume of HzO, and applied to a mixed bed ion exchange column to eliminate any peptide that was charged and therefore not blocked a t both the NH, and COOH termini. Approximately 95% of the 3H-labeled peptide did not bind to the column. The specific activity of the purified, blocked peptide was approximately 4.0 X 10' cpmlpmol.
Synthesis of Tripeptide Substrates for Oligosaccharyltransferase-The synthesis of potential substrates was accomplished using various protected amino acids and either the mixed anhydride procedure (17) or the p-nitrophenyl ester method (18) for peptide bond formation. In general, the yield obtained for peptide bond formation was between 60 and 80%. The acyl substrates were prepared by reacting a suitable p-nitrophenyl ester with the a-amino group of the corresponding tripeptide. All substrates exhibited the expected NMR resonances in dimethyl sulfoxide-& solution ( 6 values are reported with respect to trimethylsilyl derivatives), were homogeneous on thin layers of silica using butano1:acetic:acidwater (4:1:5 (v/v/v), upper layer) as the eluent, and were at least 96% pure as determined using reversed phase high performance liquid chromatography on a CIS pBondapak column (Waters Associates), with CH30H:HZ0:CF&OOH as the mobile phase. The physical constants for all substrates used in this study are summarized in Table I. Specific details for Nu-Ac-Asn-Leu-Thr-NHZ and N"-Boc-Asn(N8-Me)-Leu-Thr-NH2 are given below.' N"-Boc-Leu-Thr-NHz-To a stirred solution of Boc-Leu (2.77 g, 12 mmol) in tetrahydrofuran (25 ml) a t -15 'C, N-methylmorpholine (1.32 ml, 12 mmol) was added followed by isobutyl chloroformate (1.56 ml, 12 mmol). Stirring was continued for 7 min and a precooled (-15 " c ) solution of Thr.NH,.HCl (1.85 g; 12 mmol) and NMM (1.32 ml, 12 mmol) in DMF (14 mi) was added. After stirring for 30 min a t -15 "C and 2 h a t room temperature, the solvent was evapo-' The abbreviations used are: Ac, acetyl; Abu, L-a-aminobutyric acid; Nva, L-norvaline; DMF, dimethylformamide; NMM, N-methylmorpholine; ONp, p-nitrophenyl ester; alac17.58, tryptic peptide containing residues 17-58 of a-lactalbumin; Bz, benzoyl; Oc, octanoyl; BOC, tert-butoxycarbonyl; Endo H, endo-@-N-acetylglucosaminidase H; NP-40, Nonidet P-40, Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.   (0.93 g, 2.8 mmol) was dissolved in CHZClZ-trifluoroacetic acid (15 ml; 1:1, v/v) and allowed to stand at room temperature for 30 min. The solvent was then evaporated in uacuo and the residue was precipitated by the addition of ether. The trifluoroacetate salt was isolated and dried. It was coupled with Boc-Asn-ONp (0.99 g, 2.8 mmol) in the presence of NMM (0.31 ml, 2.8 mM) in DMF (5 ml). After a reaction period of 24 h, the solvent was evaporated in Vacuo and the residue was dissolved in aqueous citric acid (5%, 25 ml). The solution was extracted with three 25-ml portions of ethyl acetate to remove the unreacted active ester and p-nitrophenol.
N"-Boc-Asn(NB-Me)-OH-To a solution of Boc-Asn(N@-Me)-OBzl (504 mg, 1.5 mmol) in methanol (4 ml), paladium black (-100 mg) and formic acid (go%, 0.3 ml) were added. The resulting mixture was stirred at room temperature and after 30 min the catalyst was removed by filtration through Celite and the filtrate was evaporated to dryness. The residue was crystallized from tetrahydrofuran-petroleum ether.

N"-Boc-Asn(NB-Me)-Leu-Thr-NH2-To a solution
of Nu-Boc-Asn(NO-Me)-OH (246 mg, 1 mmol), 1-hydroxybenzotriazole (153 mg, 1 mmol), trifluoroacetic acid-Leu-Thr-NHz (345 mg, 1 mmol), and NMM (0.11 ml, 1 mmol) in DMF (3 ml) at 0-5 "C, dicyclohexylcarbodiimide (206 mg, 1 mmol) was added and the mixture was stirred for 4 h. The solution was then allowed to come to room temperature and the reaction continued for 20 h. The dicyclohexylurea was then filtered off, the solvent was evaporated in vacuo, and the residue was worked up in the same manner described for Boc-Asn-Leu-Thr-NHZ. . The S-carboxymethylated protein was digested with 10 mg of trypsin by two additions of the L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated enzyme at 3-h intervals and the reaction was allowed to proceed overnight in 0.2 M NH4HC03, 2 M urea at 37 "C. The digestion products were separated by gel filtration chromatography on Sephadex G-50 (2.5 X 85 cm) in 0.1 M NH4HC03. The first major product absorbing a t 280 nm and eluting after V, of the column was recovered, lyophilized, and rechromatographed on Sephadex G-50, thereby yielding a homogeneous component with an apparent molecular weight expected for the 41-residue peptide containing residues 17 through 58 of a-lactalbumin (20). The overall yield of peptide was 40 mg. The acceptor activity of this material was demonstrated by incubation with microsomes in the presence of radioactive sugar nucleotides followed by separation of the glycosylated peptide from free sugar nucleotides by gel filtration chromatography on Sephadex G-50 as previously described (21). The glycosylatable site within a-lacl-i-ss is a t Asn residue 45 in the sequence -Asn-45-Gln-Ser-.
Oviduct Microsome Preparation-The magnum portion of freshly killed laying hens was cleared of connective tissue, minced, homogenized, and centrifuged as described by Pless and Lennarz (22). Before use, microsomes were stored a t -70 "C a t a concentration of approximately 30 mg of protein/ml.
Oligosaccharyltransferase Assays Using Endogenous Oligosacchar-3 0 p~ N"[3H]Ac-Asn-Leu-Thr-NHCH3 (5 X lo5 cpm), 10 mM MnCL, ide Lipid-Standard reaction mixtures (50 p1 final volume) contained and approximately 300 pg of microsomal protein in 50 mM Tris-HC1, pH 7.4, containing 140 mM sucrose, 25 mM NaCl, and 1 mM EDTA. Reactions were started by addition of microsomes and proceeded at 30 "C for 5 or 10 min before termination by addition of 50 p1 of 20% trichloracetic acid. After 5 min on ice, samples were centrifuged in a Beckman microfuge 11 at setting 10 for 5 min; 75 pl of the supernatant was removed, backwashed three times with 1 ml of ether, and gassed with Nz to remove residual ether. The sample was then analyzed by gel filtration, affinity chromatography, or paper chromatography as described below. Any additions to the reaction mixture, such as dimethyl sulfoxide or detergent, were made prior to microsome addition.
-CH-0-). glucose were included and reactions were allowed to proceed for 15 or 30 min. For preparation of doubly labeled glycosylated product, 1 pM GDP-[14C]mannose (400,000 cpm/assay) was utilized. For preparation of glycopeptides formed by transfer of ['4C]Man-oligosaccharide to nonradioactive peptides, the assay conditions were the same as those for doubly labeled peptide except that the nonradioactive peptide was present instead of N"-[3H]Ac-Asn-Leu-Thr-NHCH3.
Competition Experiments-Competition experiments utilized endogenous oligosaccharide-lipid and were done in an manner analogous to those described above except that the indicated amounts of competingpeptides were included in the reaction mixtures before addition of microsomes.
Endogenous Oligosaccharide-Lipid Depletion Experiments-Assay conditions were the same as above except that N"-[3H]Ac-Asn-Leu-Thr-NH-CH3 was replaced by nonradioactive peptides and the reaction was allowed to proceed for 15 min before 100-fold dilution into cold oviduct buffer and immediate centrifugation at 39,000 X g for 15 min. The pellets were then washed with another 5 ml of oviduct buffer, centrifuged, and resuspended in a minimum volume (150 p l ) before addition of sugar nucleotide precursors and N"-[3H]Ac-Asn-Leu-Thr-NHCH3. After 30 min at 30 "C, assays were terminated with trichloroacetic acid and prepared as described above.
Chromatographic Procedures-Descending paper chromatography was performed in 1-butano1:acetic acidwater (12:3:5, v/v/v) on Whatman 3 " paper for 16 h. Bio-Gel P-4, 200-400 mesh, gel filtration chromatography was carried out using a column (1.6 X 110 cm) equilibrated in 0.1 M ammonium bicarbonate. Fractions of 1.4 ml were collected at a flow rate of approximately 8 ml/h. Concanavalin A affinity chromatography was done by a batchwise procedure. Samples were added to 50 p1 of concanavalin A-agarose beads prewashed in 20 mM Hepes, pH 7.5, containing 1 mM CaC1, and 1 mM MnCl,. After 1 h, the beads were washed six times with buffer and the glycosylated peptide was eluted overnight by incubation with 0.5 ml of the same buffer containing 0.5 M a-methylmannoside.

Glycosylation of N"-pH]Ac-Asn-Leu-Thr-NHCH3: Product
Characterization and Optimization of Conditions-In previous studies, the enzymatic glycosylation in hen oviduct microsomes of endogenous membrane proteins and exogenously added proteins and peptides, was quantitated by the incorporation of radioactive sugars into nonradioactive acceptors (4, 7). In this study, we have utilized nonradioactive endogenous pools of oligosaccharide-lipid present in hen oviduct microsomes as one substrate; the other substrate was exogenously added N"-[3H]Ac-Asn-Leu-Thr-NHCH3. There were a number of technical reasons for using labeled peptide instead of labeled oligosaccharide-lipid to measure the formation of glycopeptide. First, introduction of label in peptide eliminated the requirement for and variability in de novo synthesis of the oligosaccharide-lipid during transferase assays. Second, the presence of label in the peptide acceptor permitted the development of a competition assay for measurement of acceptor and inhibitor activities of a variety of unlabeled tripeptide derivatives of Asn-Leu-Thr-NH2. Third, it allowed one to measure the apparent K,,, for the acceptor. Finally, it facilitated determination of the topological orientation of the glycopeptide product within the microsomes. N"-['H]Ac-Asn-Leu-Thr-NHCH3 was chosen as the labeled acceptor because it was found to be a good substrate in a previous study (4).
The formation of glycosylated N"-[3H]Ac-Asn-Leu-Thr-NHCH3 was routinely measured by paper chromatography. Authentic standards of peptide substrate and glycopeptide product migrated near the solvent front or remained at the origin, respectively (Fig. 1, A and B). Glycopeptide formation, measured as radioactivity remaining at the origin, was time dependent (Fig. 1, C and D ) and was linear for a t least 10 min a t 30 "C (Fig. 2). As expected, treatment of the glycosylated peptide product shown in Fig. 1D with Endo H resulted in the elimination of radioactivity at the origin (Fig.lE). The recovery of radioactivity from paper was typically 35% of that applied. Unless microsomes had been predepleted of endogenous substrate, the production of glycopeptide did not require addition of exogenous oligosaccharide-lipid donor to microsomes (see below).
The 3H-glycopeptide product bound to concanavalin Aagarose and eluted from the lectin in the presence of 0.5 M amethylmannoside. Chromatography of the glycosylated product on a Bio-Gel P-4 column showed that a 3H-labeledproduct with increased molecular weight had been formed. This compound contained 14C and 3H label when 14C-sugar nucleotides were included in the reaction mixture along with 3H-peptide (Fig. 3A). As shown in Fig. 3B, treatment of the doubly labeled material with Endo H caused the 3H-component to elute at a position of apparent molecular weight close to that of unglycosylated peptide, which is consistent with cleavage of the original 3H-glycopeptide to a peptide containing one GlcNAc residue. The 14C-labeled product released by Endo H eluted in the position expected for oligosaccharide. Another 3Hproduct with a molecular weight greater than peptide was also formed during the glycosylation reaction (Fig. 3A). The structure of this product is unknown, but it was not labeled with either [14C]mannose or N-[14C]acetylglucosamine and it constituted only a small percentage of the initial 3H-peptide. This product did not bind to concanavalin A-agarose and it migrated near the solvent front on descending paper chromatography. In most experiments, glycosylation of peptides was confirmed by independent measurement of 3H-glycopeptide formation by paper chromatography, concanavalin A-agarose binding, and gel filtration on Bio-Gel P-4.
During preparation of hen oviduct microsomes, a preincubation with Mn2+ was done to increase the activity of the oligosaccharyltransferase (22). Mn2+ was then washed from microsomes by high speed centrifugation before storage of the

TABLE I1
Divalent metal requirement for N-glycosylation activity Frozen microsomes were thawed, suspended in oviduct buffer containing 20 mM EDTA, collected by centrifugation at 39,000 X g for 15 min, and then washed once in buffer containing 1 mM EDTA by resuspension and centrifugation. Incubation was carried out for 5 min at 30 "C with 3H-peptide in the presence or absence of the various metals. http://www.jbc.org/ Downloaded from microsomal pellet at -70 "C. Upon thawing the microsomes, the addition of 10 mM Mn2+ yielded transferase activity which was approximately 2-fold higher than that observed in the absence of added metal (Table 11). The presence of EDTA lowered activity considerably, indicating that the enzyme does require a divalent metal. Incubation with Mg2+ or Ca2+ rather than Mn2+ did not increase enzyme activity above the control, whereas incubation with Zn2+ or Cu2+ abolished activity. Taken together, these results indicate that oligosaccharyltransferase has a marked preference for Mn2+. Apparently, once bound, this Mn2+ is difficult to remove, even in the presence of EDTA.
The addition of 0.25 mM dithiothreitol, 5% dimethyl sulfoxide (v/v), or NP-40 up to 0.1% (v/v) to incubation mixtures did not affect glycosylation of N"-[3H]Ac-Asn-Leu-Thr-NHCH,. The rate of glycopeptide formation was dependent upon peptide concentration. The Lineweaver-Burk plot shown in Fig. 4 indicated an apparent K,,, for the peptide of 10 @M; the V,,, was found to be 3.1 pmol/mg of protein/min. Typically, 30 FM peptide was added to the reaction mixtures; as much as 20% of the peptide substrate was glycosylated in experiments containing large quantities of microsomes.
Use of a Competition Assay to Measure Acceptor or Inhibitor Activities of Unlabeled Peptides-Structures of peptides containing asparagine replacements or N"-acyl derivatives of Asn-Leu-Thr-NH, are shown in Table 111. To evaluate their ability to act as acceptors or inhibitors, an in vitro glycosylation competition assay was developed. In this assay, the glycosylation of IV-[3H]Ac-Asn-Leu-Thr-NHCH3 was measured in the presence of up to 100-fold molar excess of unlabeled competitor peptides. As shown in Fig. 5, a number of analogs inhibited the glycosylation of 3H-acceptor in a dosedependent manner. At a molar ratio of 1:1, glycosylation of the "-peptide is inhibited by nearly 50% by Ne-Ac-Asn-Leu-Thr-NHz, indicating that this compound and N"-[3H]Ac-Asn-Leu-Thr-NHCH3 are equally effective acceptors. Unblocked tripeptides have previously been shown to be inactive as acceptors in hen oviduct microsomes (4). Consistent with this finding, Asn-Leu-Thr-NHz was found to have little inhibitory effect on glycosylation of N"-[3H]Ac-Asn-Leu-Thr-NHCH3.
In contrast to the W-acyl derivatives, peptides with aspar-

FIG. 4. Lineweaver-Burk plot for glycosylation of LV"-[~H]
Ac-Am-Leu-Thr-NHCH3. Standard reaction mixtures containing 3, 10, 30, 100, or 300 PM N"-[3H]Ac-Asn-Leu-Thr-NHCH3 were incubated for 10 min at 30 "C and then analyzed for glycopeptide production by paper chromatography.  "All NH2-terminal modifications were synthesized starting with the Asn-Leu-Thr-NH2 as described under "Experimental Procedures." bThe asparagine modifications were synthesized using Leu-Thr-NH, as starting material as described under "Experimental Procedures." agine replaced by other amino acids did not inhibit production of glycosylated 3H-peptide, even at the highest concentration tested (Fig. 5 ) . Therefore, the asparagine replacements are at best very poor acceptors for or very poor inhibitors of oligosaccharyltransferase.
Studies to Determine the Acceptor or Inhibitor Properties of Unlabeled Peptides-Experiments were performed to determine whether the peptide derivatives that inhibited the glycosylation of N"-[3H]Ac-Asn-Leu-Thr-NH2 are acceptor substrates for oligosaccharyltransferase or are true inhibitors of this enzyme. Acceptor activity was evaluated by measuring the extent of depletion of endogenous oligosaccharide-lipid that occurred upon preincubation of peptide derivatives with oviduct microsomes (Table IV). Preincubation of microsomes with the known acceptor N"-Ac-Am-Leu-Thr-NH2 depleted endogenous oligosaccharide-lipid and thereby prevented the subsequent glycosylation of 3H-peptide added after removal of the unlabeled peptide (Table IV, entry 1). Addition of fresh sugar nucleotide precursors for synthesis of oligosaccharidelipid resulted in 3H-glycopeptide production, thereby indicating that Ne-Ac-Asn-Leu-Thr-NH, does not act as an irreversible inhibitor of oligosaccharyltransferase. As expected, preincubation of microsomes in the absence of any peptide derivative did not lead to the depletion of endogenous oligosaccharide-lipid because fresh sugar nucleotides were not required for formation of 3H-labeled glycopeptide (Table IV, entry 2). Preincubation with the peptide containing an octanoyl group in place of an acetyl group, a compound that competed efficiently for glycosylation of "-peptide in the Ac-Asn-Leu-Thr-NHCH3 and the indicated amounts of unlabeled tripeptide derivatives were incubated for 5 min and the supernatants were analyzed for 3H-glycopeptide production by paper chromatography or binding to concanavalin Aagarose. Abbreviations for the peptide derivatives are listed in Table  111.

TABLE IV
Endogenous oligosaccharide-lipid depletion Thawed microsomes were preincubated with the indicated tripeptide derivatives (30 p~) for 15 min at 30 "C, washed twice with oviduct buffer to remove modified peptides, and then incubated with 3H-acceptor (30 p~) in the absence or presence of sugar nucleotides as described under "Experimental Procedures." 3H-Glycopeptide formation in the absence of added sugar nucleotides serves as a measure of endogenous oligosaccharide-lipid remaining in microsomes after preincubation with various tripeptides. 3H-Glycopeptide production in the presence of sugar nucleotide serves to indicate that the abolishment of glycopeptide formation is reversible by synthesis of new oligosaccharide-lipid from precursor substrates. Values shown are the average of duplicates.  (Table IV, entry 3). Similar results were obtained for all the NHz-terminal derivatives (data not shown). Based upon these results, we conclude that the NHz-terminal modified peptides are acceptors and not inhibitors of oligosaccharyltransferase. In contrast, preincubation with a peptide containing N-methylasparagine in place of asparagine did not deplete endogenous pools of microsomal oligosaccharide-lipid (Table IV, entry 4). Analogous results were found with the other asparagine derivatives (data not shown). Therefore, compounds containing asparagine replacements are neither acceptors nor inhibitors of N-glycosylation. To confirm the above conclusions, various peptide derivatives were incubated with microsomes in the presence of 14Csugar nucleotides and the formation of glycosylated products was directly established by gel filtration on a Bio-Gel P-4 column. Incubation with W-Ac-Asn-and N"-Boc-Asn-peptides led to the formation of 14C-labeled products with apparent molecular weights virtually identical with that of glycosylated N"-[3H]Ac-Asn-Leu-Thr-NHCH3. Treatment with Endo H caused the radioactivity to elute on Bio-Gel P-4 at the position where that oligosaccharide elutes. In contrast, incubation with Asn-Leu-Thr-NHz or any of the peptides containing asparagine replacements did not lead to formation of a high molecular weight, 14C-labeled product. These observations support the conclusion that the Ne-Ac-Asn-and W -Boc-Asn-peptides are acceptors for oligosaccharyltransferase, whereas asparagine derivatives are not.
Additional evidence supports the conclusion that the N"-Oc-Asn-and N"-Bz-Asn-derivatives are very efficient acceptors. First, it was found that competition for glycosylation could be overcome by addition of increasing amounts 3Hacceptor to a fixed amount of octanoyl peptide (data not shown). This finding rules out the possibility that some contamination in the unlabeled peptide preparation causes either nonspecific or irreversible inactivation of oligosaccharyltransferase. Second, the Ne-Oc-Asn-peptide depleted more endogenous oligosaccharide-lipid when microsomes were incubated with limiting quantities of peptide than did N"-Ac-Asn-Leu-Thr-NH2 as measured by an oligosaccharide-lipid depletion experiment (Table V). Thus, the apparent K , for this peptide must be lower than the apparent K, = 10 FM measured for Ne-[3H]Ac-Asn-Leu-Thr-NHCH3. Similar results were obtained with W-Bz-Asn-peptide. The results of all of these experiments conclusively establish that these peptides are very effective acceptors.
Topological Aspects of Peptide Glycosylation-To better understand the process resulting in glycosylation of these peptides, the topological orientation of the glycopeptide product and the oligosaccharyltransferase in microsomes was examined. After reaction with 3H-peptide, the microsomes were diluted with buffer and the soluble components were separated from membrane-associated or entrapped components by high speed centrifugation The percentage of the total unglycosylated substrate and the glycopeptide product associated with the membrane pellet was then quantitated by paper chromatography. The results, shown in Table VI, indicate that most of the glycopeptide product (61%) and very little of the N"-Oc-Asn-L.eu-Thr-NH2 and N"-Ac-Asn-Leu-Thr-NH2 The rate of peptide glycosylation under conditions of increasing concentrations of N"-Ac-Asn-or N"-Oc-Asn-peptides was compared by preincubation of microsomes with peptides for 5 min, followed by centrifugation to separate microsomes from unlabeled peptide substrate. Subsequent quantitation of the amount of endogenous oligosaccharide-lipid remaining was accomplished by measuring glycopeptide formation during a second incubation for 30 min with 3H-peptide.

3H-Glycopeptide formed after Preincubation Peptide concentration dur-with ing Preincubation
N"-Oc-Am-Leu-N"-Ac-Asn-Leu-Thr-NH, Thr-NH,  After incubation of 3H-acceptor peptide (lo6 cpm) with 6 mg of microsomal membranes in a total volume of 300 p1 for 15 min a t 30 "C, two 15-pl portions were removed and treated with 10% trichloroacetic acid, and the total amount of 3H-peptide and 3H-glycopeptide was quantitated by paper chromatography. Another two 100-pl portions of the assay were diluted into 5 ml of cold oviduct buffer and spun at 39,000 X g for 15 min. The pellet was then treated with 10% trichloroacetic acid, and the 3H-peptide and 3H-glycopeptide associated with the membranes were determined by paper chromatography as above.  unglycosylated substrate (1%) remained associated with membrane. These results suggest that glycosylated product is located on the luminal side of oviduct microsomes. Support for the theory that transferase activity is present at the inner face of the endoplasmic reticulum was derived from a competition experiment utilizing a large molecular weight acceptor, the tryptic peptide encompassing residues 17 through 58 of a-lactalbumin. As seen in Fig. 6, the large peptide acceptor competes very poorly for 3H-acceptor glycosylation unless detergent is added. In contrast, the competition by N"-Ac-Asn-Leu-Thr-NHz is virtually independent of detergent concentration. These results suggest that the membranes are freely permeable to these small peptides but not to larger acceptors such as the a-lactalbumin fragment. Only under conditions in which the microsomes are made permeable can the large peptide interact with oligosaccharyltransferase and therefore act effectively as a competitor. The simplest explanation for these findings is that oligosaccharyltransferase is oriented at the luminal face of the endoplasmic reticulum, as previously proposed (7).

DISCUSSION
In earlier studies, it was established that not only unfolded proteins (8) but also peptide fragments (21,23,24) containing the sequence -Am-X-Ser/Thr-could serve as substrates for glycosylation in vitro. Subsequently, this laboratory showed that simple tripeptides of the type -Asn-X-Ser/Thr-were active acceptors if the NH, and COOH termini were blocked (4). These observations have been confirmed and extended by others (13,15,24,25). The finding that simple tripeptides are substrates for oligosaccharyltransferase raises the possibility that analogs of the tripeptide can be prepared that will serve as reversible or irreversible inhibitors of this enzyme. Indeed, one such inhibitor that acts irreversibly has been reported recently (26). Such inhibitors may be of great utility in studying the regulation of the dolichol-linked pathway and the role of glycosylation in general, and would not be expected to concomitantly inhibit protein synthesis.
As a first step in the development of inhibitors of oligosaccharyltransferase, we have synthesized several tripeptide derivatives of Asn-Leu-Thr-NHz containing different derivatives at the NH2 terminus or asparagine substitutions or modifications, and have measured their ability to act as substrates for or inhibitors of oligosaccharyltransferase. At the outset, we developed a simple and rapid assay to test peptides as possible alternative substrates or inhibitors. N"-[3H]Ac-Asn-Leu-Thr-NHCH3 was synthesized and conditions were optimized for its glycosylation by the oligosaccharyltransferase in hen oviduct microsomes. The initial screen for evaluating whether various labeled compounds were or were not acceptors or inhibitors involved a direct competition experiment with N"-[3H]Ac-Asn-Leu-Thr-NHCH3. The experiment was designed so that co-incubation of the 3H-acceptor peptide with tripeptide derivatives that were either acceptors or inhibitors would block formation of glycopeptide either by competition for endogenous oligosaccharide-lipid or by actual inhibition of oligosaccharyltransferase. Tripeptide derivatives which were not substrates or inhibitors would have no effect upon 'H-glycopeptide formation.
All the NHz-terminal derivatives of Asn-Leu-Thr-NH, that were tested inhibited 3H-glycopeptide production in a dosedependent manner. They were subsequently shown to be acceptors for oligosaccharyltransferase because they either served as acceptors of radioactive oligosaccharide and/or they depleted pools of endogenous oligosaccharide from oviduct microsomes. None of the peptides were irreversible inhibitors as they were readily washed from microsomes and did not inhibit subsequent glycosylation of 'H-acceptor. As expected, the NO-Ac-Asn-compound inhibited glycosylation by 50% at a concentration equivalent to that of the 'H-peptide, whereas the nonacylated peptide had no inhibitory activity. TWO of the hydrophobic NH,-terminal derivatives tested, W-Oc-Asn-Leu-Thr-NHn and P-Bz-Asn-Leu-Thr-NH2, inhibited glycosylation of the "-peptide by approximately 50% a t a concentration 10-fold below that of the "-substrate. The P -Boc-Asn derivative was slightly poorer as a substrate than the 'H-peptide, as measured by the competition assay. The decreased acceptor activity of this compound may be due to some hydrolysis of the NH2-terminal protecting group by enzymes in oviduct microsomes. Alternatively, it could be because the urethane linkage at the NHz terminus is sterically and electronically different from the simple amide bond in the other acceptors. In any case, the fact that the N"-Boc-Asn derivative is an acceptor implies that oligosaccharyltransferase does not require an amide bond at the NH2-terminal position and that other derivatives that neutralize the positive charge can be used. A similar observation has been reported using dinitrophenylated and dansylated tetrapeptide substrates which contain carbon-nitrogen linkages rather than the typical amide bond (26).
In contrast, a methylamide derivative of asparagine, which contains a single methyl substitution for hydrogen in the asparagine side chain, was neither an acceptor substrate nor an inhibitor of oligosaccharyltransferase. It did not inhibit in the competition assay, nor did it deplete endogenous oligosaccharide-lipid or accept radioactive oligosaccharide. Furthermore, when the carboxyamide group of the side chain was replaced by a H or a CH, group, the resulting peptide was not an inhibitor. These results suggest that recognition by oligosaccharyltransferase is extremely dependent upon the precise structure of the asparagine side chain and that modifications in this side chain abolish binding to the active site. Based upon previous work demonstrating that tripeptides alone contain all the necessary information for glycosylation, it has been suggested that accessibility of this sequence to oligosaccharyltransferase is the major factor controlling glycosylation of -Am-X-Thr/Ser-sites i n vivo (4). Several other results point also to this conclusion. First, the oligosaccharide chains of N-linked glycoproteins generally are found a t pturns (27). Second, the presence of proline in the X amino acid position of peptide substrates results in a loss in acceptor activity (9, 10). Third, only denatured, and therefore unstructured, proteins are glycosylatable in vitro (8). Fourth, disulfide bonds limit glycosylation of intermediate size peptides i n vivo (11). Finally, dimethyl sulfoxide and phospholipid alter the circular dichroism profiles of certain peptide acceptors as well as increase their activity as substrates (12,13).
The increased acceptor activity observed with Ne-Oc-Asnand N"-Bz-Asn-peptides, which were insoluble in water in the absence of dimethyl sulfoxide, suggests that peptide hydrophobicity increases acceptor activity. A role for hydrophobicity is consistent with the biology of the process of glycosylation. I n vivo, the nascent chains attached to membranebound polysomes (1, 2) are glycosylated by the integral membrane protein, oligosaccharyltransferase. Glycosylation is believed to occur at or near the luminal face of the endoplasmic reticulum where the oligosaccharide-lipid is synthesized ( 5 , 6), while the protein is passing through the membrane on its way to the lumen. The findings of the current study indicate that glycosylation of small peptide acceptors in oviduct microsomes occurs by a similar intraluminal mechanism. The majority of the 3H-glycopeptide produced remains associated with the microsomes, whereas virtually all of the unglycosylated peptide substrate is found in the supernatant after the microsomes are washed. Furthermore, the presence of the nonionic detergent NP740 has no stimulatory effect on the acceptor activity of the %-peptide. In contrast, a large peptide acceptor peptide containing 41 amino acid residues is a very poor acceptor unless NP-40 is added. These results indicate that the small peptides can freely enter and exit from the microsomes and that their glycosylation, like the glycosylation of nascent chains, occurs at or near the luminal face.
The finding that a variety of substitutions are permissible at the NH2-terminal end of the peptide and that hydrophobic substitutions increase the efficiency of the peptide as a substrate for oligosaccharyltransferase indicates that this would be a suitable site for introduction of chemically reactive or photoreactive groups designed to generate an irreversible inhibitor of oligosaccharyltransferase. Furthermore, the hydrophobic nature of such compounds raises the possibility that they could be delivered to cells either directly or indirectly via liposomes. If this is feasible, such peptides could be valuable tools to study the consequences of i n vivo inhibition of oligosaccharide transfer to protein as well as the mechanism of N-glycosylation. In fact, we have recently found that the tripeptide substrates described in this report inhibit the cotranslational glycosylation of ovalbumin and vesicular stomatitis virus G protein i n vitro (28).