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Blood, 1 April 2006, Vol. 107, No. 7, pp. 2713-2719. Prepublished online as a Blood First Edition Paper on November 22, 2005; DOI 10.1182/blood-2005-07-2990. This Article Abstract Full Text (PDF) All Versions of this Article: 2005-07-2990v1 107/7/2713    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Mitchell, W. B. Articles by Coller, B. S. Search for Related Content PubMed PubMed Citation Articles by Mitchell, W. B. Articles by Coller, B. S. Related Collections Gene Expression Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY IIb3 biogenesis is controlled by engagement of IIb in the calnexin cycle via the N15-linked glycan W. Beau Mitchell, JiHong Li, Deborah L. French, and Barry S. Coller From Mount Sinai School of Medicine; and the Laboratory for Blood and Vascular Biology, Rockefeller University, New York, NY.    Abstract Top Abstract Introduction Materials and methods Results Discussion References   Although much is known about IIb3 structure and function, relatively little is understood about its biogenesis. Thus, we studied the kinetics of pro-IIb production and degradation, focusing on whether proteasomal degradation or the calnexin cycle participates in these processes. In pulse-chase analyses, the time to half-disappearance of pro-IIb (t1/2) was the same in (1) HEK293 cells transfected with (a) IIb plus 3, (b) IIb alone, (c) mutant V298FIIb plus 3, or (d) I374TIIb plus 3; and (2) murine wild-type and 3-null megakaryocytes. Inhibition of the proteasome prolonged the t1/2 values in both HEK293 cells and murine megakaryocytes. Calnexin coprecipitated with IIb from HEK293 cells transfected with IIb alone, IIb plus 3, and V298FIIb plus 3. For proteins in the calnexin cycle, removal of the terminal mannose residue of the middle branch of the core N-linked glycan results in degradation. Inhibition of the enzyme that removes this mannose residue prevented pro-IIb degradation in 3-null murine megakaryocytes. IIb contains a conserved glycosylation consensus sequence at N15, and an N15Q mutation prevented pro-IIb maturation, complex formation, and degradation. Our findings suggest that pro-IIb engages the calnexin cycle via the N15 glycan and that failure of pro-IIb to complex normally with 3 results in proteasomal degradation.    Introduction Top Abstract Introduction Materials and methods Results Discussion References   The platelet IIb3 complex is a member of the integrin family of receptors, each of which is composed of an and a subunit derived from separate genes. IIb3 is important in platelet function, and both qualitative and quantitative disorders of IIb3 result in the bleeding disorder Glanzmann thrombasthenia.1,2 The biogenesis of IIb3 in megakaryocytes is complex. Studies of human erythroleukemia (HEL) cells, megakaryocyte-lineage cells derived from peripheral blood progenitor cells of patients with chronic myelogenous leukemia (CML), and CHO and HEK293 cell lines transfected with IIb and 3 cDNAs have provided the following model. In the endoplasmic reticulum (ER), asparagine-linked (N-linked) glycans are attached to both the pro-IIb and 3 subunits, the glycans undergo carbohydrate modifications, and the subunits complex with one another, although the precise sequence of these events is not established; the complexes are then transported to the Golgi for further carbohydrate modification and cleavage of the pro-IIb (relative molecular mass [Mr] 140 000) into its mature 2-chain form (Mr 120 000 + 20 000); the mature receptor is then transported to -granule membranes and the plasma membrane of developing platelets.3-7 A large number of naturally occurring and site-directed missense mutations in IIb or 3 result in markedly decreased IIb3 surface expression, attesting to the presence of stringent quality control systems.8,9 The ER-resident chaperone calnexin has recently been demonstrated to play an important role in the folding of a number of glycoproteins, controlling their partitioning to either the Golgi or to a degradation pathway via a series of carbohydrate modifications referred to as the calnexin cycle.10 In addition, proteasomal degradation of newly synthesized proteins after retrotranslocation from ER to cytoplasm has been demonstrated to limit the length of time proteins remain in the ER.11,12 To assess whether the calnexin cycle or proteasomal degradation contributes to IIb3 biogenesis, we have performed pulse-chase and steady-state analyses in cells transfected with 3 in combination with normal IIb or IIb subunits containing mutations, some of which are associated with Glanzmann thrombasthenia. In addition, to overcome the limitations associated with studying recombinant or naturally expressed proteins in cell lines, we studied IIb3 biogenesis in megakaryocytes derived from the bone marrow of wild-type (WT) and 3-null mice. We have focused our studies on the following questions: (1) Is pro-IIb processing in the ER controlled by proteins of the calnexin cycle? (2) Does the proteasome participate in the degradation of pro-IIb? (3) Is the amount of pro-IIb a limiting factor in IIb3 complex formation? (4) And, finally, are there differences in the kinetics of pro-IIb production and degradation between normal and mutant IIb subunits, or between HEK293 cells and murine megakaryocytes?    Materials and methods Top Abstract Introduction Materials and methods Results Discussion References   Antibodies The antibodies used in this study were monoclonal antibodies (mAbs) B1B513,14 (IIb-specific; kindly provided by Dr Mortimer Poncz, University of Pennsylvania, Philadelphia, PA), M-148 and H-160 (IIb-specific; both from Santa Cruz Biotechnology, Santa Cruz, CA), 10E515 (IIb3-specific), PMI-116,17 (II3-specific; kindly provided by Dr Mark Ginsberg, Scripps Research Institute, La Jolla, CA), CD41 clone MWreg30 (anti–mouse IIb-specific; PharMingen, San Diego, CA), AP318 (3-specific; kindly provided by Dr Peter Newman, The Blood Center of Southeastern Wisconsin, Milwaukee, WI), 1B519 (anti–mouse IIb3-specific), AF820 (murine anticalnexin; kindly provided by Dr Michael Brenner, Harvard Medical School, Boston, MA), SPA-865 (rabbit anticalnexin; Stressgen, Victoria, BC, Canada), FITC-labeled goat anti–mouse immunoglobulin (IgG) F(ab')2 (Jackson Research Labs, Bar Harbor, ME), TRITC-labeled goat anti–rabbit IgG (Sigma-Aldrich, St Louis, MO), and horseradish peroxidase (HRP)–labeled goat anti–mouse light chain and HRP-labeled goat anti–rabbit IgG (both from Southern Biotechnology Associates, Birmingham, AL). Generation of cDNA constructs Human 3 and IIb cDNAs were generously provided by Dr Peter Newman; these were expressed in either pcDNA3.0 or pcDNA3.1 (both from Invitrogen, Carlsbad, CA).An IIb-N15Q construct was generated from pcDNA3.0/IIb by using the splice by overlap extension polymerase chain reaction (PCR) method, as previously described.21 The generation of pcDNA3.0/IIb-V298F and pcDNA3.0/IIb-I374T was described previously.21 Murine megakaryocyte culture Megakaryocytes were generated from the bone marrow of WT and 3-null mice. Bone marrow was flushed from femurs and tibiae with Iscove Dulbecco modified Eagle medium (DMEM) containing 2% fetal bovine serum (FBS), and mononuclear cells were separated on a Ficoll-Hypaque gradient by centrifugation at 450g for 30 minutes at room temperature (RT). The mononuclear cells were plated at 1 x 106 cells/mL in Iscove DMEM containing 30% serum-free medium (StemCell Technologies, Vancouver, BC, Canada), 1% bovine serum albumin, 10–4 M 2-mercaptoethanol, 10 ng/mL murine thrombopoietin (kindly provided by Kirin Brewery, Kirin, Japan), as well as 10 ng/mL IL-6 and 10 ng/mL IL-11 (both from R&D Systems, Minneapolis, MN). Cells were grown in 6-well plates and harvested for analysis on days 6 to 8. Immunoprecipitation and immunoblot analyses Samples were prepared as previously described.21 Briefly, at 48 hours after transfection, cells were lysed in 1% Triton X-100 buffer containing 20 mM N-methylmaleimide (NEM). Lysates were precleared with protein-G Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ) and equivalent amounts of protein were incubated with the anti-IIb mAbs B1B5 and M-148 overnight at 4°C. Protein-G Sepharose beads were added to the samples, incubated for 1 hour at 4°C, washed, and incubated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer at 100°C for 10 minutes. Samples were subjected to SDS-PAGE, transferred onto PVDF membranes, and immunoblotted with the indicated mAbs. Secondary labeling was performed with an appropriate HRP-conjugated secondary antibody, and membranes were developed using the enhanced chemiluminescence (ECL) method (Amersham Pharmacia Biotech), and exposed to film (Blue Sensitive, Denville Scientific, Metuchen, NJ). Loading controls were whole cell lysates directly subjected to SDS-PAGE and immunoblotted with the antibodies of interest. Nonspecific binding was controlled for by performing immunoprecipitation on whole cell lysates of nontransfected and mock transfected (empty vector only) HEK293 cells. Biosynthetic labeling and immunoprecipitation Samples were prepared as previously described.21 Briefly, cells were incubated for 30 minutes at 37°C in methionine/cysteine-free media, followed by incubation for 15 minutes at 37°C in medium containing 35S-methionine/cysteine (300 µCi[11.1 MBq]/10-cm plate). The pulse was terminated by incubation in media containing unlabeled methionine/cysteine (1 mg/mL each). Following cell lysis, supernatants were precleared with protein-G Sepharose beads, and samples containing equivalent amounts of trichloroacetic acid–precipitable radioactivity ( 5-6 x 106 counts/sample) were immunoprecipitated using one or more of the antibodies listed (4 µg/reaction; see "Antibodies"). Samples were incubated with protein-G Sepharose beads for 1 hour at 4°C, washed twice, and incubated with SDS sample buffer for 10 minutes at 100°C. Samples were then subjected to SDS-PAGE and the gels were dried and exposed to film. Loading was controlled for by either running a simultaneous immunoblot using equivalent amounts of protein from each sample, or by stripping the membrane and reprobing for IIb to confirm that equivalent amounts of IIb protein were immunoprecipitated and loaded on the gels. Metabolic analysis of IIb3 biosynthesis in the presence of chemical inhibitors In some experiments, pulse-chase analyses were performed in the presence of the proteasome inhibitors MG132 or PSI (both 10 µM; both from Sigma-Aldrich), or the diluent used to dissolve the inhibitors (DMSO). In other experiments, pulse-chase analysis was performed in the presence of deoxynojirimycin (DNJ), which inhibits ER glucosidases I and II; 1, deoxy-mannojirimycin (DMJ), which inhibits ER mannosidases I and II; 1,4-dideoxy-1,4-imino-D-mannitol (DIM), which inhibits ER mannosidase II (all from Sigma-Aldrich); kifunensine (KIF; Biomol Research Laboratories, Plymouth Meeting, PA), which inhibits ER mannosidase I (all at 1 mM), or the vehicle control DMSO. Cells were preincubated at 37°C with the inhibitor for 30 to 60 minutes before the metabolic pulse, and the inhibitor was present in all subsequent media and washes.    Results Top Abstract Introduction Materials and methods Results Discussion References   Normal and mutant pro-IIb subunits exhibit similar dynamics of production and disappearance in transfected HEK293 cells In HEK293 cells transfected with normal IIb and 3, pro-IIb was observed in immunoprecipitates obtained immediately after the pulse was initiated (0 hour; Figure 1A); peak pro-IIb intensity was observed at 2 hours, and then the amount of pro-IIb decreased with a half-life (t1/2) of 5 plus or minus 2 hours (mean ± SD; n = 5), consistent with previous reports in the literature.4-6,21 The band corresponding to mature IIb heavy chain was first observed at 2 hours and it increased in intensity over 24 hours, concomitant with the loss of pro-IIb. Trace amounts of 3 were immunoprecipitated by the antibodies to IIb at 0 hour, suggesting the presence of a small amount of pro-IIb3 complex; the intensity of the 3 band precipitated by antibodies to IIb increased over time, indicating ongoing production of pro-IIb3 or mature IIb3 complexes. Similarly, immunoprecipitation studies using antibodies to 3 demonstrated coprecipitation of mature IIb at 2 hours, consistent with the mature IIb being complexed with 3 (data not shown). In the same assays, immunoprecipitation with the IIb3 complex-specific mAb 10E5, other mAbs to IIb or 3, or with an isotype control antibody revealed no nonspecific bands (data not shown). When IIb was transfected alone, the pro-IIb disappeared with a t1/2 of 6 plus or minus 1 hours (n = 3), and there was no detectable mature IIb (Figure 1B). Pulse-chase analysis of cells transfected with 3 in combination with IIb containing either of 2 mutations, V298F (VF) or I374T (IT), which cause Glanzmann thrombasthenia as a result of decreased IIb3 surface expression,21 demonstrated that neither of the pro-IIb subunits was processed to mature IIb, and that the disappearance rates of both mutant pro-IIb subunits from their times of maximum intensity were essentially the same as that of the pro-IIb expressed in cells transfected with normal IIb and 3, 6 plus or minus 3 hours and 7 plus or minus 1 hours, respectively (n = 3) (Figure 1C-D). The IIb-specific immunoprecipitates of these cells contained trace amounts of 3, indicating that a small percentage of mutant pro-IIb was in complex with 3. However, the intensity of the 3 bands did not increase over time. View larger version (22K): [in this window] [in a new window]   Figure 1.. Pro-IIb containing either of 2 Glanzmann thrombasthenia mutations is degraded at the same rate as normal pro-IIb. Cells were transiently transfected with cDNA constructs expressing both normal IIb and 3 (Nl) (A), IIb alone (B), or the IIb mutants V298F (VF; C) or I374T (IT; D) in combination with 3. At 36 hours, cells were metabolically labeled with 35S-methionine/cysteine and then harvested at the indicated time points. Radiolabeled protein was immunoprecipitated using a combination of the IIb-specific mAbs, B1B5 and M-148; the samples were then subjected to SDS-PAGE under reducing conditions, and dried gels were exposed to film. Arrowheads indicate bands representing pro-IIb, mature IIb, and 3; the migration of molecular weight standards is shown on the right. (E) Band intensities representing each subunit were measured by densitometry and plotted as a percentage of maximum band density of pro-IIb (at 2 hours for Nl and at 0 hours for VF and IT). The times to half-maximum appearance of mature IIb and 3 were both 2 plus or minus 1 hours (n = 5).   Both normal and mutant pro-IIb subunits are degraded by the proteasome To assess whether pro-IIb subunits are degraded by a proteasomal mechanism, we analyzed the effects of 2 proteasome inhibitors, MG132 and PSI, on IIb disappearance in HEK293 cells transfected with IIb alone, IIb and 3, and VFIIb and 3. In cells transfected with IIb alone (Figure 2), the t1/2 of pro-IIb in the presence of the vehicle (DMSO) was 5 ± 3 hours (n = 6), which is similar to the t1/2 observed in cells without DMSO (Figure 1). In sharp contrast, in the presence of either proteasome inhibitor, there was virtually no proteolysis of the pro-IIb during the first 9 hours, and the calculated t1/2 values were markedly prolonged (MG132 median t1/2 85 hours, data not normally distributed, signed ranked test used [P = .03], PSI t1/2 14 ± 5 hours [P = .02]). In cells transfected with both IIb and 3, the formation of mature IIb and the IIb3 complex proceeded normally in the presence of either DMSO or MG132, but the t1/2 of pro-IIb was prolonged in the presence of the proteasome inhibitor, from 5 ± 2 hours to 11 ± 2 hours (n = 3; P = .03; Figure 3A-B). Despite the prolongation of the pro-IIb t1/2 by MG132, there was no detectable increase in the formation of IIb3 complexes. In cells expressing VFIIb and 3, the t1/2 of pro-VFIIb was also prolonged from 5 ± 3 hours to 14 ± 10 hours (n = 5; P = .05) by MG132 (Figure 3C). Despite the prolonged survival of the mutant pro-IIb subunit, there was no detectable increase in either pro-VFIIb3 formation or conversion of pro-VFIIb into mature VFIIb. View larger version (47K): [in this window] [in a new window]   Figure 2.. The half-life of pro-IIb is prolonged in the presence of proteasome inhibitors. (A-C) Pulse-chase analyses were performed as described in Figure 1 on HEK293 cells stably expressing IIb alone in the presence of DMSO (vehicle control; A) or either of the proteasome inhibitors MG132 (B), or PSI (C). (D) The densities of the pro-IIb bands from a representative experiment are plotted as a percentage of the density at 3 hours after the chase.   Pro-IIb is degraded by the proteasome in WT and 3-null murine bone marrow megakaryocytes Because IIb is normally made in megakaryocytes, we assessed the degradation of IIb in megakaryocytes derived from the bone marrow of normal mice and mice we previously characterized with a targeted deletion of 3 (3-null).22 Since in the latter cells' murine IIb has no 3 partner, they recapitulate the effect of transfecting IIb alone into HEK293 cells, but have the advantage of being the native cell of IIb3 biogenesis. In the WT murine megakaryocytes, pro-IIb was observed at maximal intensity in immunoprecipitates obtained 3 hours after the chase, and then the intensity decreased with a t1/2 of 4 ± 2 hours (n = 3) (Figure 4A). 3 was also immunoprecipitated at the 3-hour time point, when there was little mature IIb, indicating the presence of pro-IIb3 complexes. Subsequently, both the 3 band and the mature IIb band increased in intensity, with both reaching maximum density by 7 hours. Incubation with MG132 did not affect either the appearance rate or the total amount of mature IIb3 receptor complex, but the t1/2 of pro-IIb was increased to 9 ± 5 hours (n = 3; P = .2). The IIb from the 3-null megakaryocytes was not processed to mature IIb, and exhibited a t1/2 of 3 ± 2 hours (n = 6), comparable to that in the WT megakaryocytes (Figure 4A,C). In the presence of the proteasome inhibitor, the t1/2 was prolonged to 7 ± 2 hours (n = 6; P = .01). Thus, normal pro-IIb is degraded in a proteasome-dependent manner in megakaryocytes from both WT and 3-null animals. View larger version (45K): [in this window] [in a new window]   Figure 3.. The t1/2 values of normal pro-IIb and VFIIb subunits expressed with 3 are prolonged in the presence of a proteasome inhibitor. Pulse-chase analyses were performed as described in Figure 1 on HEK293 cells transiently transfected with 3 and either normal IIb or mutant VFIIb in the presence of either DMSO (A,C), or MG132 (B,D). (E) The density of the pro-IIb bands of one representative experiment are plotted as a percentage of their 3-hour postchase density. The normal pro-IIb and pro-VFIIb subunits disappeared from the cells cultured in DMSO with t1/2 values of 5 ± 2 (n = 3) and 5 ± 3 (n = 5) hours, respectively. Disappearance of both the normal and mutant pro-IIb subunits was delayed in the presence of MG132 to 11 plus or minus 2 hours (n = 3, P = .03), and 14 plus or minus 10 hours (n = 5, P = .05), respectively.   View larger version (44K): [in this window] [in a new window]   Figure 4.. The half-lives of murine pro-IIb are prolonged in the presence of a proteasome inhibitor in both wild-type and 3-null murine megakaryocytes. Megakaryocytes generated from the bone marrow of WT (A-B) and 3-null mice (C-D) were cultured in the presence of DMSO or MG132, and the cells were lysed at the times indicated. Samples were analyzed as described in Figure 1. (E) The densities of the pro-IIb bands from a representative experiment are plotted as a percentage of the density at 0 hours. The disappearance of pro-IIb subunits was delayed in WT megakaryocytes and significantly delayed in 3-null megakaryocytes in the presence of MG132 4 plus or minus 2 hours versus 9 plus or minus 5 hours (n = 3), and 3 plus or minus 2 hours versus 7 plus or minus 2 hours (n = 6; P = .01), respectively. This indicates that pro-IIb is degraded by a proteasomal mechanism in WT and 3-null murine megakaryocytes.   View larger version (52K): [in this window] [in a new window]   Figure 5.. Normal and mutant IIb subunits associate with calnexin. (A) Whole-cell lysates of cells either transiently or stably expressing normal IIb3, VFIIb plus 3, or IIb alone were immunoprecipitated with antibodies against IIb, subjected to SDS-PAGE, and then immunoblotted with mAbs against IIb (PMI-1, top left panel, or H-160, top right panel) or calnexin (AF8, bottom left panel, or SPA-865, bottom right panel). Calnexin was coprecipitated from cells expressing normal IIb3, either transiently or stably, VFIIb plus 3, and cells expressing IIb alone, but not from mock (vector-only)–transfected cells. As a loading control, equivalent amounts of whole-cell lysate from each cell type were subjected to SDS-PAGE and immunoblotted with anticalnexin, revealing that equal amounts of total calnexin had been loaded (data not shown). (B,C) Pulse-chase analysis demonstrated a markedly prolonged t1/2 for pro-N15QIIb (17 ± 2 hours) as compared with normal pro-IIb (5 ± 2 hours, n = 3, P < .001), and failure of pro-N15QIIb to undergo normal processing to mature N15QIIb or to form the N15QIIb-3 complex normally. (D) The top panel shows the sequence alignment of the first 60 amino acids of human IIb with IIb from mouse, rat, pig, horse, rabbit, and dog. The bottom panel shows the sequence alignment of the corresponding residues of human V, 8, 5, 3, 7, and 4. N-linked glycosylation consensus sequences at position 15 are highlighted in yellow, and those at other positions are in cyan. Residues comprising strands are indicated in magenta. (E) Location of N15 in the IIb propeller, and the relative location of N45 in V. Propeller blades are numbered 1 through 7 and the Cap domain is indicated. In the crystal structures of both subunits, residues at position 15 are located at the top outer corner of the first propeller blade, whereas residues at approximately position 45 lie at the apex of the next upward-facing loop of the same blade, thus occupying a position adjacent to residue 15 on the propeller's surface.23,24   Calnexin associates with IIb expressed with and without 3, and with VFIIb expressed with 3 Association between calnexin and IIb was assessed by coimmunoprecipitation analysis on whole cell lysates of HEK293 cells following transient or stable transfection with pro-IIb alone, or 3 in conjunction with normal IIb or mutant VFIIb (Figure 5). In cells transiently transfected with normal IIb plus 3 or VFIIb plus 3, immunoprecipitation with an antibody to IIb also coprecipitated calnexin, which was detected by the anticalnexin antibodies AF8 and SPA-865. In contrast, calnexin was not detectable or only minimally detectable when mock (vector only) transfected HEK293 cell lysates were immunoprecipitated with an antibody to IIb. In general, the amount of calnexin precipitated was in proportion to the amount of pro-IIb expressed. Similarly, calnexin also coprecipitated with IIb from cells stably transfected with normal IIb plus 3, or normal IIb alone, but not mock-transfected cells. Disruption of the IIb N15 glycosylation consensus sequence prevents normal IIb3 biogenesis N-glycosylation within the first 50 amino acids of a nascent protein has been proposed to result in preferential interaction with calnexin and calreticulin over other chaperones.5,26 Cross-species sequence alignment of 7 IIb chains identified a highly conserved N-linked glycosylation sequence at N15 in all of the IIb sequences, but there were no comparable sites at this location in 6 other human chain sequences analyzed (Figure 5D). The N15 glycosylation consensus sequence was disrupted by an NQ mutation (N15QIIb). In cells transfected with N15QIIb and normal 3, there was no maturation of pro-IIb to mature IIb (Figure 5) and no formation of the IIb3 complex. Pulse-chase analysis of cells transfected with N15QIIb and 3 revealed that the t1/2 of the pro-N15QIIb was prolonged compared with normal pro-IIb (17 ± 2 hours versus 5 ± 2 hours; n = 3; P < .001; Figures 5C and 1E). Carbohydrate modification targets IIb for degradation The effects of inhibitors of the glycan modification enzymes of the calnexin cycle on IIb biogenesis and degradation were studied in 3-null megakaryocytes by pulse-chase analysis (Figure 6). Both DMJ, which inhibits ER-mannosidases I and II, and KIF, which inhibits ER-mannosidase I,27 decreased the rate of pro-IIb degradation from 3 plus or minus 2 hours to 25 hours (median of t1/2 values, data not normally distributed, signed ranked test; n = 3; P = .02) and 27 hours (n = 3; P = .05), respectively, suggesting a role for the terminal mannose of the middle branch of the core N-linked glycan in pro-IIb degradation. Similar data were obtained when DMJ and KIF were used in HEK293 cells transfected with normal IIb and 3 (data not shown). In contrast, inhibition of the glucosidases I and II with DNJ,28 had no discernible effect on the rate of pro-IIb degradation in 3-null megakaryocytes (Figure 6). View larger version (21K): [in this window] [in a new window]   Figure 6.. Inhibition of N-linked core oligosaccharide processing impairs degradation of normal pro-IIb in murine megakaryocytes. (A) The N-linked core oligosaccharide that is added to nascent polypeptide chains in the ER contains 14 saccharide units in a branched configuration. Enzymes that are involved in the modification of this glycan are shown, along with inhibitors of these enzymes. represents the mannose that is removed by ER mannosidase I. The checkered diamond represents the single glucose in the oligosaccharide structure that is recognized by calnexin. (B) Pulse-chase analyses were performed as described in Figure 1 on bone marrow megakaryocytes from 3-null mice in the presence or absence of the inhibitors 1, deoxy-mannojirimycin (DMJ), deoxynojirimycin (DNJ), or kifunensine (KIF). Whole cell lysates were immunoprecipitated with a combination of mAbs to IIb (B1B5 and M-148). ER Man I indicates ER mannosidase I; ER Man II, ER mannosidase II; UGGT, UDP-glucose:glycoprotein glucosyltransferase.      Discussion Top Abstract Introduction Materials and methods Results Discussion References   Much is known about the structure and function of IIb3, whereas relatively little is understood about its biogenesis. Jennings and Phillips first reported that GPIIb and GPIIIa (IIb and 3) were present as a calcium-dependent complex on the platelet surface, and they hypothesized that assembly of the complex was a prerequisite for surface expression.29 Bray et al3 studied RNA derived from the megakaryocyte-like HEL cell line in a cell-free system and determined that IIb and 3 were translated from separate mRNAs, and that the heavy and light chains of IIb were derived from a single pro-IIb precursor chain. Duperray et al4 performed pulse-chase metabolic analysis of IIb3 biogenesis in megakaryocytes cultured from cryopreserved progenitor cell concentrates of patients with CML. Their findings established that N-linked glycosylation was associated with conversion of pro-IIb to mature IIb. Using the carbohydrate-processing enzyme endo-H, which trims the unprocessed high-mannose carbohydrate moieties found in the ER, but not the fully processed carbohydrates found in the Golgi, they found that virtually all detectable pro-IIb was sensitive to endo-H cleavage, whereas virtually none of the mature IIb was sensitive to endo-H. The 3 subunit, in contrast, was found to have unprocessed N-linked carbohydrates even when complexed with mature IIb on the cell surface. They also reported that 60% of the 3 was in a free pool, whereas the detectable IIb was present in complex with 3, indicating an excess of 3 subunits. Rosa and McEver also analyzed IIb3 biogenesis in the HEL cell line and reported that degradation contributed significantly to pro-IIb disappearance.5 Contrary to Duperray's findings, however, they reported a 5-fold excess of pro-IIb over 3 in HEL cells, the majority of which was degraded without further processing. O'Toole et al, using cDNA transfection into COS cells, demonstrated that both the IIb and the 3 subunits were required for pro-IIb processing into mature IIb and for surface expression of IIb3.6 A model of strict compartmental sorting emerged from these studies, in which pro-IIb is confined to the ER and mature IIb is confined to the Golgi and post-Golgi compartments. This model is supported by (1) observations that all detectable pro-IIb is endo-H sensitive, whereas all detectable mature IIb is endo-H resistant,4 (2) observations that even though the entire pool of mature IIb3 appears to pass through the pro-IIb3 intermediate during biogenesis, only small amounts of pro-IIb3 are detectable at any time, and (3) the absence of any pool of free mature IIb. Our studies were designed to extend these observations by using transfected HEK293 cells, 2 IIb mutants that cause Glanzmann thrombasthenia as a result of decreased IIb3 surface expression,21 and murine megakaryocytes from WT and 3-null animals. Our major new findings are that uncomplexed IIb, but not 3, is degraded by a proteasomal mechanism, and that both IIb maturation and degradation are controlled by the calnexin cycle, with the glycan at IIbN15Q a key moiety in these processes. We found that pro-IIb is degraded in a proteasome-dependent manner in both transfected HEK293 mammalian cells and in murine megakaryocytes. Moreover, because disappearance of pro-IIb reflects both degradation of uncomplexed pro-IIb and conversion of pro-IIb to mature IIb, the profound effect of proteasome inhibition suggests that the majority of pro-IIb undergoes degradation rather than maturation. Data on the relative effects of proteasome inhibition on the t1/2 values of pro-IIb in cells expressing IIb alone (where no maturation occurs), IIb plus 3 (where maximum maturation occurs), and V298FIIb plus 3 (where intermediate amounts of maturation occur) are consistent with this interpretation. The rate of pro-IIb degradation is not dependent on the presence or absence of 3, which implies that both the degradation and maturation pathways follow first-order kinetics. Similarly, IIb containing either of 2 Glanzmann thrombasthenia mutations, V298FIIb and I374TIIb, that prevent normal IIb3 maturation but do not prevent pro-IIb3 complex formation, do not significantly alter the rate of pro-IIb degradation by the proteasome. Thus, even though dissociation of the IIb3 complex in vitro enhances its susceptibility to proteolytic degradation,29 it does not appear that failure of pro-IIb to complex with 3 results in more rapid proteolytic degradation, and mutations of IIb that prevent IIb3 maturation do not necessarily result in more rapid turnover of the mutant subunits. In the ER, N-linked glycans function as sorting signals within the calnexin cycle, a protein folding and quality control system.10 The core N-linked glycan, Glc3Man9GlcNac2 (Figures 6A and 7) is added to nascent glycoproteins at N-linked glycosylation consensus sequences, N-X-S/T, as they pass through the translocon complex into the ER during biosynthesis. The presence of an N-linked glycan within the first 50 amino acids of a protein is a signal for entry into the calnexin cycle.25,26 Immediately after the core glycan is transferred to the protein, it is trimmed by glucosidases I and II to the monoglucosylated form, Glc1Man9GlcNac2, which binds to calnexin. When the final glucose is trimmed off, leaving Man9GlcNac2, the protein can no longer bind to calnexin, but the glycan is now a potential substrate for UDP-glucose: glycoprotein glucosyltransferase (UGGT). This enzyme binds to partially folded glycoproteins and adds back a single glucose to the core glycan, reverting the glycan to its calnexin-binding state, and thus preventing protein egress from the ER (Figure 7C-D).30,31 Once the glycoprotein has attained its native folding state, however, UGGT cannot bind to it, even if it has the UGGT-recognized glycan structure, and the folded protein exits the calnexin cycle (Figure 7E). The terminal middle-branch mannose residue of the core glycan is necessary for high-affinity binding to both calnexin and UGGT (Figures 6-7).32 Removal of this mannose residue by ER mannosidase I decreases protein binding to calnexin and eliminates protein binding to UGGT. Therefore, glycoproteins in the calnexin cycle that have not successfully folded by the time this mannose residue is removed from the core N-linked glycan also exit the calnexin cycle (Figure 7F). These misfolded proteins, however, bind to the mannosidase-like protein, ER degradation enhancer, mannosidase -like (EDEM), which facilitates retrotranslocation of glycoproteins, leading to degradation by the proteasome.11,12 View larger version (30K): [in this window] [in a new window]   Figure 7.. Model of pro-IIb interactions with the calnexin cycle. (A) The core N-linked glycan, Glc3Man9GlcNAc2, is attached to the Asn at position 15 (N15) of pro-IIb during translocation into the ER.(B) The glucosidases I and II (Gluc I + II) cleave off the glucose moieties, forming Glc1Man9GlcNAc2, which is the glycan form recognized by calnexin (CNX). (C) Pro-IIb remains bound to calnexin until the third glucose moiety is cleaved off by glucosidase II. (D) The glycan formed by removal of the third glucose, Man9GlcNAc2, is a substrate for UDP-glucose:glycoprotein glucosyltransferase (UGGT), which then reattaches a glucose moiety, recreating the Glc1Man9GlcNAc2. Because incompletely folded glycoproteins (shown as squiggle) are substrates for UGGT, partially folded pro-IIb subunits cycle between calnexin and UGGT, which provides additional time to achieve its native fold. There are 2 exits from the cycle: (E) Pro-IIb may achieve its native fold (shown as spiral), at which point it is no longer a substrate for UGGT, and thus continues on the biogenesis pathway to complex formation with 3, or (F) the slowly active mannosidase I (Man I) may cleave the terminal mannose from the middle branch of the glycan, creating the structural signal that targets pro-IIb to the proteasome for degradation via binding to the mannosidase-like protein EDEM and retrotranslocation out of the ER (not shown). Of note is the finding that proteasomal degradation of pro-IIb continues at the normal rate in the presence of glucosidase inhibitors; thus pro-IIb may proceed directly from step A to step F, without interaction with calnexin.   The IIb N15 glycosylation consensus sequence is the most proximal of 5 such sequences in pro-IIb, and the only one within the first 50 amino acids. Because disrupting the N15 glycosylation consensus sequence by the N15Q substitution not only resulted in profound inhibition of IIb3 complex formation, but also led to a marked decrease in the rate of pro-IIb degradation, we conclude that the N15 glycan is important for pro-IIb entering the calnexin cycle, and that the calnexin cycle is important in pro-IIb folding, pro-IIb3 complex formation, and degradation of misfolded pro-IIb via the proteasome. In the crystal structure of IIb,24 N15 lies at the upper, outside corner of blade 1 of the -propeller, exposed to solvent (yellow sphere in Figure 5E). Of note, the glycosylation consensus sequence at N15 is conserved in IIb across species (Figure 5D), whereas some other human subunits (eg, V, 3, 4, 5, 7, 8) have their first N-linked glycosylation sequence at approximately amino acid 45, which lies at the apex of the next upward facing loop of blade 1 and occupies a position adjacent to residue N15 on the propeller's surface (blue sphere in Figure 5E). Further support for the importance of the pro-IIb N15 glycan comes from the finding that the elimination of 2 of the other 4 N-glycosylation consensus sequences in IIb by the mutations N249W (W.B.M., J.L., D.L.F.; unpublished data, December 2001) and N931Q33 have no effect on biogenesis. Additional support for the importance of the calnexin cycle in controlling pro-IIb degradation comes from our finding that inhibition of ER-mannosidase I dramatically reduced the rate of degradation of normal pro-IIb. Thus, removal of the terminal mannose from the middle branch of the core glycan appears to target pro-IIb for degradation. In the same cells, inhibition of mannosidase I had no apparent effect on the rate of disappearance of the 3 subunit (data not shown), which is also glycosylated, thus demonstrating that the effect on pro-IIb was not a nonspecific effect of disrupting glycoprotein processing in the ER, or more global cellular damage. These findings indicate that normal pro-IIb degradation is dependent, in part, on the calnexin cycle. In contrast to the data with inhibitors of mannosidase I, however, inhibition of glucosidases I and II did not affect the rate of degradation of pro-IIb. Although these enzymes are important for processing proteins so that they can enter the calnexin cycle (Figure 7A,B), they are not required for mannosidases to trim mannose residues from the core N-linked glycan.34,35 Hence, even in the absence of glucose trimming, removal of the middle branch terminal mannose residue by mannosidase can still occur, targeting the glycoprotein to proteasomal degradation.36-39 Finally, of particular interest is our finding that prolonging the time that pro-IIb remains in the ER (by decreasing its rate of proteasomal clearance, and thus perhaps increasing its concentration), does not appear to affect either the rate of production or the final amount of mature IIb3 formed. Thus, the limiting factor in IIb3 production appears to be the mechanism controlling the formation of pro-IIb3 complexes suitable for transport to the Golgi for further processing. We suspect that one or more additional chaperones participate in this process.    Acknowledgements   We gratefully acknowledge Rona Weinberg for culturing the murine megakaryocytes.    Footnotes   Submitted July 26, 2005; accepted November 13, 2005. Prepublished online as Blood First Edition Paper, November 22, 2005; DOI 10.1182/blood-2005-07-2990. Supported by grants HL68622 and HL19278 from the National Heart, Lung and Blood Institute; grants from