Biosynthesis of a Variant Surface Glycoprotein of Trypanosoma brucei

Vol. 263,No. 33, Issue of November 25, pp. 17697-17705,1988 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Biosynthesis of a VariantSurface Glycoprotein of Trypanosoma brucei PROCESSING OF THE GLYCOLIPID MEMBRANE ANCHOR AND N-LINKED OLIGOSACCHARIDES* (Received for publication, June 6, 1988)

James D. Bangs$, Tamara L. Doerings, Paul T. Englund, and GeraldW. Hartll From the Departmentof Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland21205

In recent years a novel class of eukaryotic membrane proteins, characterized by covalently attached phosphatidylinositol glycan anchors, has been identified (1, 2). While the number of proteinsinthis class is growing, and much is known about the structure of these anchors, little is known about the biosynthesis of these important glycolipids.

The variant surface glycoprotein (VSG)’ of Trypanosoma brucei is the best characterized example of this group of membrane proteins. The bloodstream form of this parasite is covered by a surface coat composed of lo7 molecules of VSG (3, 4). The phosphatidylinositol glycan of VSG has a linear tetrasaccharide core composed of 3 mannosyl residues and a non-acetylated glucosamine (5-7). Dimyristoyl phosphatidylinositol is glycosidically linked to glucosamine at the reducing is linked by a end of the core ( 5 , 6),andethanolamine phosphodiester to the other end (7). This structure, which also contains side chain galactosyl residues (7,8), is linked to the protein by an amide bond between ethanolamine and the a-carboxyl group of the carboxyl-terminal amino acid (9). The glycolipid moiety is susceptible toan endogenous, membrane-bound phospholipase C (VSG lipase; Refs. 10-14) that is activated when cells are disrupted under nondenaturing conditions. The activated lipase removes dimyristoylglycero1 from the glycolipid of membrane form VSG releasing soluble VSG from trypanosoma1 membranes. In addition to theglycolipid, all VSGs contain at least one N-linked oligosaccharide attached at Asn-X-Ser/Thr sites(8, 15).Compositional studies of isolated VSG glycopeptides (15) and lectin-binding studies with intact VSG (16) indicate that these oligosaccharides can be of the high mannose or complex type. The presence of complex-type oligosaccharides is consistent with suggestions that oligosaccharide processing occurs during VSG biosynthesis (17-19). Previously, using the T. brucei variant ILTat 1.3, we have studied the biosynthesis and post-translational processing of VSG (18, 19). ILTat 1.3VSG has an intracellular 58-kDa biosynthetic intermediate that contains the glycolipid as well as N-linked oligosaccharides at each of two potential glycosylation sites (Fig. l).Modification of the N-linked oligosaccharide(s), and possibly the glycolipid, results in the conversion of immature 58-kDa VSG to a mature 59-kDa form as VSG is transported (tlIz= 15 min) to the cell surface. We report here on the analysis of the three individual glycosylation sites of both the 58- and the 59-kDa forms of ILTat 1.3 VSG. Our studies indicate that each of the two N linked glycosylation sites of mature VSG contain markedly different types of oligosaccharides. Those at Asn-419 appear

* This work was supported by National Institutes of Health Grant A121334 and a grant from the MacArthur Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact. $. Current address: Dept. of Microbiology and Immunology, Stanford University School of Medicine, Fairchild Bldg., Stanford, CA 94305. I Supported by Medical Scientist Training Grant5T32GM07309. 7 Established Investigator of the American Heart Association.

The abbreviations used are: VSG, variant surface glycoprotein; sVSG, soluble form of VSG; mfVSG,membrane form of VSG; Hepes, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonicacid TLCK, N-ap-tosyllysine chloromethyl ketone; Bicine, N,N-bis(2-hydroxyethy1)glycine; BBS, Bicine-buffered saline; CRD, cross-reacting immunological determinant; HPLC, high performance liquid chromatography; Endo H, endo-@-N-acetylglucosaminadaseH; PNGase F, peptide-N-glycanase F; SDS, sodium dodecyl sulfate; TPCK, L-1tosylamido-2-phenylethylchloromethyl ketone; ConA, concanavalin A gp, glycoprotein; NP-40, Nonidet P-40; PAGE, polyacrylamide gel electrophoresis; GC-MS, gas chromatography-mass spectrometry.

The variant surfaceglycoprotein (VSG) of the ILTat 1.3 variant of Trypanosoma brucei has two asparagine-linked glycan moieties, as well as a phosphatidylinositol glycan membrane anchor. We have investigated the structure and processing of each of these oligosaccharides through analysisof the intact protein and of glycopeptides. Processing hasbeen examined by comparing glycan structures purified from an immature intracellularform (58 kDa) of VSG with those of the mature form (59 kDa) found on the parasite surface. We find exclusively high mannose oligosaccharides (Man4-7-GlcNAcz) at Asn-432 in both the immature58kDa and mature 59-kDaforms. In contrast, the“core” oligosaccharide of Asn-419 (Mans-GlcNAcz)appears to be nearly quantitatively processed to a complex biantennary structure ((Gal-GlcNAc-Man)2-ManGlcNAc2) during VSG maturation.Theasparaginelinked structures at Asn-419, but not those at Asn-432, are resistantto endo-8-N-acetylglucosaminidaseH within 30 s of biosynthesis. This suggests possible novel and selective mechanisms for glycosylation in African trypanosomes. Finally, we show that the carboxyl-terminal glycolipid is galactosylated (3-4 residues) relatively late in VSG biosynthesis. Phosphatidylinositol glycans have been identified on a growing number of eukaryotic membrane proteins. This reportprovides a directdemonstration of the processing of such a glycolipid anchor following its attachment to protein.

17697

17698

Processing of VSG Glycans

PEAK II

PEAK 111

PEAK I

FIG. 1. Diagram of ILTat 1.3 VSG glycosylation sites. The sequence a t each site is derived from the ILTat 1.3 cDNA sequence (20);residues are numbered from the first in-frame methionine codon. Positions of each site within the primary sequence are not to scale. Arrowheads denote the tryptic (upward) and chymotryptic (downward) cleavages that generate the Peak I, 11, and 111 glycopeptides (gpCT, -419, and gp432, respectively; see text).

1

2

.

-

3

4

._

5

.

6

6659\ 5a

I

45FIG.2. Purification of 68- and 59-kDa VSG. [3H]Mannoselabeled (60 min) trypanosomes were lysed and fractionated as described under "Materials and Methods." A fluorograph of an SDSgel is shown. Marker lanes 1 and 6 (1.5 X lo6 cell equivalents) contain immunoprecipitated [3H]mannose-labeledsoluble VSG polypeptides. Cell fractions (10' cell equivalents), as defined under "Materials and Methods," are: lane 2, whole cell lysate; lane 3, lysis supernatant; lane 4, release supernatant; lane 5, crude membrane extract. Scale refers to apparent molecular mass (kilodaltons).

to be almost exclusively complexbiantennary, whereas those at Asn-432 are all high mannose species. Surprisingly, the oligosaccharides at Asn-419 (but not Asn-432) are resistant to Endo H within 30 s ofVSG synthesis. These data imply the operation of novelmechanismsfor the synthesis and attachment of N-linked oligosaccharides in trypanosomes. Furthermore, we find that thecarboxyl-terminal glycolipid is galactosylated late in VSG synthesis. This observation is a direct demonstration of the post-translational processing of a phosphatidylinositol glycan anchor. MATERIALS AND METHODS~

Trypanosomes, Antibodies,and Immunoprecipitation-The source of the cloned ILTat 1.3 variant of T. brucei, the growth and isolation of bloodstream trypanosomes, and theproduction of affinity-purified rabbit anti-VSG and anti-CRD antibodies have been described (19). Mouse monoclonal anti-ILTat 1.3VSG antibody (mAb68.63.1, a generous gift of Dr. Klaus Esser, SmithKline & French Laboratories) was purified from ascites fluid using DEAE-Affi-Gel Blue (Bio-Rad) (21) and coupled to Sepharose 4B with cyanogen bromide (22). Radiolabeled soluble VSG polypeptides were immunoprecipitated from trypanosome extracts, as described previously (19). Purification of58- and 59-kDa VSG-In this procedure, the scheme of Cross (23) was modified to exploit the differential susceptibility of 59- and 58-kDa VSG to VSG-lipase when trypanosomes are lysed hypotonically (see below and Ref. 19). All operations were a t 0 "C unless stated otherwise and all buffers except BBS contained 0.1 mM N-a-p-tosyl-L-lysine chloromethyl ketone (TLCK) and 1 pg/ml leupeptin to minimize proteolysis. Samples of fractions were retained for analysis by SDS-polyacrylamide gel electrophoresis (Fig. 2).

* Portions of this paper (including part of "Materials and Methods" and Tables I and 11) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal thatis available from Waverly Press.

[3H]Mannose-labeledtrypanosome cultures (100 ml) were diluted with 100 ml of BBS (50 mM Bicine sodium salt, 70 mM glucose, 50 mM NaCI, 5 mM KCl, pH 8.0) and centrifuged (2500 rpm, 10 min, 4 "C, Sorvall HS-4 rotor). The cells were washed twice in BBS and lysed in 0.5mlof H20 for 5 min a t 0 'C. This whole cell lysate, containing VSG polypeptides (Fig. 2, lane 2) with the accelerated electrophoretic mobility characteristic of membrane form VSG, was centrifuged (6000 rpm, 5 min, 4 "C, Sorvall HB-4 rotor). Few VSG molecules were detected in the resultant lysis supernatant (Fig. 2, lane 3 ) .The pellet was resuspended in 10 mM sodium phosphate, pH 8.0(0.5 ml), and incubated 5 min a t 37 "C to allow release of the mature 59-kDa VSG from the plasma membrane byVSG lipase. Intracellular 58-kDa VSG is unaffected by this treatment (19). The suspension was diluted with 1.5 ml of 10 mM sodium phosphate, pH 8.0, layered on a 0.2-ml cushion of 60% sucrose in TEN buffer (50 mM Tris-HCl,150 mM NaCl, 5 mM EDTA, pH 7.5), and centrifuged (100,000 X g, 30 min, 4 "C,Beckman SW Ti-60 rotor). The clarified release supernatant, containing soluble mature 59-kDa VSG (Fig. 2, lane 4 ) , was removed and lyophilized. Immature 58-kDa VSG remained in the membrane fraction. The cushion and interface, containing the crude trypanosoma1 membranes, were resuspended in 5.0 ml of 10 mM sodium phosphate, pH 8.0, and centrifuged (lO0,OOO X g, 30 min, 4 'C, Beckman SW 50.1 rotor). The washed membrane pellet was solubilized in TEN containing 1%Nonidet P-40 (0.5 ml) and incubated at 37 "C for 5 min to allow cleavage of the glycolipid on the 58-kDa VSG. Insoluble debris were removedby centrifugation (2 min, Microfuge B, Beckman) to yield a crude membrane extract, containing soluble 58-kDa VSG (Fig. 2, lane 5 ) . Although free of 59-kDa VSG, the crude membrane extract contained other radioactive contaminants that were evident in longer exposures (not shown). Therefore, 58-kDa VSG was further purified by immunoaffinity chromatography. A 33% slurry of anti-VSG-Sepharose (1.5 ml in TEN containing 1% Nonidet P-40, 0.1 mM TLCK, and 1 pg/ml leupeptin) was mixed with 5 X lo8 cell equivalents of crude membrane lysate (0.5 ml) and gently agitated a t 4 "C for 4 h. The slurry was loaded in a minicolumn and packed with 2.0 ml of wash buffer (TEN, 0.5% Nonidet P-40). The pooled flow-through was passed over the column 5 times, and thecolumn was washed with 32 ml of wash buffer followed by 8.0 ml of TEN buffer. Bound 58-kDa VSG was eluted with boiling 1%SDS, and fractions (2.0 ml) containing radioactivity were pooled and lyophilized. The sample, in 0.3 ml of H20, was precipitated with acetone using 20 pg of cytochrome c as carrier. In a typical preparation, 7.4 X 10' cpm of 59-kDa and 4.3 X 10' cpm of 58-kDa soluble VSG were recovered from 5 X 10' trypanosomes. RESULTS

Endo H Treatment of Mature VSG-To evaluate whether the two N-linked oligosaccharides on mature ILTat 1.3 VSG (19) are of the high mannose or complex type, [35S]methionine-labeled 59-kDa solubleVSG was denatured and treated with either PNGase F, an enzyme with broad specificity for N-linked oligosaccharides (24), or Endo H, an enzyme with specificity for high mannose-type oligosaccharides (25). The reaction products were analyzed by SDS-polyacrylamide gel electrophoresisand fluorography (Fig.3). Consistent with previousresults (19), PNGase F treatment of mature 59-kDa VSG (lanes 1 and 6 ) generates a single polypeptide (lane 4 ) that comigrates with radiolabeled VSG polypeptide from trypanosomes that were treated with tunicamycin (lanes 2 and 5 ) , an inhibitor of N-linked glycosylation (26). In contrast, Endo H treatment generates a doublet of lower relative molecular mass polypeptides (lane 3). The major species is of an intermediate relative molecular mass, suggestingthat most mature VSG polypeptides haveone Endo H-resistant and one Endo H-sensitive oligosaccharide. However, the presence of a less abundant polypeptide that comigrates with the tunicamycin control (lanes 2 and 5 ) suggests that a small fraction of mature VSG polypeptides has two Endo H-sensitive oligosaccharides. These data are consistent with the presence of a single high mannose and asingle complex oligosaccharide on most mature

Processing of VSG Glycans 1

2

3

4

5

6

A

17699

TOTAL

B 1

gpCT

45

66-

59-

4

I

45-

FIG.3. Deglycosylation of mature VSG. Anti-VSG antibody was used to immunoprecipitate '%labeled VSG polypeptides from lysates of cells that were pulse-labeled for 2 min and chased for 58 min in the presence (lanes 2 and 5 ) or absence (lanes 1 , 3 , 4 , and 6 ) of tunicamycin. Labeled VSG polypeptides were eluted from protein A beads, treated with either Endo H (lane 3 ) or PNGase F (lane 41, and then fractionated by SDS-polyacrylamide gel electrophoresis as described in the Miniprint. All lanes contain lo6 cell equivalents. A fluorograph of an SDS gel is shown. Scale refers to apparent molecular mass (kilodaltons). ILTat 1.3VSG molecules. However, these resultsdo not indicate whetherattachment of the two forms of oligosaccharides is site-specific or random between the two available N glycosylation sites. Identification of VSG Glycopeptides-We wished to analyze the glycoconjugatesof ILTat 1.3 VSG individually in order to answer the following questions: 1) is the Endo H-resistant oligosaccharide localized at a specific asparagine residue, and 2) are the two N-linked moieties and the carboxyl-terminal glycolipid modified during maturation of 58-kDa VSG to 59kDa VSG? Therefore, VSG tryptic/chymotryptic glycopeptides were purified at both the radiochemical and preparative levels. [3H]Mannose glycopeptides from radiochemically pure 58and 59-kDa VSG (see Fig. 2) were fractionated by C18reversephase HPLC. Three labeled glycopeptides, Peaks I, 11, and 111, in order of increasing retention time, were resolved (Fig. 4A). An identical pattern of glycopeptides was obtained for 58-kDa VSG (data not shown). Peak I1 is actually a double peak that is not fully resolved by the analytical elution program. However, preparative reverse-phase HPLC of a large scale digest of 59-kDa VSG resolved Peak I1 into two discrete peaks (IIa andI b , data not shown). The glycopeptides from preparative fractionationof 59-kDa VSG were subjected to amino acid analysis (Table I, Miniprint) allowing alignment of these glycopeptides with the ILTat 1.3 VSGsequence (Fig. 1). PeakI1 and I11 glycopeptides derive from the N-linked glycosylation sites a t Asn-419 and Asn-432, respectively. The heterogeneity in Peak I1 (IIa and IIb differ by a single lysine) results from the presence of consecutive lysine residues allowing alternative tryptic cleavage at thecarboxyl end of this peptide. The order of retention of Peaks IIa and IIb is consistent with the predicted relative retention times of peptides that differ by a single lysine (27). Peak I1 is designated glycopeptide 419 (gp419) and Peak I11 is designated glycopeptide 432 (gp432). Peak I contains a single Asx residue indicating that this is the carboxyl-terminal glycopeptide ( ~ P C T derived )~ from a tryptic cleavage adjacent to the carboxyl-terminal aspartate residue. During VSG isolation, VSG lipase cleaves dimyristoylglycerol from the carboxyl-terminal glycolipid ( l l ) , exposing an immunologically cross-reacting determinant (CRD, Ref. 28). Glycopeptides from 59-kDa [3H]mannose-VSGwere tested in a fluid-phase immunoassay for reactivity with antiCRD antibody (29). Only gpCT was capable of binding to This structure is analagous to thestructure designated sCT-gp in Ref. I .

/

F gpd32 EndoH

0 0

15

30 45 (MIN)

TIME

0

11

, , '

15

_-

30

TIME

.45

60

(MIN)

FIG.4. Endo H treatment of 59-kDa [SH]mannose-labeled VSG tryptic/chymotryptic glycopeptides. Radiolabeled tryptic/ chymotryptic glycopeptides of mature VSG from 60-min labeled trypanosomes were purified, treated with Endo H, and analyzed by Cla reverse-phase HPLC. Panel A, total digest; Panel B, gpCT; Panel C, gp419; Panel D,gp432; Panel E, Endo H-treated gp419; Panel F, Endo H-treated gp432. Samples contained 25,000 cpm (Panel A ) or 10,000 cpm (Panels B-F). Closed circles represent radioactivity; the solid line represents percent acetonitrile. anti-CRD antibodies (data not shown), verifying that this glycopeptide is thecarboxyl-terminal glycopeptide. The carbohydrate composition of VSG glycopeptides was also determined (Tables I and 11, Miniprint). The sugars in gp419 (GlcN, Man, Gal)are detected in ratios consistent with the presence of a complex (Endo H-resistant)oligosaccharide at this site. The sugar content ofgp432 (GlcN, Man) is consistent with the presence of high mannose (Endo Hsensitive) oligosaccharides at this site. The composition of gpCT (ethanolamine,inositol, Man, GlcN, Gal) confirmsthat this glycan is derived from the carboxyl-terminal glycolipid. The mannose composition of gpCT is in agreementwith published compositions from many variants (2 residues, Ref. 8), but is lower than expected from the published structure of this moiety (3 residues, Ref. 7). This discrepancy is probably due to themannose at thenonreducing end of the glycan core, which is ina phosphodiesterlinkage with ethanolamine. Acid hydrolysis of this linkage is known to generate mannose 6phosphate (7), not detected by the analytical protocol used here. Endo H Treatment of VSG Glycopeptides-To confirm that the high mannose and complex oligosaccharideswere attached at specific sites, the EndoHsensitivity of isolated [3H] mannose-labeled glycopeptides was assessed. Aliquots of gp419 and gp432 from 59-kDa VSG were treated with Endo H and reanalyzed by C18 HPLC to measure release of oligosaccharides, as indicated by appearance of radioactivity in the column void volume (30). Endo H released all of the radioactivity from gp432 (Fig. 4, compare Panels D and F ) but had little effect on gp419 (Fig. 4, compare Panels C and E ) . These data indicate that the complex and high mannose oligosaccharides are attachedspecifically to gp419 and gp432, respec-


17700

tively. The small amount of radioactivity released from gp419 (Panel E ) is consistent with the detection of a small amount of completely deglycosylated VSG following Endo H treatment of intact 59-kDa VSG (Fig. 3); these data suggest that a minor population of high mannose oligosaccharides are also attached at this site. Identical results were obtained with gp419 and gp432 derived from immature 58-kDa VSG (data not shown). Preparation of Free N-Linked Oligosaccharides-Endo H was used to prepare free high mannose oligosaccharides from gp432 for further biochemical characterization. Free oligosaccharides from gp419 were generated with PNGase F.However, this enzyme requires a blocking moiety on the a-aminogroup of the glycosylated asparagine residue (31). Thus, PNGase F, which can deglycosylate intact VSG (Fig. 2), is unable to release oligosaccharides from purified gp419 (data notshown) because the glycosylated asparagine is at theamino terminus; the glycopeptide must be modified. gp419from both 58- and 59-kDa VSG was dansylated, treated with PNGase F, and analyzed for release of incorporated radioactivity (Fig. 5). Dansylation resulted in a marked increase in retention time of gp419 due to the incorporation of hydrophobic dansyl moieties (data not shown). PNGase F treatment of dansyl-gp419 from 58- and 59-kDa VSG completely released the incorporated radioactivity (Fig. 5, Panels A and B ) . A portion of released oligosaccharide, 16%of gp419 from 58-kDa VSG and 64% of gp419 from 59-kDa VSG, eluted after the void volume (5 min) with a retention time of 9.5 min. Retention of free complex-type oligosaccharides on Cle resin has been observed in other systems (30) and probably results from an increased terminal N-acetylglucosamine content in these oligosaccharides. The increased fraction of retained oligosaccharide in gp419 from mature VSG suggests that theoligosaccharides at this site aremodified, possiblyby the incorporation of additional N-acetylglucosamine, as 58kDa VSG is processed to 59-kDa VSG. Size Analysis of N-Linked Oligosaccharides-Free radiolabeled N-linked oligosaccharides from 58- and 59-kDa VSG were analyzed by gelfiltration chromatography (Fig. 6). gp419

0 (MATURE)

45

z

300

U

m

30

2

""

+I

a

f 150 u

15

a 0

0

0

15

30 60 45 TIME WIN)

FIG. 5. Release of oligosaccharides from dansyl-gp419. gp419 glycopeptides from [3H]mannose-labeled 58-kDa (Panel A ) and 59-kDa (Panel B ) VSG weredansylated and treated with PNGase F. Samples (10,000 cpm) were analyzed by ClS reverse-phase HPLC. Closed circles represent radioactivity; the dashed line represents percent acetonitrile.

800 600

400 200 E a

U

0 800

O

.

C

.

0

600

400 200 "

0.0

0.2

0.4

0.6

0.8

1.0

K av FIG. 6. Size fractionation of N-linked oligosaccharides from [3H]mannose-labeledVSG glycopeptides. Free oligosaccharides from PNGase F-treated dansyl-gp419 (Panel A ) and Endo H-treated gp432 (Panel B ) glycopeptides were fractionated on a Bio-Gel P-4 (-400 mesh) column. Oligosaccharides were from 59-kDa VSG (open circles) and 58-kDa VSG(closed circles). The elution positions of GlcNAcl-6standards are indicated by diamonds.

oligosaccharides from 58- and 59-kDa VSG were detected as major peaks of K., 0.46 and 0.28, respectively (Fig. 6, Panel A). These data indicate that gp419 oligosaccharides are modified during the processing of 58-kDa VSG to 59-kDa VSG. The presence of overlapping leading and lagging shoulders on the column profiles of the immature and mature forms is consistent with a precursor/product relationship. gp432 oligosaccharides from 58- and 59-kDa VSG were resolved as peaks with Kayvalues of0.46 and 0.48, respectively (Fig. 6, Panel B ) , indicating that no major size modifications of the high mannose oligosaccharides on gp432 are apparent during VSG processing. The structures of the N-linked oligosaccharideswere investigated by exoglycosidase digestion and gel filtration chromatography. Following a-mannosidase treatment, all the radioactivity from both forms ofgp432 oligosaccharides, and from immature gp419, eluted from a P-4 column as free mannose (data not shown). Digestion of mature gp419 oligosaccharide (Fig. 7, Panel A) with @-galactosidasegenerated a smaller molecule (Fig. 7, Panel B, K., 0.33) consistent with the removal of 2 hexoses (46). A shoulder and small peak (Kav 0.31) probably result from incomplete digestion. Subsequent treatment with @-hexosaminidase(Fig. 7, Panel C ) converted a major fraction of the degalactosylated oligosaccharide to a size (Kav 0.55) consistent with the removal of 2 GlcNAc residues. Further treatment of this product with a-mannosidase resulted in release of all the radiolabel as free hexose (data not shown). The other major peak (Kav0.38) in the @hexosaminidase-treated sample may arise from digestion of the partial products of the @-galactosidasetreatment. These data suggest that mature gp419 oligosaccharides probably have 2 terminal galactose residues in @-linkageto internal GlcNac residues. Both forms of gp432, and theimmature form of gp419, have mannose as terminal sugars. Acquisition of Endo H Resistance in gp419"In higher eukaryotic cells N-linked oligosaccharides are generally attached to protein in the endoplasmic reticulum as Endo Hsensitive high mannose forms (32). Subsequently, these oligosaccharides may be processed to Endo H-resistant forms, by the removal of terminal sugar residues, as glycoproteins are transported to the Golgi apparatus (32). We investigated whether trypanosomes can process N-linked oligosaccharides


300

A

17701

66'

1

581 56""

54/

45-

U

B

1

2

3

4

5

6

45-

K av FIG. 7. Sequential exoglycosidasedigestion of mature gp419 oligosaccharides. [3H]Mannose-labeled oligosaccharides from mature gp419 were fractionated as in Fig. 6;the main radioactive peak was pooled and lyophilized for these experiments. All samples were analyzed on a Bio-Gel P-4 column as in Fig. 6; radioactive fractions were pooled and lyophilized for subsequent overnight treatments (see the Miniprint). Panel A, untreated oligosaccharides.Panel B, oligosaccharides from Panel A treated with @-galactosidase.Panel C, oligosaccharides from Panel B treatedwith @-hexosaminidase.

in a similar manner by assaying the appearance of Endo Hresistant oligosaccharides on Asn-419. ["SIMethionine-soluble VSG from pulse-labeled cultures was treated with PNGase F or Endo H. As previously demonstrated (18,19), a doublet of newly synthesized VSG polypeptides, 56 and 58 kDa, is detected immediately following VSG synthesis (Fig. 8, Panels A and B, lanes 1 and 6).The 56-kDa polypeptide,containing a single N-linked oligosaccharide, is a precursor of the 58-kDa polypeptide; both forms have the glycolipid (18,19). The higher ratio of 56- to 58-kDa polypeptide at 30 s, relative to 2 min (compare Panels A and B, lane 1 or 6 ) , is consistent with a precursor/product relationship. PNGase F treatment of newly synthesized VSG generates a single 54-kDa VSG polypeptide that comigrates with newly synthesized VSG from tunicamycin-treated cells (Panels A and B, compare lanes 4 and 5). In contrast, EndoH converts the 56/58-kDa doublet to a 54/56-kDa doublet (PanelsA and R, lane 3). Endo H remained active throughout the reaction period and was capable of completely deglycosylating RNase B that was added during the final4 h of incubation (data not shown). In addition,a 10-fold excess of Endo H (660 milliunits/ml) was alsoincapable of removing the remaining Nlinked oligosaccharides onthese polypeptides (datanot shown). These data strongly indicate the presence of Endo H-resistant oligosaccharides on Asn-419 within 30 s of synthesis, suggesting that novel site-selective mechanism(s) of N-linked oligosaccharide addition or processing may be operative in trypanosomes. Size Analysis of gpCT-All the componentsof the glycolipid anchor of VSG are present infixed molar ratios except galactose, which can vary both between different VSGs (0-8 residues) and within a single variant (7,8). Galactose is attached

FIG. 8. Deglycosylation of newly synthesized VSG. AntiVSG antibody was used to immunoprecipitate "S-labeled VSG polypeptides from lysates of cells that were pulse-labeled for 30 s (Panel A ) or 2 min (Panel B ) in the presence (lanes 2 and 5 ) or absence (lanes I , 3 , 4 , and 6)of tunicamycin. Labeled VSG polypeptides were eluted from protein A-beads, treated with either Endo H (lane 3) or PNGase F (lane 4 ) and analyzed as in Fig. 3. All lanes contain lo6 cell equivalents except lanes 3 and 4 in Panel A which contain 2 X IO6 cell equivalents. A fluorograph of an SDS gel is shown. Scale refers to apparent molecular mass (kilodaltons).

"

0.2

0.4

0.6

Kav

0.8

FIG.9. Exoglycosidase analysis of gpCT. Panel A, [3H]mannose-labeled gpCT from mature (closed circles) and immature (open circles) VSG were fractionated by Bio-Gel P-6 gel filtration chromatography. A high salt buffer was used to minimize potential charge interactions of gpCT with the resin. Panel B, mature gpCT was treated with a-galactosidase as described in the Miniprint. An aliquot of treated gpCT was desalted, lyophilized, and retreated with agalactosidase. Once treated (closed circles) and twice treated (open circles) gpCT was fractionated as in Panel A. The data arepresented as radioactivity versus Kav.The elution positions of internal ["C]GalGICNAC*-~, ['4C]Gal-GlcNAc,, and ["C]Gal-GlcNAcs standards are denoted by diamonds.

as an a-linked side chain (7), and it has been suggested that these residues may be incorporated following the attachment of the glycolipid to VSG (8,19,33,34). To test this possibility, radiolabeled gpCT from 58- and 59-kDa VSG was analyzed by gel filtration chromatography, before and after a-galactosidase treatment (Fig. 9). The mature and immatureforms of gpCT were resolved as separate peaks (Panel A ) of K., 0.46 and 0.54, respectively. A

17702


single treatment with a-galactosidase converts mature gpCT This structure is supported by the fact that this oligosaccharide is a substrate for the rat liver a2-6Gal01-4GlcNAc siato a heterogeneous form that elutes between matureand apparent additionof Gal0l-4GlcNAc terimmature gpCT (Panel B ) . A second treatment generates a lyltran~ferase.~ The form that co-elutes with immature gpCT (Panel B ) . a-Galac- mini accounts for both the larger size of mature gp419 oligotosidase, 0-galactosidase or a-mannosidase had no effect on saccharide and its weak interaction with CISresin. The rapid development of EndoH resistance in gp419 the elution profile of gpCT from immature 58-kDa VSG (data not shown). These data demonstrate that the glycolipid is oligosaccharides is unprecedented. In mammalian cells, the indeed processed, probably by incorporation of 3-4 galactosyl final trimming stepsthat alter high mannose oligosaccharides to Endo H-resistant structures occur in the Golgi apparatus residues, during the conversion of 58-kDa to 59-kDa VSG. A minor peak (Kav 0.48) in the immature gpCT profile (32). However, resistance within 30 s of VSG synthesis sug(Panel A ) may represent an intermediate in the formation of gests thatthis oligosaccharide is already resistant in the mature gpCT. A similar peak in the degalactosylated mature endoplasmic reticulum, since transport of VSG to the site of gpCT profile (Panel B ) may represent incomplete digestion, conversion of 58- to 59-kDa VSG (presumably the Golgi) has although a-galactosidase remained greater than 90% active a tllz of 15 min (19). Any explanation of this phenomenon throughout the digestion period (standard assay, Ref. 35). must account for the fact that only one of two available N Alternatively, heterogeneity in the glycolipid core could ac- linked oligosaccharides in ILTatl.3VSG is Endo H-resistant. count for these minor species of both untreated immature and One possibility is that Endo H-sensitive oligosaccharides are transferred to both sites andthe oligosaccharides at Asna-galactosidase-treated maturegpCT. To minimize the possibility that contaminating activities 419, but not Asn-432, are then rapidly trimmed by endoin a-galactosidase were responsible for the decrease in the plasmic reticulum a-mannosidases such as those in higher eukaryotes (32). The time required for trimming could be apparent size of mature gpCT, the treated and untreated forms of mature and immaturegpCT were analyzed by anion reduced if an abbreviated high mannose structure were iniexchange chromatography. gpCT, generated by the combined tially transferred from dolichol pyrophosphate. Transfer of action ofVSG lipase and trypsin, has aspartate and phos- nonglucosylated structures assmall as MansGlcNAcnhas been phoinositol at opposite ends of the glycolipid core; any deg- reported in related trypanosomatid species (36-39) and the radation of the core would alter theanionic properties of this predominant dolichol-linked oligosaccharide detected in t3H] molecule. All forms of gpCT tested eluted from Mono Q resin mannose-labeled ILTat 1.3 trypanosomes is Man7GlcNA~~.~ (Pharmacia LKB Biotechnology Inc.) at the same salt con- In thisscenario, the site selectivity of Endo Hresistance could centration (data not shown) suggesting that the action of a- be explained by the inaccessibility of oligosaccharides on Asn432 to trimming enzymes due to protein folding (40,41) and/ galactosidase is peripheral to the glycolipid core. or VSG dimerization (42). Alternatively, trypanosomes may specifically transfer Endo DISCUSSION H-resistantand -sensitive oligosaccharides, from distinct Processing of N-Linked Oligosaccharides-Endo H treatlipid-linked pools, to Asn-419 and Asn-432, respectively. The ment of [35S]methionine-labeled mature 59-kDa VSG and oligosaccharidestransferred to Asn-419would beso truncated [3H]mannose-labeledglycopeptides derived from 59-kDa VSG in size that no trimming would be necessary to produce an indicates that most ILTat 1.3 VSG molecules contain one Endo H-resistant structure. That truncated Endo H-resistant high mannose and one complex oligosaccharide at different oligosaccharides can be transferred to protein has been demN-linked glycosylation sites. Amino acid compositional analyonstrated in mutant Chinese hamster ovary cells (43). The sis of purified VSG glycopeptides has allowed the assignment Endo H-resistantpool would have to be an intermediate, or a of the complex and high mannose Oligosaccharides to Asnside product, of the general biosynthetic pathway for N-linked 419 and Asn-432, respectively. The carbohydrate composition oligosaccharidessince tunicamycin, which blocks the first step of the oligosaccharides at each of these sitesis consistent with in the synthesis of dolichol-linked oligosaccharides (26), inthese designations. hibits glycosylation at both Asn-419 and Asn-432 (18, 19). A temporal order for N-glycosylation can be inferred. About Trypanosomes do contain a distinct population (15-20% of half of the VSG polypeptides in a 30-s pulse label have only the total) of dolichol-linked oligosaccharides that are Endo one N-linked oligosaccharide (Fig. 8A, lane 1 or 6) yet most H-resistant andwhose sizeis consistent with a Man4GlcNAcz of the polypeptides already have the Endo H-resistant oligo- structure! This scenario would implythe existence of multiple saccharide (Fig. 8A, lane 3 ) .Thus, attachmentof the resistant site- and saccharide-specific oligosaccharyltransferases. oligosaccharidegenerally precedes attachment of the sensitive Processing of the Glycolipid-Several lines of evidence conoligosaccharide. firm that gpCT is derived from the glycolipid anchor: it has From the size analyses, before andafter exoglycosidase the amino acid composition expected from a tryptic cleavage digestions, structures can be proposed that are compatible event at thecarboxyl terminus; it contains ethanolamine and with both the composition and theEndo Hsensitivity of these inositol; and it reacts specifically with antibodies directed at oligosaccharides. The oligosaccharides on Asn-432, from 58- the CRD. and 59-kDa VSG, are probably Man4-7GlcNAczmoieties, senThe apparent size difference between gpCT from mature sitive to both Endo H and a-mannosidase. Oligosaccharide and immature VSG indicates that the glycolipid is modified processing at this site plays no apparent role in VSG matu- during VSG maturation. Reduction of mature gpCT to the ration. size of immature gpCT by a-galactosidase is consistent with In contrast, processing of the oligosaccharides on Asn-419 both the published structure of the glycolipid (7) and the does contribute to thematuration of 58- to 59-kDa VSG. The sugar composition of mature ILTat 1.3 gpCT, suggesting that size of the immature oligosaccharide at Asn-419 is compatible processing of the glycolipid involves a-galactosylation (3-4 with a small (Mana-GlcNAcp-Asn)structure that is Endo H- residues) of a core glycolipid structure. This galactosylation resistant (25),yet sensitive to a-mannosidase. The pattern of probably accounts for a previously described ILTat 1.3 VSG exoglycosidase digestion and the size of the mature oligosaccharide are consistent with an Endo H-resistant asialobianK. Smith, S. Whiteheart, and J. Bangs, unpublished observations. T. Doering, unpublished observations. tennary ((Gal-GlcNAc-Man)2-Man-GlcNAc2-Asn) structure.

Processing of VSG Glycans processing event, detected as a small increasein VSG relative molecular mass during pulse-chase experiments in the presence of tunicamycin, that is unrelated to either N-linked oligosaccharide (19). The finding that glycolipid attachment immediately (