Differential fate of glycoproteins carrying a ... · proteins occurs at the level of the cis-Golgi network, also referred to as the intermediate compartment. Properly folded proteins

323Journal of Cell Science 110, 323-336 (1997)Printed in Great Britain © The Company of Biologists Limited 1997JCS9548

Differential fate of glycoproteins carrying a monoglucosylated form of

truncated N-glycan in a new CHO line, MadIA214, selected for a

thermosensitive secretory defect

Myriam Ermonval1,*, René Cacan2, Karin Gorgas3, Ingrid G. Haas4, André Verbert2 and Gérard Buttin1

1Unité de Génétique Somatique, URA CNRS 1960, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France 2Laboratoire de Chimie Biologique, UMR CNRS 111, Université des Sciences et Technologies de Lille, 59655 Villeneuve d’AscqCedex, France3Institut für Anatomie und Zellbiologie II der Universität Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany4Institut für Biochemie I der Universität Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany

*Author for correspondence (e-mail: [email protected])

A temperature sensitive secretory line, MadIA214, wasselected from mutagenized Chinese hamster ovary cellsthat express two heterologous export marker proteins: asecretory form of the human placental alkaline phos-phatase (SeAP), and the Kd heavy chain of mouse MHCclass I. SeAP secretion in MadIA214 was extremelyreduced at elevated temperature (40°C), while the exportof functional H-2Kd molecules to the plasma membranewas only slightly affected. This mutant constitutively trans-ferred onto newly synthesized proteins a truncatedoligosaccharide core, Man5GlcNAc2, which was monoglu-cosylated in the protein-bound form. Nevertheless, the finaloligosaccharide-structures associated to mature SeAP andH-2Kd were similar in mutant and wild-type glycoproteins.The inaccessibility in MadIA214 endoplasmic reticulum(ER) of one or more components required for oligosaccha-ride chain elongation is supported by the reconstitution of

a correct core structure, obtained after disruption ofcellular compartments, but not after cell permeabilisationor blocking ER-to-Golgi transport. The increased associa-tion of the ER-chaperone BiP with immature SeAP corre-lated with the thermodependent decrease in SeAPsecretion. The retention of incompletely folded polypep-tides in MadIA214 parallels both a marked ER-dilationand an important glycoprotein degradation documented bythe formation of soluble oligomannosides with one GlcNAcresidue. Our data provide the first in vivo evidence that theinitial step in N-glycosylation differentially governs glyco-protein maturation, transport and degradation.

Key words: CHO, Thermosensitive secretion, MHC class I, Secretoryalkaline phosphatase, N-glycosylation, Glycoprotein degradation, ERretention

SUMMARY

INTRODUCTION

The transport through the eukaryotic cells of secreted ormembrane glycoproteins is widely thought to be mediated byvesicular budding and fusion between the different organellesof the secretory pathway (Pelham, 1989; Rose and Doms,1988; Rothman, 1994). Protein trafficking starts in the ER intowhich the newly synthesized proteins are translocated andshaped to secretion-competence. A first sorting of secretoryproteins occurs at the level of the cis-Golgi network, alsoreferred to as the intermediate compartment. Properly foldedproteins are conveyed through the Golgi apparatus (cis-,medial- and trans-), from which sorting to lysosomes or tosecretory vesicles of the constitutive or regulated pathwaytakes place. Fusion of these vesicles with the plasma membraneleads to either surface deposition of membrane proteins or tothe secretion of soluble components. The maturation of N-gly-cosylated proteins includes sequential post-translational mod-ifications each occurring within a specific compartment. Most

recent data provide evidence for N-glycosylation beingimportant for correct folding, assembly, and transport as wellas for the acquisition of glycoprotein functions (reviewed byFiedler and Simons, 1995), but the precise modalities of car-bohydrate involvement in these important biological processesremain to be elucidated.

The use of conditional secretory mutants offers the possibil-ity to analyze in vivo the molecular mechanisms underlyingsecretion abnormalities (reviewed by Colbaugh and Draper,1993). As yet, many secretory mutants have been phenotypicallycharacterized as impaired in endocytosis and/or exocytosis buttargets of the mutations often remained elusive. Genetic studiesin yeast (Huffaker and Robbins, 1983; Orlean, 1992; Roos et al.,1994) and in mammalian cells (Brändli, 1991; Stanley, 1987)have in contrast contributed to the determination of a greatnumber of the enzymatic reactions participating in N-glycosyla-tion. Considering the enormous complexity of this process, rel-atively little is known about the molecular basis of the individ-ual steps interfering with glycoprotein maturation and export.

324 M. Ermonval and others

We established conditional secretory mutants (our unpub-lished data) after mutagenesis of our Cl42 parental clonederived from the functionally pseudohaploid CHO cell line.This Cl42 cellular model stably expresses two exogenousexport marker proteins: a modified form of human placentalalkaline phosphatase (SeAP), and the mouse MHC class Imolecules, H-2Kd. The soluble SeAP is secreted and enzy-matically active as a monomer whereas the transmembraneprotein H-2Kd is a heterotrimer made of non-glycosylated β2-microglobulin (β2m), the Kd heavy chain and an endogenouslyprocessed peptide. The MHC complex usually requires aproper assembly in the ER in order to be exported to the cellsurface and to be active in antigen presentation to T lympho-cytes. A temperature sensitive (ts) clone, MadIA214, wasselected, which exhibited a strong reduction in SeAP secretionat elevated temperature (40°C) but was only slightly affectedin the delivery of functional MHC class I molecules to theplasma membrane. The fact that the export of only one of themarker proteins is drastically impaired renders this cellularmodel particularly well-suited to gain insight into the exportrequirements of individual proteins in the same cell.MadIA214 exhibits a constitutive defect in the N-glycosylationpathway, which affects transport of various proteins to differentextents and in a temperature sensitive manner. Our studiesreveal the importance and differential role of N-glycosylationin glycoprotein folding, secretion, and degradation inmammalian cells.

MATERIALS AND METHODS

Reagents and antibodiesRPMI and alpha-MEM culture medium as well as additives (Hepes,glutamine, G418) were purchased from Gibco-BRL, 2-β-mercap-toethanol (2-βME) and puromycin from Sigma and the FCS,mycoplasma free, from PBS Orgenics.

Monoclonal antibodies (mAbs) specific to H-2Kd class I MHC werethe 20-8-4S (Ozato and Sachs, 1981), the SF1.11 (ATCC) and the 31-3-4S (ATCC), for immunofluorescence, and the SF1.11, for immuno-precipitation. Human placental alkaline phosphatase (AP) andhamster BiP (grp-78) proteins were immunodetected with rabbit poly-clonal Abs, respectively, from Dako and StressGen. We used assecond Abs, goat anti-mouse or anti-rabbit Igs either unlabelled(Southern Biotechnology), labelled with FITC (Amersham), or withHRP or biotin (Southern Biotechnology). Immunoprecipitation ofimmune-complexes was done with Protein A coupled to SepharoseCL4B beads (Pharmacia).

Cell linesThe Cl42 parental clone was obtained after electroporation of CHO-K1 (ATCC) cells with three plasmids: pKCKdwt (a kind gift from J.-P. Abastado, Institut Pasteur, Paris, France) encoding the entire heavychain of H-2 Kd (Jaulin et al., 1992); pBC12RSVSeAP (Berger et al.,1988) encoding a modified form of the human placental AP lackingthe GPI-anchoring signal and pSV2Neo to confer cell resistance toG418 antibiotic.

The MadIA214 ts clone was obtained after ethyl-methyl-sulfoxidemutagenesis of the Cl42 clone, using a strategy of enrichment for cellswith an adhesion defect to plastic dishes at 40°C (designated as thenon-permissive temperature). The MadIA214 clone with a clear tsSeAP secretory defect was selected in the course of the screening ofthis cell population (M. Ermonval, unpublished).

The Cl42cw3-D3.41 and MadIcw3-D54 clones were derived from

Cl42 and MadIA214 clones, respectively, in which two plasmids wereintroduced by electroporation: p42 (a kind gift from H. Gourmier,Institut Pasteur, Paris, France) containing the gene encoding the entirehuman HLA-Cw3 class I heavy chain and pLXSP (a kind gift fromM. Mehtali, Transgene, Strasbourg, France) conferring puromycinresistance.

All the CHO derived cells were grown in alpha-MEM medium sup-plemented with 5% FCS and 10 mM Hepes. When necessary, antibi-otics were added for selection (1 mg/ml for G418 and 10 µg/ml forpuromycin).

9-4 (a kind gift from J.-P. Abastado) is a murine T cell hybridomathat expresses a T cell receptor specific for the peptide p-Cw3 (170-179) restricted to H-2Kd (Bellio et al., 1994). The IL-2 dependentCTLL-2 (ATCC) T cell clone was maintained in 20 i.u./ml of mouserecombinant rIL-2 (Genzyme). These two lines were grown in RPMIsupplemented with 5% FCS, 10 mM Hepes, 2 mM L-glutamine and50 µM 2-βME.

Immunofluorescence analysis of H-2Kd transport at theplasma membrane H-2Kd molecules were first removed from the cell surface by inducedendocytosis after cross-linking of the complex in the presence ofcycloheximide (cycloH) at 50 µg/ml to avoid new protein synthesis.Cells (5×106) in a volume of 200 µl were incubated for 1 hour at 33°Cwith 400 µg/ml of an Ig-enriched fraction of 20-8-4S. After washing,the cells were incubated for 1 hour with anti-mouse IgG2a Abs at 10µg/ml. CycloH and Abs were discarded and the cells were split intothree groups to be incubated for 5 hours either on ice, or at the per-missive or non-permissive temperature to allow for H-2Kd newsynthesis. H-2Kd reexpression was then measured by immunofluo-rescence. Briefly, cells in suspension (5×105-106) were incubated for30 minutes on ice in 50 µl of purified 20-8-4S anti-Kd mAb directlycoupled to FITC (diluted 1/500 in PBS/BSA, 1%), in the presence ofunlabelled IgG2a as competitor for the anti-IgG2a remaining bindingsites. After 3 washes, propidium iodide was added at 10 µg/ml to labeldead cells and analysis was done with a Facscan programme on aBecton Dickinson FACS apparatus.

Enzymatic detection of alkaline phosphataseAll the enzymatic tests were performed using conditions that selec-tively inactivate some cellular or serum phosphatases without inhibit-ing the human placental AP activity (65°C heated samples and 10 mMof L-homoarginine). An amplification microassay (NAD/NADHcycling) was performed using the NADP as substrate of the AP(Johannsson et al., 1986; Self, 1985). Briefly, 0.1 mM of NADP(Boehringer) was added to the samples in a final volume of 100 µl at50 mM diethanolamine (DEA), pH 9.5, 1 mM MgCl2, 0.1 mM ZnCl2and incubated for 45 minutes at room temperature. Then added was200 µl of amplifying buffer containing 0.1 mg/ml of alcohol dehy-drogenase (Sigma) and 0.075 mg/ml of diaphorase (Boehringer) in a20 mM phosphate buffer, pH 7.2, containing 5 mg/ml of BSA and0.55 mM of p-iodonitrotetrazolium violet as indicator of the reaction.Absorption at 492 nm was measured on an LP500 reader plate (Diag-nostic Pasteur). For highly sensitive detection, supernatants of cellsgrown in Soft Cell CHO synthetic medium (TCS Biological) wereincubated with the lumigen PPD chemiluminescent substrate(Boehringer) at 0.1 mg/ml in 0.1 M DEA. Light emission keptconstant after 30 minutes was monitored in a microbeta counter(Wallac/Berthold).

Assay for antigen presentationThe activation of the 9-4 T cell hybridoma in response to its specificantigen p-Cw3 (170-179) restricted to H-2Kd was quantitated bymeasuring IL-2 secreted in the supernatant. Antigen presenting cells(2.5×104) were cocultured in 96-well microplates with 9-4 T cells(5×104), for 48 hours at the indicated temperatures, in the presence ofp-Cw3 synthetic peptide (a kind gift from H. Gourmier, Institut

325A CHO thermosensitive secretory cell line

Pasteur, France) at concentrations ranging from 10−7 to 10−11 M. Forthe assay of endogeneously processed peptide, different ratios ofHLA-Cw3 transfected cells (3×101-3×104) to 9-4 T cell hybridoma(3×104) were cultured for 48 hours at different temperatures.Activated T cell supernatant (20 µl) was added to 104 CTLL2indicator cells. The IL-2 dependent CTLL2 proliferation wasevaluated after 48 hours of culture at 37°C, as described by Landegren(1984) by measuring the hexosaminidase cellular amount which isproportional to cell numbers.

Immunoprecipitation of metabolically labelled proteinsand SDS-PAGE analysisCells (2.5×105) were cultured overnight in 24-well culture plates atthe indicated temperatures. After 30 minutes pre-pulsed with Met/Cysfree medium (ICN-Flow), cell cultures were pulsed for 15 minuteswith 100 µCi of radioactive [35S]Met-[35S]Cys mixture (NEN). A 4hour labelling with 25 µCi was used for steady-state analysis. After0 hours or 3 hours of chase in 500 µl of medium containing an excessof unlabelled Met and Cys amino acids, supernatant was recovered,and cell lysates were prepared by a 5 minutes treatment on ice, in NETbuffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 5 mM EDTA and1% Triton X-100). Specific immunoprecipitations were performed for30 minutes at 4°C with 25 µl per sample of Protein A-Sepharose beads(Pharmacia) precoated with 2 µg of Abs. After 4 washes in high salt(0.5 M NaCl) NET buffer, followed by 2 washes in 40 mM Hepes,pH 7.4, the immunoprecipitates were eluted in reducing sample buffercontaining SDS, boiled, then run on an SDS-PAGE gel. Amersham’sHyperfilm was used for autoradiography of intensified gels.

For endo-β-D-glucosaminidase (endoH) digestion, immunoprecip-itates were eluted from the beads with 50 µl of reducing buffer con-taining 5% 2-βME and 0.5% SDS, then 20 µl of eluted sample weretreated or not with 5 mU of endoH (Boehringer) for 18 hours at 37°C.Sialic acids were directly released from immune-complexes attachedto the beads by 1.5 hours treatment at 37°C with 100 mU of neu-raminidase (Nase: Sigma) in 0.1 M citrate buffer, pH 5.5.

Western blot analysis of total or immunoprecipitatedcellular extractsProteins from total extracts corresponding to 5×104 cells lysed in NETbuffer or anti-SeAP immunoprecipitates of unlabelled cells preparedas above but treated or not for 15 minutes at 25°C with 5 mM ATP(in 50 mM Tris-HCl, pH 7.5, buffer containing 50 mM NaCl and 5mM MgCl2), were run on an 8% SDS-PAGE gel. Proteins were trans-ferred from the gel onto nitrocellulose membranes. After blocking inPBS/1% gelatin/0.1% Tween-20, the membranes were incubated withanti-BiP (1/500) or anti-SeAP (1/2,000) rabbit Abs. The detection oflight emission on film was achieved after reacting the membrane withgoat anti-rabbit Abs coupled with HRP (1/4,000), using the ECLchemiluminescent procedure (Amersham).

Oligosaccharide fractionation and HPLC analysisCells (5-10×106) were metabolically labelled for 1 hour at 34°C inalpha-MEM medium containing 5% of dialysed FCS, 0.5 mM Glc and100 µCi of 2-[3H]Man (42.9 GBq/mmole; Amersham). Castanosper-mine or deoxynojirimycin glucosidase inhibitors (Boehringer), wereadded at 50 µg/ml. When indicated, cells were pretreated for 15minutes with 5 µg/ml of brefeldin A (BFA, Epicentre Technologie)and labelled for 1 hour in the presence of BFA. Sequential extractionprocedure was performed as already described (Cacan et al., 1993).Briefly, PBS washed cell monolayer was harvested with 1.1 ml of amixture of methanol (8 vols) and of a solution (3 vols) containing 10%of IgG in cacodylate buffer. After adding 1.2 ml of CHCl3, threephases were separated by centrifugation, the lower organic phase con-taining the Man-P-Dol, the interphase, and the upper aqueous phasecontaining the soluble metabolic precursors as well as the solubleoligosaccharides. The oligosaccharide-P-P-Dol were extracted fromthe interface formed by a mixture of chloroform/methanol/water

(10/10/3, by vol.), then glycans were released from this lipid extractby mild acid treatment (0.1 N HCl in tetrahydrofuran) for 2 hours at50°C. Newly glycosylated proteins remaining in the pellet afteroligosaccharide-P-P-Dol extraction were digested overnight at roomtemperature with 300 µg of trypsin, then treated with 0.5 units ofpeptide-N-glycanase F (PNGase-F, Boehringer) to release their Asn-linked oligosaccharides.

The different fractions were first purified by gel filtration on aBiogel P2 column (desalting and elimination of the unreacted radioac-tive metabolic precursors). The soluble fraction was also passedthrough a QAE-Sephadex column to separate the neutral materialfrom the charged. Size fractionation of oligosaccharide material wasperformed by HPLC on a ASAHIPAK-NH2 column (Asahi,Kawasaki-Ku) by elution with an acetonitrile/water gradient (70/30 to50/50, v/v), at a flow rate of 1 ml/minute over an 80 minute timeinterval. The elution profile of incorporated radioactivity associatedwith the different oligomannosides was monitored with a Flo-one βdetector (Packard), using as standard a mixture of radiolabelled oligo-mannosides (Cacan et al., 1993).

Oligosaccharide biosynthesis in disrupted or semi-permeabilized cellsOligosaccharides from disrupted or semi-intact cells were metaboli-cally labelled with GDP-[14C]Man (1.4 GBq/mmole; Amersham).Microsomal preparations were obtained by Dounce homogeneizationafter the cells were swelled in a 10 mM Hepes, 15 mM KCl buffer.Microsomes were recovered by 10 minutes spinning at 30,000 g ofthe supernatant from a first 1,500 g centrifugation. Samples were thenincubated for 30 minutes at 37°C with 5 µCi of the GDP-[14C]Manin the presence of 50 µg/ml of castanospermine and 50 µM UDP-GlcNAc, 50 µM UDP-Glc, 5 mM AMP, 2 mM MnCl2, 5 mM MgCl2in a 100 µl final volume of TKM buffer (30 mM Tris-HCl, pH 7.5,120 mM KCl, 4 mM Mg-acetate).

Semi-intact (permeabilized) cells were prepared as described byPlutner et al. (1992). Adherent cells grown in 10 cm diameter Petridishes were treated for 5 minutes on ice with 40 µg/ml of digitonin.Digitonin was washed out and semi-intact cells were radioactivelylabelled with 4 ml of the same TKM incubation medium. Oligosac-charides from disrupted or permeabilized cells were analyzed asdescribed for intact cells.

Electron microscopy analysis Ultrastructural analysis was performed as already published (Gorgas,1985) on cells grown at 33°C or 40°C before being fixed for 15minutes in 2.5% glutaraldehyde in 0.1 M Pipes buffer, pH 7.4, con-taining 2% sucrose. Briefly, pelleted cells solidified in agar wereincubated in the alkaline DAB medium to visualize the peroxisomalmarker enzyme, catalase, as well as to enhance the staining oforganelle membranes. After a 45 minute incubation at 37°C, thesamples were postfixed for 60 minutes at 4°C in 1% osmium ferro-cyanide then stained en bloc for 45 minutes at 4°C with 1% uranylacetate. After ethanol dehydration, samples were embedded in Epon812. Thin sections were stained with alkaline lead citrate for 45seconds and examined in a Zeiss EM10 electron microscope.

RESULTS

SeAP secretion, but not H-2Kd export, isthermodependent in the MadIA214 lineWe established the Cl42 line as a cellular model to isolate tssecretory mutants. Cl42, a CHO-K1 derivative, expresses twoheterologous proteins as markers for protein export: the H-2Kd

haplotype of the mouse MHC class I at the cell surface and asecretory form of the human placental alkaline phosphatase(SeAP). We isolated the MadIA214 line from mutagenized


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Fig. 1. Growth properties of parental and mutantcells and enzymatic detection of alkalinephosphatase secretion in their supernatant.Properties of Cl42 (left panels) and MadIA214(right panels) were compared both at permissive(33°C; j). and non-permissive (40°C; u)temperatures. The growth curves (A) wereobtained by plotting the number of cells as afunction of time in culture. The SeAP activity inthe culture supernatant was expressed either as afunction of the number of plated cells grown for18 hours (B) using a chemiluminescent detectionmicroassay in which the LCPS (luminescentcounts per second) were emitted afterdephosphorylation of the lumigen PPD, or inshort time kinetics (C) of the SeAP activityreleased in the supernatant by 5×104 cells usinga NAD/NADH amplification assay in whichoptical density at 492 nm is expressed as afunction of the incubation time. Note that theLCPS scales are different.

Cl42 cells screened for a reduced SeAP activity in their super-natant at 40°C (non-permissive temperature) as compared to33°C (permissive temperature).

An impairment of protein export in eukaryotic cells couldelicit severe effects on cell growth, which might interfere withnumerous metabolic processses. Therefore, mutant growthcharacteristics were primarily evaluated. The MadIA214growth was only slightly reduced at 40°C in comparison to itsgrowth at permissive temperature or to the Cl42 growth (Fig.1A). The SeAP activities released in the supernatant of cellsgrown for short periods of time varying either the cell number(Fig. 1B) or the time (Fig. 1C) in culture was thus measuredby sensitive assays (enzymatic amplification assay or chemi-luminescent procedure). Raising the temperature from 33°C to40°C resulted in a dose- and time-dependent decrease in SeAPactivity in the parental clone (approx. 3-fold). This was not dueto an intrinsic thermosensitivity of the enzyme since heatstability is a distinctive feature of human placental APexploited in our assays to exclude other endogenous phos-

phatase activities. The higher level of active SeAP found at33°C in MadIA214 as compared to Cl42 probably resultedfrom the selection of a high SeAP producer clone and is con-sistent with the higher steady state levels revealed by westernblot (see Fig. 8, lanes 2 and 3). However, at the non-permis-sive temperature, reduction in SeAP activity was much morepronounced in the mutant (9.6-fold versus 3-fold in the parent).These findings indicate that SeAP export is slightly tempera-ture sensitive per se but is more drastically affected at 40°C inthe MadIA214 mutant.

We examined whether temperature sensitivity in secretionwas a general feature of MadIA214 also applying to the exportof the second marker H-2Kd. Cell surface expression of mouseMHC class I molecules was assessed by FACS analysis usingdifferent mAbs (20-8-4s, SF1-11, 31-3-4s) interacting withconformational determinants of the assembled H-2 Kd

complex. No difference in cell surface expression of these classI molecules was seen with Cl42 and MadIA214 even whencells were grown at the non-permissive temperature (data not


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Fig. 2. FACS analysis of H-2Kd MHC class I reexpression at the cellsurface of parental and mutant cells. H-2Kd molecules were firstremoved from the cell surface of Cl42 and of MadIA214 byendocytosis induced by cross-linking mouse MHC with Abs. Newlysynthesized H-2Kd exported at the cell surface were monitored byFACS using the 20-8-4s Ab directly labelled with FITC and thepercentage of class I reexpression was calculated from the formula(I′−Io/Imax−Io)×100: I′ is the mean value of the fluorescent peakobtained with the sample to be analyzed, Io is the mean value of thenegative control given by incubating untransfected CHO cells with20-8-4s-FITC and Imax is the mean of the fluorescent peak of H-2Kd

at the cell surface of non-manipulated mutant or parental cells. Thehistograms represent the percentage of H-2Kd present at the cellsurface at time 0 after class I endocytosis (black bars), after 5 hoursof incubation at 33°C (hatched bars) or at 40°C (dotted bars).

shown). Growth in synthetic medium allowed us to rule outthat MHC staining with the 20-8-4s mAb revealed complexesstabilized at the cell surface by association of free H-2 Kd

heavy chains (Towsend et al., 1990) with FCS β2m rather thanwith the endogenous hamster β2m (data not shown). Thisindicates that both Cl42 and MadIA214 are capable of assem-bling and exporting class I molecules. Since these analysesonly reflect a steady state distribution, the export rate of newlysynthesized molecules was determined by measuring H-2 Kd

reexpression in a 5 hour interval after induced endocytosis.Class 1 endocytosis was performed in the presence of cyclo-hexamide to avoid H-2 Kd new synthesis during the internal-ization process, then the drug was discarded to allow for classI reexpression. When cyclohexamide was maintained duringthe reexpression step, H-2 Kd detected at the cell surface ofCl42 grown at 37°C or 40°C for various periods of time, didnot increase over the background level. In contrast, a time-dependent increase of membrane class I was observed in theabsence of the drug (data not shown). This control experimentexcluded the H-2 Kd molecules detected in these conditions asbeing the result of a recycling of the internalized class Imolecules. Both Cl42 and MadIA214 reexpress a higheramount of H-2 Kd molecules at the elevated temperature ascompared to their respective levels reexpressed at 33°C (Fig.2). However, the rate of MHC class I reexpression is dimin-ished in the mutant, both at 33°C and at 40°C, to around 25%of the reexpression rate in the parental clone.

Thus, in contrast to the slow down of SeAP secretionobserved at 40°C in Cl42, class I export is accelerated atelevated temperature in these cells and in MadIA214 as well.Furthermore, while SeAP secretion is drastically reduced inMadIA214 in a thermodependent manner, export of MHC classI is only slightly diminished in this line whatever the temper-ature.

SeAP and H-2Kd glycoproteins are functional inMadIA214 Our results favour a mutation effect on protein export ratherthan on protein function as shown by the significant decreasein SeAP secretion observed in biosynthetic analyses (seebelow).

That H-2Kd molecules present at the cell surface ofMadIA214 were functional was assessed by an antigen pre-sentation assay using the p-Cw3/H-2Kd specific T cellhybridoma (9-4) as responding cells. When cocultured in thepresence of exogenously added p-Cw3 peptide, Cl42 andMadIA214 were both competent to 9-4 T cell activation (Fig.3A) while, as expected, CHO cells not expressing H-2Kd wereunable to activate 9-4 T cells (data not shown). At non-per-missive temperature, both Cl42 and MadIA214 were slightlyless efficient in peptide presentation. This contrasts with theincreased rate of MHC export at elevated temperature, butcould be explained by stabilization of the class I complex atlower temperature (Rock et al., 1991). We also tested parentaland mutant cells ability to present endogenously processedantigen. Cells in which H-2Kd and HLA-Cw3 class I moleculesare coexpressed present endogenously processed p-Cw3peptide in an H-2Kd restricted manner (Casanova et al., 1992).Therefore, we transfected both Cl42 and MadIA214 with thegene encoding the HLA-Cw3 class I heavy chain. Parental andmutant clones transfected with Cw3 (Cl42cw3-D3.4 and

MadIcw3-D53) activated the 9-4 T cells (Fig. 3B), while non-transfected cells did not (data not shown). However, theparental-Cw3 transfectant was more efficient than the mutantin T hybridoma activation, especially at the non-permissivetemperature. Thus, when compared to Cl42, MadIA214 isgenerally less efficient (approx. 10 times) in presenting bothexogenously added peptides and endogenously processedantigen, a phenomenon which remains to be elucidated.

Functional SeAP and H-2Kd molecules are thus found in theMadIA214 mutant.

Maturation of both SeAP and H-2Kd glycoproteinsare affected in MadIA214We then examined possible effects of the mutation onsynthesis, maturation and transport of glycoproteins. SeAP(Fig. 4A) and H-2Kd (Fig. 4B) were immunoprecipitated fromextracts or culture supernatant of cells metabolically labelledeither at 33°C (lanes a) or at 40°C (lanes b), and analyzed byelectrophoresis.

In Cl42, newly synthesized SeAP (Fig. 4A) appeared aftera 15 minute radioactive pulse at 33°C as an immature form(lane 1a) of 67 kDa further trimmed after a 3 hour chase in a65 kDa intermediate (i, lane 2a). The SeAP mature form (m)detected in the culture supernatant had an apparent size of 71kDa (lane 3a). The same patterns were obtained at 40°C(compare lanes b to lanes a), except that more immature formsaccumulated intracellularly (lanes 1b and 2b). This correlatedwith the slight reduction in both SeAP secretion (lane 3b) andSeAP activity (see Fig. 1). In MadIA214, newly synthesized


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effector cells / APC ratio

Fig. 3. Antigen presentation by H-2Kd moleculesexpressed at the cell surface of parental and mutantcells. The capacity of mouse MHC class I to presentantigen was evaluated either by adding variousconcentrations of exogeneous p-Cw3 peptide to thecells (A), or by testing presentation ofendogenously processed peptide at different ratiosof effector cells to parental or mutant cells thatstably express the HLA-Cw3 antigen (B). The leftpanels correspond to presentation by the parentalcells and their Cw3-derivative and the right ones tothose of the mutant cells and their Cw3 derivative.Antigen presentation was measured at 33°C (j) and40°C (h) by quantitating the IL-2 released afteractivation of the 9-4 T cell hybridoma specific forH-2Kd/p-Cw3 antigen. The titration of IL-2 in thesupernatant of activated cells was performed by acolorimetric proliferation assay of the CTLL-2 line,measuring its cellular hexosaminidase content.Activation is therefore expressed as the absorbence(A) at 405 nm (CTLL-2 proliferation IL-2dependent) in relation to exogenous or endogenousp-Cw3-peptide presentation.

Fig. 4. SDS-PAGE analysis of SeAP and H-2Kd biosynthesized inparental and mutant cells. Proteins from Cl42 and MadIA214 cellswere metabolically labelled either at 33°C (lanes a) or at 40°C (lanesb). SeAP was specifically immunoprecipitated using polyclonal Absto human placental AP (A) and assembled H-2Kd using the SF1.11mAb (B). Immunoprecipitations were performed either from lysatesof cells pulsed for 15 minutes (lanes 1), or chased for 3 hours afterthe pulse (lanes 2) or from cell culture supernatant recovered afterthe chase (lanes 3). Immunoprecipitated material was analysed bySDS-PAGE and autoradiography. The position of intermediate (i) andmature (m) forms of SeAP and H-2Kd are indicated both for theparent and for the mutant on the left and on the right side,respectively, of the gel autoradiograph. The arrow indicates theposition of the 78 kDa protein which is co-immunoprecipitated withSeAP. As some background was obtained using the anti-Kd Abs, wealso show the non-specific products immunoprecipitated from nontransfected CHO cell lysates.

and trimmed SeAP appeared as doublets (65-62 kDa in lane1, 63.5-62 kDa in lane 2, respectively) and all species migratedmore rapidly than the respective parental forms. The secretedmaterial (lanes 3) also consisted of two bands (71 kDa and66.5 kDa), the upper band having the same molecular mass asthe secreted form in Cl42. The constant ratio between thebands of the doublets indicated that both glycoprotein specieswere exported with the same efficiency. Protein patterns werecomparable at both temperatures, however, at the non-per-missive temperature, SeAP intermediates accumulated inmutant lysates whereas secreted protein only appeared as afaint band. Similar observations were obtained from steady-state analysis after a 4 hour labelling (Fig. 5). Immature SeAPintermediates found in the lysate of mutant cells grown at33°C, 37°C or 40°C, appeared as doublets migrating fasterthan the corresponding parental form (Fig. 5A). A gradualreduction in the amount of mature SeAP secreted inMadIA214 supernatant was observed when the temperaturewas raised (Fig. 5B).

Interestingly, the H-2Kd immature forms (see Fig. 4B) syn-thesized by the mutant both at 33°C (lanes a) and 40°C (lanesb) also displayed a decrease in molecular mass (approx. 2kDa). As seen for SeAP, the mature form of H-2Kd moleculesexhibited the same molecular mass in mutant and parentalcells. In agreement with FACS analysis (Fig. 2) the export ofH-2Kd at the cell surface was accelerated at 40°C both in Cl42and MadIA214 as shown by the higher amount of mature formsfound at this temperature as compared to 33°C (compare lanes2b with 2a). Also in line with FACS results, this analysis showsat both temperatures a reduction of the H-2Kd export rate inMadIA214 as compared to Cl42. Indeed, after a 3 hour chase,the protein distribution was in favour of the mature form at33°C in Cl42 (lane 2a) with a weak amount of immature inter-mediates at 40°C (lane 2b), while in MadIA214 the immature


Fig. 5. SDS-PAGE analysis of SeAP labelled to steady state in theparental and in the mutant clones. Cells were submitted to a longpulse labelling of 4 hours either at 33°C, 37°C or 40°C. HumanSeAP was immunoprecipitated from the cell lysates (A) or from thecorresponding supernatants (B) and analyzed by SDS-PAGE. Notethat whereas both the trimmed immature (i2) and the mature (m)form of SeAP appear as a single band in Cl42, a doublet is seen withthe immature (i2a and i2b) as well as with the mature (ma and mb)forms of SeAP in the mutant. The co-immunoprecipitated 78 kDaprotein is indicated by an arrow.

forms were predominant at 33°C (lane 2a) and still present athigh levels at 40°C (lane 2b).

These results show that, independent of the temperature,MadIA214 newly synthesized glycoproteins exhibit a reducedmolecular mass whereas exported proteins acquire the correctsize typical for mature proteins. Furthermore, intracellularaccumulation of immature SeAP observed at 40°C indicatesthat the mutation can interfere with glycoprotein export in atemperature sensitive fashion.

Fig. 6. Comparison of the EndoHand Nase sensitivity of SeAP in theparent and in the mutant.Immunoprecipitates obtained fromcell lysates prepared directly after15 minutes of pulse or after 3hours of chase at 33°C as well asfrom the culture supernatant afterthe chase were treated (+) or not (−) with either EndoH or with Naseas indicated. The digestionproducts were analyzed by 8%-SDS-PAGE: i1, i2 and mcorrespond to the parental earlyimmature, trimmed immature, andmature forms, respectively. Theposition of the non-glycosylatedSeAP obtained after EndoHdigestion is indicated (NG).

MadIA214 glycoproteins are altered at an early stepof N-glycosylationIn a first attempt to elucidate the nature of the modificationaffecting the glycoproteins in MadIA214, we used reagentsinterfering with N-glycosylation. Tunicamycin cell treatment,which inhibits transfer in vivo of the N-glycan core onto newlysynthesized proteins, as well as endoglycosidase (PNGase-F)digestion of immunoprecipitated glycoproteins, generated non-glycosylated SeAP and H-2Kd proteins of the expectedmolecular mass (data not shown) both in mutant and parentalclones. Thus, the reduction in apparent size of SeAP and H-2Kd glycoproteins in the mutant was not due to an alterationof polypeptide biosynthesis but resulted from a modification ofthe oligosaccharide structures.

N-glycans attached to early and mature forms of proteinssynthesized at permissive temperature were further analyzedfor their susceptibility to various glycosidases. SeAP (Fig. 6)and H-2Kd (Fig. 7) immunoprecipitates were treated withendoH, active on oligomannoside structures (unsubstitutedα1-6 linked core mannose) generated during the early stepsof N-glycosylation. As expected, in Cl42 the immature formsof SeAP and H-2Kd exhibited endoH sensitivity whereas themature forms were endoH resistant. In MadIA214, however,all the SeAP and H-2Kd intermediates were endoH resistant,a result consistent with an alteration of an initial N-glycosy-lation step. Immunoprecipitates were also digested with neu-raminidase (Nase) releasing sialic acid added in the TGN ata final stage of glycoprotein maturation. The effect of Nasewas similar on both mutant and parental mature forms ofSeAP and H-2Kd indicating that MadIA214 glycoproteinswhich leave the ER acquired the glycan structure typical fornormal maturation. The two mature SeAP forms detected inthe supernatant of the mutant migrated faster after Nasetreatment indicating that both species were sialylated.According to their molecular mass, these two species maycorrespond to SeAP molecules carrying one or two N-glycanmoieties, respectively.

These data demonstrate that MadIA214 is affected in aconstitutive way at an early step of the N-glycosylationpathway.


Fig. 7. Comparison of EndoH and Nase sensitivityof H-2Kd in the parent and in the mutant. Metaboliclabelling as well as EndoH and Nase treatment ofH2-Kd immunoprecipitated by SF1.11 mAb wereperformed as described in the legend to Fig. 6,except that the digestion products were analyzed by12% SDS-PAGE. The asterisks indicate themigration position of the immature H-2Kd formappearing in the mutant.

Truncated oligomannoside structures aretransferred onto Asn-sites of the MadIA214 proteinsThe reduced molecular mass and endoH resistance of theimmature forms of SeAP and H-2Kd synthesized in MadIA214were consistent with the presence of a truncated oligosaccha-ride on the Asn-sites of these proteins. To define the exact N-

Oligosaccharide-P-P-Dol

M5

M9

G3M9A

M5D

Glycop

3H-M

anno

se in

corp

orat

ion(

dpm

)

M4M5

M6

B

M4

M3

M5

G1M5

E

AAtimFig. 8. HPLC analysis of oligosaccharides present in different fractions metabolically labelled with 2-[3H]Man for 1 hour at 34°C. Soluble, lipidchloroform/methanol/water mixtures as indicated in Materials and MethDol: A, D) and from the glycoprotein (glycoproteins: B,E) fractions werthe soluble phase of chloroform/methanol/water extracts prior to HPLC oligomannoside species (dpm) are eluted by acetonitrile-water gradient The oligomannoside peak corresponding to carbohydrates with one attacunmarked. The abbreviations used are the following: M, Man; G, Glc.

glycan composition of the altered glycoproteins, we performed1 hour oligosaccharide labelling with radioactive 2-[3H]Man inthe presence of castanospermine as inhibitor of glucosidases.Dolichol-associated oligosaccharide precursors were separatedfrom glycoproteins and the fractions were analyzed by HPLC.Fig. 8 shows that the lipid intermediates (oligosaccharide-P-P-

roteins

M7

M8

M9

G1M9

G2M9

G3M9

AAAe (min)

AAAAAAAAAANeutral soluble oligosaccharides

AAM4

AAM5

AAG1M5

AAG3M5

AAF

AAAAA

AAAAAAAA

AAAAAAAAAAAA

AA

AAM4

AAM5

AAAAAAAAAG1M5

orM6

AAAAAAAAAG3M5

orM8

AAAAAAG1M9

AAAAAAG3M9

AAC

of parental and mutant cells. Cl42 (A-C) and MadIA214 (D-F) were and protein fractions were partitioned by extraction with differentods. The oligosaccharides released from the lipid (oligosaccharide-P-P-e analyzed by HPLC. Phosphorylated sugar species were removed fromanalysis of the neutral soluble sugar fraction (C,F). The radioactivefrom an AASAHiPak-NH2 column and recorded as a function of time.hed GlcNAc are hatched, whereas the GlcNAc2 derivatives are let


Dol) of the parental clone were composed of completeGlc3Man9GlcNAc2 and Man9GlcNAc2 (G3M9 and M9) highmannose products and of the non-glucosylated Man5GlcNAc2(M5) intermediate (Fig. 8A), while only the incomplete M5intermediate was found in the mutant (Fig. 8D). This truncatedcore was efficiently transferred onto Asn-acceptor sites of gly-coproteins synthesized in the mutant (Fig. 8E), while mainlyMan9 derivatives were found in the parental glycoproteinfraction (Fig. 8B). It should be noted that all the expected glu-cosylated Man9 forms (G3M9, G2M9 and G1M9) weredetected in Cl42 whereas the predominant glucosylated Man5species contained only one glucose (G1M5) in MadIA 214.Interestingly, when oligosaccharide synthesis was performedin the presence of another glucosidase II inhibitor (deoxyno-jirimycin), the major glucosylated form associated with theprotein in the mutant was also the Glc1Man5GlcNAc2 (G1M5:see Fig. 9C,D).

This analysis reveals that the block at an early N-glycosyla-tion step in MadIA214 leads to the biogenesis of glycoproteinsloaded with a monoglucosylated truncated sugar core and thatthis block is complete and remains stable.

The glycosylation block is released in MadIA214 cellextracts but not in semi-intact or BFA-treated cellsConsidering the complexity of the N-glycosylation pathway(Kornfeld and Kornfeld, 1985), a block leading to a truncatedsugar core structure could occur at various levels. The Man5 toMan9 elongation steps take place in the lumen of the ER,although the Man5GlcNAc2-P-P-Dol precursor is synthesizedon the cytosolic side of the ER membrane. Thus, this Man5lipidic intermediate must first be reoriented towards the ERlumen. To ensure oligosaccharide elongation Man-P-Dol andGlc-P-Dol also made up on the cytosolic side of the ERmembrane has to be turned to the lumenal side as well.

The level of mannose incorporation (see HPLC profiles)

Man

nose

inco

rpor

atio

n(dp

m)

A

CFig. 9. HPLC analysis of oligosaccharides synthesizedin the mutant subjected to different treatments.Carbohydrate moieties of oligosaccharide-P-P-Dolextracted from MadIA214 disrupted cells (A)radiolabelled with GDP-[14C]Man or from MadIA214digitonin-permeabilized cells (B) radiolabelled with 2-[3H]Mannose were analyzed by HPLC.Oligosaccharides were also prepared from theglycoproteins of the MadIA214 mutant cellsradioactively labelled in the presence of glucosidaseinhibitor (deoxynojirimycin) (C) or in the presence ofBFA and glucosidase inhibitor (D) and analyzed byHPLC. The same abbreviations as in Fig. 9 are used.

indicates that N-glycan synthesis is as efficient in the mutantas in its parent. Furthermore, thin layer chromatography andchemical characterization demonstrated that comparableamounts of Man-P-Dol were produced in both lines (data notshown). Therefore MadIA214 is neither defective in dolicholsynthesis nor in Man-P-Dol synthase activity as described forthe CHO glycosylation mutants of the Lec9 (Rosenwald andKrag, 1990) and Lec15 (Chapman et al., 1980; Stoll et al.,1982) complementation groups, respectively. PIR mutantsbelonging to a third complementation group (Lec35), in whichMan5 to Man9 elongation steps were also inhibited, restorednormal N-glycosylation when oligosaccharide synthesis wasanalyzed in disrupted cells (Zeng and Lehrman, 1990). Wetested whether reconstitution was possible in MadIA214 cellsdisrupted by Dounce homogenization then labelled with GDP-[14C]Man in the presence of castanospermine. HPLC analysisof lipid associated oligosaccharides showed that Man5 to Man9elongation occurs in the MadIA214 homogenate (Fig. 9A).

Thus, all compounds required for Man5 to Man9 elongationare present and potentially functional in MadIA214 but do notgenerate the expected high mannose structure in intact cells.The glycosylation block might be due to the action of a putativesoluble inhibitor which is washed off from the microsomalpreparation. Alternatively, all components may not be accessi-ble at the site of mannose elongation and cell homogenizationcould allow normal N-glycosylation as a result of reorientationor redistribution of some compounds. To test these two possi-bilities, we used digitonin permeabilized cells washed of theircytosol. The permeability of Cl42 and MadIA214 plasmamembranes was assessed by trypan blue uptake and themembrane integrity of the ER by measurement of the latencyof SeAP release from the ER to the medium. The latency wasof 95% indicating that no more than 5% of the ER wasdisrupted (data not shown). Permeabilized and washedMadIA214 cells did not reconstitute synthesis of a complete

M5

G3M9

M5

G3M9

B

M2

M5

G1M5G3M5

G1M9G3M9

M4

M5

G1M5 DM3

M4

M5

G1M5

AAAAAAAAtime (min)


Fig. 10. Western blot analysis of the 78 kDa protein coprecipitated inSeAP immunoprecipitates. SeAP was immunoprecipitated from thepost-nuclear supernatant prepared from the same number of CHO(1), Cl42 (2) or MadIA214 (3) cells cultured at 40°C and eithertreated (+) or not (−) with ATP. Total or immunoprecipitated materialwas separated by 8% SDS-PAGE and analyzed by western blottingusing rabbit anti-SeAP (A) or anti-BiP (B) Abs and ECL procedurefor immunodetection.

Man9 core as demonstrated by HPLC analysis (Fig. 9B).Indeed, 95% of the mannose derivatives consisted of Man5species and only 3% corresponded to Man9 species, a levelaccounted for by the 5% ER leakage. Under the same con-ditions a major peak of Glc3Man9GlcNAc2 was identified inthe parent (data not shown). Thus, the MadIA214 glycosyla-tion block is most likely not due to the action of a cytosolicinhibitor and depends on the integrity of ER membranes.

It is known that intracellular transport is blocked in perme-abilized and washed cells because this procedure removescytosolic compounds needed for vesicular budding. If theMadIA214 phenotype was due to the lack of a compoundabberantly leaving the ER, a block in ER-to-Golgi transportshould have restored mannose elongation. However, the blockat Man5 was maintained in semi-intact cells, or when intracel-lular transport was interrupted by BFA treatment (Fig. 9D).This drug not only blocks vesicular transport in intact cells butalso leads to recycling of Golgi constituents back to the ER.

In conclusion, the block at Man5 is abolished in MadIA214microsomes while it is maintained in semi-intact cells as wellas under different experimental conditions that disturb intra-cellular trafficking. These results favour an abnormal structuralorganization of the N-glycosylation pathway in the mutant.

Immature forms of SeAP accumulating in MadIA214are bound to the ER chaperone BiP As shown in Fig. 5, the reduced SeAP secretion in the super-natant of MadIA214 and the corresponding accumulation ofimmature SeAP, was accompanied by an increase in the copre-cipitation of a 78 kDa protein. A weak band of the same sizewas only visible in Cl42 cells when cultured at 40°C. This 78kDa protein was insensitive to endoH treatment and was notsialylated (Fig. 6). Consistently, tunicamycin and PNGasetreatments revealed that this protein was not N-glycosylated(data not shown). This is characteristic of a protein with thesame molecular mass, BiP, an ER resident hsp70 chaperone.Western blot analysis of SeAP immunoprecipitates revealedeither with an anti-SeAP (Fig. 10A) or an anti-BiP (Fig. 10B)serum, shows that the 78 kDa protein reacted with the BiPspecific antiserum (Fig. 10B). BiP was present in comparableamounts in CHO, Cl42 and MAdIA214 total extracts but wasdetected only in MadIA214 SeAP immunoprecipitates. Asexpected for a specific BiP/ligand complex, this interaction wasreleased upon ATP treatment. This points to the fact that SeAPintermediates with altered carbohydrate moiety are bound tothe ER-chaperone BiP.

Degradation in MadIA214 is increased Incompletely folded proteins retained in the ER via BiP inter-action have been described to undergo ER degradation(Knittler et al., 1995). In order to determine whether this alsoapplies for abnormal glycoproteins produced by MadIA214,we analyzed the neutral soluble oligosaccharides as possiblemarkers of intracellular degradation. As previously demon-strated (Cacan et al., 1992), soluble oligosaccharides obtainedafter a 2-[3H]Man pulse consist of oligomannosides with 2GlcNAc residues at the reducing end, arising from oligosac-charides-P-P-Dol hydrolysis by an oligosaccharidyl transferasewhen water is used as acceptor substrate. After a long chaseperiod, oligomannosides with only one GlcNAc residue at thereducing end begin to appear and gradually accumulate as a

result of glycoprotein degradation (Villers et al., 1994). Anenzyme with a chitobiase cytosolic activity was shown to beimplicated in the generation of oligomannoside with only oneGlcNAc residue (Kmiécik et al., 1995). A comparison ofneutral soluble oligosaccharides generated in the parental andmutant clones is shown in Fig. 8C,F. Only oligomannosideswith 2 GlcNAc residues were observed in Cl42 while inMadIA214, oligomannosides with one GlcNAc residue at thereducing end were also formed in addition to the GlcNAc2species.

These results reveal that the degradation rate of glycopro-teins is enhanced in MadIA214.

MadIA214 morphology is different from Cl42Compared to the parental clone (Fig. 11A), the most distinc-tive feature of MadIA214 morphology is a remarkable dilationof the RER both at 33°C and 40°C as seen in electron micro-graphs (Fig. 11B,C). Particularly at the non-permissive tem-perature distended ER cisternae are most abundant and exhibita striking accumulation of finely granular and flocculent pre-cipitates of stored proteins (Fig. 11C). Near the nucleus as wellas at the cell periphery the branching and anastomosingcisternae tend to be stacked in parallel arrays and occupy mostof the cytoplasm, whereas adjacent to the Golgi as well as tothe microtubule organizing center the RER forms a meander-ing tubular network displaying a conspicuous variation in thediameter of individual profiles (data not shown).

These observations reveal an ER dilation in MadIA214which is more pronounced at non-permissive temperature.

DISCUSSION

The impact of N-glycosylation on glycoprotein maturation andfunction was often investigated by using modified polypeptidesequences lacking specific glycosylation sites or in mutant cellsselected for defective glycosylation (reviewed by Varki, 1993).In this study, we present a novel cellular model that permits usto compare in vivo the export requirements of two differentprotein types, a secreted monomeric glycoprotein (SeAP) anda plasma membrane heterotrimeric complex (H-2Kd). We have


Fig. 11. Ultrastructural analysis of parental andmutant cells. Compared to the parental CHO-derived Cl42 cell line (A, 33°C) a prominentand characteristic finding in the mutantMadIA214 (B, 33°C and C, 40°C) was thedilation of the RER which was mostpronounced at 40°C. The dilated cisternaecontain conspicuous amounts of a finelygranular material. Bar (A-C): 500 nm.

isolated a clone, MadIA214, exhibiting a severe ts defect inSeAP secretion and identified in this cell line a constitutivealteration of an early step of the N-glycosylation pathway: atruncated Man5GlcNAc2 (instead of Glc3Man9GlcNAc2) coreunit is transferred onto the Asn-acceptor sites of newly syn-thesized proteins.

One of the most interesting features of the MadIA214 cloneis that the two glycoproteins used as export markers appear tobe differentially affected by the Man5 block. At 33°C as wellas at 40°C, the altered glycosylation impaired neither H-2Kd

assembly and export to the cell surface nor the ability of H-2Kd to present antigen, although these specific functions wereslightly decreased in efficiency. The SeAP activity was alsomaintained. However, SeAP maturation which occurrednormally at 33°C was considerably affected at non-permissivetemperature where the SeAP secretion was almost unde-tectable. Interestingly, the transfer of a truncated sugar wasshown to interfere with the surface expression of the influenzaviral hemaglutinin in another ts-secretory mutant (Hearing etal., 1989).

The major intracellular SeAP form detected in the Cl42parental cell lysate at steady state after biosynthetic labellingor in western blot (data not shown) is an immature endoHsensitive form, i2, produced as shown by pulse chase experi-

ments, after trimming of the early immature endoH sensitivei1 form synthesized in the ER. This i2 intermediate is furtherprocessed into a mature endoH resistant form which is neu-raminidase sensitive and detected only in the cell supernatant.In the MadIA214 mutant, each of these species (i1, i2 andmature) appears as a mixture of two glycoprotein forms con-taining either one or two truncated oligomannosides, all ofthem being endoH resistant.

Our data support a model in which the striking tempera-ture-dependency of SeAP secretion points to the truncated N-glycan moiety affecting glycoprotein folding in the ER, par-ticularly of the i2 intermediate: (i) in the mutant, SeAPimmature forms are associated with the ER chaperone BiPknown to assist folding and to retain malfolded proteinsinside the ER until the correct conformation is achieved(Hammond and Helenius, 1994a). The association of the i2intermediate with the ER marker BiP supports the hypothe-sis that the trimmed i2 form is a folding intermediateproduced in the Golgi apparatus and recycling between theER, the intermediate compartment and the cis-Golgi cisternaeas described for a folding intermediate of the G protein oftsO45 vesicular stomatitis virus (Hammond and Helenius,1994a). The involvement of cis-Golgi enzymes is indeedindicated by the fact that Man4GlcNAc2, the major oligosac-


charide structure associated with MadIA214 proteins, shouldderive from the cleavage of Man5GlcNAc2 or ofGlc1Man5GlcNAc2 by the cis-Golgi mannosidase I or the cis-Golgi endomannosidase (Lubas and Spiro, 1987), respec-tively; and (ii) in Cl42, BiP is also found in association withSeAP but at the elevated temperature only. These observa-tions strongly suggest a transient interaction of BiP withSeAP which is prolonged under unfavourable folding con-ditions such as a temperature shift or the addition of atruncated oligosaccharide as it is the case in MadIA214.Prolonged BiP-interaction has previously been described forimmunoglobulin light chains (Knittler and Haas, 1992) thatremain incompletely folded when oligomeric assembly isprohibited (Leitzgen et al., 1997). (iii) Altered SeAP foldingmay also account for the finding that, in the mutant, approx-imately half of the newly synthesized SeAP molecules usedonly one of two potential N-glycosylation sites. The firstaddition of a truncated N-glycan may well render the secondsite in SeAP less accessible to the action of the oligosaccha-ryl transferase. A structural control of protein glycosylationis documented by the finding that under conditions prevent-ing disulfide bond formation, the tissue plasminogen activatorexhibited complete glycosylation of an Asn-acceptor site thatis variably glycosylated in untreated cells (Allen et al., 1995).These findings demonstrate that the initial N-glycosylationstep can interfere with very early cotranslational foldingevents. This can lead to the retention of some folding inter-mediates by interaction with ER chaperones and couldaccount for the cytological modification observed inMadIA214 (see below).

Still, this situation does not apply to all proteins synthesizedby the mutant cells, as seen with the MHC class I molecules.The limited effect of an altered glycosylation on H-2Kd prop-erties is consistent with reports demonstrating that MHC classI molecules are transported to the cell surface when glucosetrimming is blocked (Ballow et al., 1995) and even when N-glycosylation is prohibited. In the latter case, however, thesurface molecules were no longer functional in antigen pre-sentation (Rubocki et al., 1991).

How could the altered N-glycosylation in MadIA214account for the differential effect on glycoprotein maturation?Protein folding in the ER seems to imply sequential interac-tions with different chaperones (Hammond and Helenius,1994b; Kim and Arvan, 1995). The ordered transfer of foldingintermediates to ER chaperones might thus determine thetransport competence of a glycoprotein in MadIA214. On theone hand incorrectly folded proteins retained in the ER by theirassociation with chaperones could undergo degradation (Boni-facino and Lippincott-Schwartz, 1991; Knittler et al., 1995);neutral soluble oligosaccharide analysis indicates that glyco-protein degradation is indeed enhanced in MadIA214. On theother hand, prolonged ER retention may also allow improperlyfolded glycoproteins to reach a secretion competent state by analternative pathway. It has recently been demonstrated that theER resident chaperone calnexin assists folding and oligomer-ization of some glycoproteins through interactions involvingmonoglucosylated oligosaccharide side chains (Hammond etal., 1994; Ware et al., 1995). In this context, it is worth men-tioning that UDP-Glc:glycoprotein glucosyltransferase gluco-sylates misfolded but not native glycoproteins. This glucosyl-transferase catalyzes the addition of one glucose residue to

protein-linked oligomannosides then producing Glc1Man7-9side chains and has been proposed to act in concert withcalnexin in ER quality control (Sousa and Parodi, 1995). TheGlc1Man5 structure linked to MadIA214 proteins could reflecta low affinity of the glucosyl transferases I and II for Man5,these enzymes carrying out the three glucose addition on theoligomannoside-P-P-Dol core. This is indeed suggested by thefact that in conditions where a complete Glc3Man9 core is syn-thezised like in Cl42 (Fig. 8) or in other wild-type CHO cells(Cacan et al., 1992), a non glucosylated Man5 intermediate isfound in the dolichol fraction. However, Man5 glucosylationby these two transferases is possible as demonstrated in anothermutant, B3F7, blocked at Man5 as a result of a defect in Dol-P-Man synthase, but transferring Glc3Man5 sugar moietiesonto proteins (Cacan et al., 1992). These observations are con-sistent with the hypothesis that the Glc1Man5 species associ-ated with MadIA214 proteins but not found in dolicholfractions originate from the reglucosylation of immature gly-coproteins by the UDP-Glc:glucosyltransferase. Glc1Man5binds poorly to calnexin in vitro (Ware et al., 1995). We arethus investigating whether calnexin could accept Man5 deriv-atives in intact cells. If so, it will be of interest to study theassociation of this chaperone with class I in MadIA214 sinceclass I assembly in the ER has been shown to depend oncalnexin interaction.

Like SeAP, many proteins in MadIA214 are presumablyretained in the ER by association with chaperones and arethereby brought into line for degradation or secretion. Theobserved dilation of the ER at elevated temperature could be amorphological manifestation of the increased retention andaccumulation of newly synthesized glycoproteins. Interestingly,the phenotype of MadIA214 is reminiscent of a human heredi-tary glycosylation disease (carbohydrate deficient glycosylationsyndrome, CDGS) of still unknown origin. Morphologicalstudies revealed a fibroblast RER dilation in CDGS (Marquardtet al., 1995). The serum transferrin of CDGS patients exhibitssialylated sugar moieties but, like for MadIA214 SeAP, not allof the N-glycosylation sites usually utilized are occupied(Yamashita et al., 1993). Moreover, an alteration in oligosac-charide-P-P-Dol synthesis has recently been described infibroblasts of some CDGS patients (Krasnewich et al., 1995).More generally, several human diseases have been shown toresult from a defect in protein secretion (Amara et al., 1992)and evidence accumulates that alterations in protein foldingcould be the basis of some of these diseases (Thomas et al.,1995).

Another part of the study presented here attempted to definethe molecular basis of the altered N-glycosylation inMadIA214. A number of glysosylation mutants blocked at aMan5 step of oligosaccharide elongation have been assigned tothree different complementation groups (Beck et al., 1990).Our results excluded the MadIA214 mutant from being amember of the Lec9 (isoprenol reductase deficient) or of theLec15 (Dol-P-Man synthase deficient) groups while the abilityof disrupted cells to reconstitute a normal Glc3Man9GlcNAc2oligosaccharide core is a characteristic shared with PIR, a gly-cosylation mutant defining the Lec35 complementation group(Zeng and Lehrman, 1990). Therefore, all the enzymesnecessary to build up the N-glycan structure are present andpotentially functional in MadIA214 as they are in PIR mutant.However, MadIA214 exhibits differences with PIR and in par-


ticular: (i) the block at Man5GlcNAc2 is not leaky inMadIA214; (ii) MadIA214 proteins contain monoglucosylatedN-glycan (Glc1Man5GlcNAc2) moieties whereas the truncatedsugar precursors associated with PIR proteins do not containglucose (Lehrman and Zeng, 1989). Complementation groupanalysis will be necessary to solve the question of a geneticlink between MadIA214 and PIR.

Several hypotheses have been proposed to account for thePIR defect (Zeng and Lehrman, 1990) and the recent cloningof a suppressor for the two different Lec15 and Lec35mutations shows that the mannosylation process could be evenmore complex than expected (Ware and Lehrman, 1996). Addi-tional experiments were performed with MadIA214 to furtherdetermine the conditions required to reconstitute a normal N-glycan synthesis in these cells and distinguish between: (i) theaction of a cytosolic inhibitor of the N-glycosylationmachinery; or (ii) the inaccessibility of enzymes or substratesnecessary for high mannose synthesis. Digitonin-permeabi-lized mutant cells in which cytosol has been washed off did notrestore normal N-glycosylation, arguing against the existenceof a cytosolic inhibitor. Preliminary reconstitution experimentsin which cytosol of MadIA214 was added to digitonin perme-abilized Cl42 are consistent with this conclusion (data notshown). Thus, only membrane disorganization (cellular dis-ruption) restored a complete N-glycosylation. BFA treatmentof MadIA214 which inhibits vesicular ER-to-Golgi transportand redistributes Golgi compounds back to the ER (Doms etal., 1989), did not release the block at Man5. This indicatedthat the defect is not due to a mislocalization of an intermedi-ate essential for Man9 synthesis along the secretory pathwayand pointed to a misorientation of intermediates outside theER. However, the N-glycosylation machinery may require adefined spatial organization and we cannot exclude a mislo-calization of one component to a specific ER subcompartment(Sitia and Meldolesi, 1992), insensitive to BFA redistribition.Thus, the most attractive hypotheses to account for theMadIA214 phenotype are either a misorientation of the Man-P-Dol caused by a deficiency in a postulated ‘flipping’ activity(Hirschberg and Snider, 1987), or the mislocalization in adistinct ER subcompartment of a compound responsible forMan5 elongation. Identification and structural analysis of themutation target in MadIA214 should help in understanding thespatial organization of the N-glycan biosynthesis at the ERmembrane.

In conclusion, our cellular model allowed us to characterizean N-glycosylation defect differentially affecting glycoproteinmaturation. Investigation of specific chaperone interactions inMadIA214 should provide more information on the role of gly-cosylation in the mechanism of ER quality control and mightalso add to the understanding of the molecular basis of foldingpathology. In addition, the well characterized properties of thiscellular system offer the possibility to identify novel compo-nents or factors that are important in processes of retention ordegradation of incorrectly folded proteins.

The authors thank J.-P. Abastado, F. Lemonnier and H. Gourmierfor helpful discussions and for gifts of antibodies, plasmids and celllines. We express our thanks to F. Wieland for a critical review of themanuscript. We are also indebted to W. Houssin for careful readingof the manuscript. Part of this work was supported by the DeutscheForschungsgemeinschaft through SFB352, and the University Pierreet Marie Curie.

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(Received 11 September 1996 - Accepted 25 November 1996)

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Differential fate of glycoproteins carrying a ... · proteins occurs at the level of the cis-Golgi network, also referred to as the intermediate compartment. Properly folded proteins