Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M502160200 on May 10, 2005

J. Biol. Chem., Vol. 280, Issue 27, 25881-25886, July 8, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/27/25881    most recent
M502160200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, B.
Right arrow Articles by Ginsburg, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, B.
Right arrow Articles by Ginsburg, D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

LMAN1 and MCFD2 Form a Cargo Receptor Complex and Interact with Coagulation Factor VIII in the Early Secretory Pathway*

Bin Zhang{ddagger}§, Randal J. Kaufman¶||**, and David Ginsburg{ddagger}||**{ddagger}{ddagger}§§¶¶

From the {ddagger}Life Sciences Institute, the Departments of Biological Chemistry,{ddagger}{ddagger} Internal Medicine,§§ Human Genetics, and the|| Howard Hughes Medical Institute, University of Michigan, Ann Arbor, Michigan 48109

Received for publication, February 25, 2005 , and in revised form, April 25, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutations in LMAN1 (ERGIC-53) and MCFD2 are the causes of a human genetic disorder, combined deficiency of coagulation factor V and factor VIII. LMAN1 is a type 1 transmembrane protein with homology to mannose-binding lectins. MCFD2 is a soluble EF-hand-containing protein that is retained in the endoplasmic reticulum through its interaction with LMAN1. We showed that endogenous LMAN1 and MCFD2 are present primarily in complex with each other with a 1:1 stoichiometry, although MCFD2 is not required for oligomerization of LMAN1. Using a cross-linking-immunoprecipitation assay, we detected a specific interaction of both LMAN1 and MCFD2 with factor VIII, with the B domain as the most likely site of interaction. We also present evidence that this interaction is independent of the glycosylation state of factor VIII but requires native calcium concentration in the endoplasmic reticulum. The interaction of MCFD2 with factor VIII appeared to be independent of LMAN1-MCFD2 complex formation. These results suggest that LMAN1 and MCFD2 form a cargo receptor complex and that the primary sorting signals residing in the B domain direct the binding of factor VIII to LMAN1-MCFD2 through calcium-dependent protein-protein interactions. MCFD2 may function to specifically recruit factor V and factor VIII to sites of transport vesicle budding within the endoplasmic reticulum lumen.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Correctly folded proteins destined for secretion by antero-grade transport toward the Golgi are packaged in the ER1 into COPII-coated vesicles (1). These vesicles then uncoat and fuse with each other to form the ER-Golgi intermediate compartment (ERGIC). Resident proteins recycle from the ERGIC back to the ER in COPI-coated vesicles. Many transmembrane cargo proteins, such as the SNAREs, bind to Sec24p in the COPII coat through ER exit motifs in their cytoplasmic tails. The transport mechanism for the exit of soluble cargo proteins from the ER has been explained by two distinct models. Secretion of certain abundant proteins is consistent with a bulk flow model in which cargo moves by default and requires no export signals (2, 3). In contrast, the receptor-mediated export model envisions selective packaging of secreted proteins into budding vesicles with the help of membrane-anchored cargo receptors that interact with COPII coat proteins and is supported by recent observations of sorting in ER-derived transport vesicles (4–7). In yeast, a heteromeric complex of Emp24p and Erv25p was shown to directly cross-link with Gas1p and to be required for its efficient packaging (8). Similarly, the ER-localized membrane protein Erv29p functions by binding and directing glycosylated pro-{alpha}-factor into COPII-coated transport vesicles (9, 10).

Our recent genetic studies identified mutations in LMAN1 (also referred to as ERGIC-53) and MCFD2 as the causes of an inherited human bleeding disorder, combined deficiency of coagulation factor V and factor VIII (F5F8D) (11, 12). This disorder is associated with plasma levels of factor V (FV) and factor VIII (FVIII) in the range of 5–30% of normal, without obvious reduction in other known proteins. LMAN1 is a type 1 transmembrane protein with homology to leguminous mannose-binding lectins (13–15). It cycles between the ER and the ERGIC through a C-terminal diphenylalanine (FF) ER exit motif that binds to the Sec24 subunit of the COPII coat (16, 17) and a dilysine (KK) ER retention motif (18). MCFD2 is a soluble EF-hand-containing protein that is retained in the ER through a calcium-dependent interaction with LMAN1 (12). The involvement of LMAN1 and MCFD2 in F5F8D suggests that they function as a cargo receptor for the ER-to-Golgi transport of FV and FVIII. Both LMAN1 and MCFD2 are essential for this transport receptor function as mutations in either LMAN1 or MCFD2 result in indistinguishable clinical manifestations. All LMAN1 mutations identified to date are predicted to result in complete loss of protein function. However, two missense mutations were identified within the second MCFD2 EF-hand domain. These mutations disrupt LMAN1-MCFD2 interaction (12), underscoring the importance of this interaction for the cargo receptor function.

LMAN1/MCFD2-dependent secretion of FV and FVIII provides a unique system to study receptor-mediated ER-to-Golgi transport in higher eukaryotes. FV and FVIII are large homologous proteins that share a similar domain structure (A1-A2-B-A3-C1-C2). The B domains of FV and FVIII share no sequence identity but are both heavily glycosylated and encoded by single large exons (19). Deletion of the B domain of FVIII results in a 20-fold increase in the levels of mRNA and the primary translation product. However, B-domain-deleted FVIII (BDD-FVIII) is poorly secreted, due to either reduced folding or inefficient ER-to-Golgi transport (20). FVIII with a short B-domain sequence containing five putative N-glycosylation sites can secrete as efficiently as full-length FVIII (21). Paradoxically, in cells overexpressing an ER exit-deficient LMAN1, secretion of FV and FVIII is impaired, whereas secretion of B-domain-deleted FV and FVIII is unaffected (22).

We now further examine the interaction of LMAN1 and MCFD2 and the role of this complex in the ER-to-Golgi transport of FVIII. Our results demonstrated a calcium-dependent interaction of FVIII, but not a B domain-deleted form of FVIII, with endogenous LMAN1 and MCFD2. The interaction of MCFD2 with FVIII was independent of the binding of MCFD2 to LMAN1 or the glycosylation state of FVIII. In contrast, the interaction of LMAN1 with FVIII, although also independent of FVIII glycosylation, appeared to require LMAN1-MCFD2 complex formation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Anti-heavy chain factor VIII monoclonal antibody conjugated to CL-4B-Sepharose was a gift from D. Pittman (Genetics Institute Inc.). Anti-ERGIC-53 monoclonal antibody (G1/93) and rabbit anti-rat P58 antibodies were gifts from H.-P. Hauri (University of Basel) and R. Petterssen (Ludwig Institute for Cancer Research, Stockholm, Sweden), respectively. Rabbit anti-MCFD2 was prepared as described previously (12). Rabbit anti-TRAP{alpha} was a gift from R. Hegde (NICHD, National Institutes of Health). Rabbit anti-myc was purchased from Santa Cruz Biotechnology. Thapsigargin, tunicamycin, castanospermine, and deoxymannojirimycin were purchased from Sigma.

Transfection and Metabolic Labeling—For transient expression, COS1 cells were transfected using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions and metabolically labeled at 26 h after transfection for 45 min with [35S]methionine/cysteine (250 µCi/ml in methionine/cysteine-free Dulbecco's modified Eagle's medium) (ICN) followed by a 30-min incubation in complete medium (except in pulse-chase experiments in which cells were pulse-labeled for 15 min followed by incubation for various times in complete medium containing 100-fold excess methionine). Chinese hamster ovary cell lines that stably express FVIII (10A1) (23) or BDD-FVIII (LA3–5) (24) were similarly labeled at ~70% confluence. Steady-state labeling was performed in methionine/cysteine-free Dulbecco's modified Eagle's medium with 1% serum and 1% complete medium.

Cross-linking and Immunoprecipitation—To perform cross-linking using the thiol-cleavable cross-linker, DSP (dithio-bis(succinimidyl propionate)), cells were washed three times with ice-cold phosphate-buffered saline, pH 7.4, with 2 mM CaCl2, 250 mM sucrose, and incubated with 1 mM DSP for 20 min on ice with gentle agitation. Cross-linking reactions were quenched in 20 mM Tris-HCl, pH 7.5. Immunoprecipitations were performed in cell lysates in Nonidet P-40 buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.05% SDS, 2 mM CaCl2) plus protease inhibitor mixture (EDTA-free, Roche Applied Science) as described previously (12). Unless specified, immunoprecipitates were washed three times in Nonidet P-40 buffer. Proteins were analyzed by SDS-PAGE under reducing conditions and visualized by fluorography. Quantification of radiographic band intensities was performed using a Typhoon PhosphorImager (Amersham Biosciences) and ImageQuant software.

Glycosylation Studies—COS1 cells were transfected with a myc-tagged MCFD2 and metabolically labeled at 24 h after transfection. Cells were then incubated in serum-free medium containing 1 µg/ml aprotinin for 12 h. Cell lysates were immunoprecipitated with rabbit anti-MCFD2, and the conditioned media were immunoprecipitated with rabbit anti-MCFD2 IgG conjugated on Sepharose beads (12). Digestions of the immunoprecipitates with N-glycosidase F and O-glycanase were performed using an enzymatic deglycosylation kit (Prozyme) according to the manufacturer's recommendation.

Western Blot Analysis—Epstein-Barr virus-immortalized lymphoblasts derived from two patients with MCFD2-null mutants (families 11 and 12) (12) as well as two normal controls were lysed in Nonidet P-40 buffer. Equal amounts of cell lysate (10 µg) were separated by SDS-PAGE under reducing and non-reducing conditions, transferred to nitrocellulose membranes, and probed with a monoclonal antibody against LMAN1 (G1/93).

N-terminal Protein Sequencing—Lysates of COS1 cells transfected with myc-tagged MCFD2 were immunoprecipitated with rabbit anti-MCFD2 IgG conjugated on Sepharose beads. The immunoprecipitates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane in 10 mM CAPS, 10% methanol, pH 11. Protein bands were visualized by staining with 0.1% Amido Black in 10% acetic acid, 2% ethanol and then excised. Peptide sequencing was performed by the Protein Structure Facility at the University of Michigan.



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 1.
Pulse-chase and steady-state labeling. a, COS1 cells were pulse-labeled for 15 min and chased for the indicated times. Cell lysates were immunoprecipitated with antibodies against LMAN1 or MCFD2. The intensities of LMAN1 and MCFD2 bands were quantitated using a PhosphorImager and plotted as a bar graph. b, COS1 cells were labeled for 30 h to approximate steady-state. Equal aliquots of cell lysates were immunoprecipitated with the indicated antibodies. L, anti-LMAN1; M, anti-MCFD2; myc, anti-myc.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
MCFD2 and LMAN1 Have Similar Half-lives and Form a Stable 1:1 Stoichiometric Complex—To measure the half-life of LMAN1 and MCFD2, COS1 cells were pulse-labeled with [35S]methionine/cysteine and chased with excess unlabeled methionine. Equal amounts of cell lysates were immunoprecipitated with either anti-LMAN1 or anti-MCFD2 antibodies. Intensities of LMAN1 and MCFD2 bands were quantified using a PhosphorImager. Both proteins have long but similar half-lives (~26 h) (Fig. 1a), consistent with previous studies that suggested a long half-life for LMAN1 (25).

To approximate conditions for steady state, we performed a 30-h labeling of COS1 cells with [35S]methionine/cysteine. Cell lysates were sequentially immunodepleted with a rabbit anti-MCFD2 antibody followed by immunoprecipitation with a monoclonal anti-LMAN1 antibody or vice versa (Fig. 1b). Co-immunoprecipitation (co-IP) of LMAN1 and MCFD2 is evident using either antibody. Nearly all LMAN1 and MCFD2 appear to be located in these stable complexes, as demonstrated by sequential immunodepletions. Similar results were observed in HeLa and 293T cells (data not shown). The ratio of labeling intensities for the LMAN1 and MCFD2 bands as determined by PhosphorImager analysis was ~2.6:1. Mature LMAN1 contains 9 methionines and 4 cysteines, whereas mature MCFD2 contains 5 methionines. Taken together with the apparently similar half-lives for both proteins by pulse-chase analysis (Fig. 1a), these results suggest that LMAN1 and MCFD2 are associated in a 1:1 stoichiometry.

MCFD2 Is Not Required for Oligomerization of LMAN1— LMAN1 is known to exist in the cell as both disulfide-linked homodimers and disulfide-linked homohexamers (15). We prepared cell lysates from lymphoblasts derived from normal controls and patients with MCFD2-null mutations. Western blotting for LMAN1 in cell lysates electrophoresed under denaturing, but non-reducing conditions revealed no significant difference in dimer and hexamer formation between normal and MCFD2-deficient lymphoblasts (Fig. 2). These results demonstrate that MCFD2 binds to LMAN1 non-covalently and that it is not required for normal LMAN1 oligomerization.



View larger version (52K):
[in this window]
[in a new window]
  		FIG. 2.
MCFD2 does not affect oligomerization of LMAN1. Cell extracts from two wild-type lymphoblast lines (wt) and two MCFDF2 mutant lines (mt) were incubated with SDS sample buffer at 100 °C with or without 5% {beta}-mercaptoethanol (BME) and then run on a 4–12% gradient gel. LMAN1 was detected by Western blot analysis.

 
FVIII Interacts with the LMAN1-MCFD2 Complex—Although FVIII was previously reported to co-immunoprecipitate with overexpressed LMAN1 (26), we were unable to demonstrate a consistent interaction of FVIII with endogenous LMAN1 or MCFD2 by direct immunoprecipitation. We therefore performed chemical cross-linking in live cells. COS1 cells were transfected with wild-type FVIII and labeled with [35S]methionine/cysteine at 26 h after transfection. Cells were then incubated with the membrane-permeable, thio-cleavable cross-linker, DSP. Cell lysates were immunoprecipitated with antibodies against MCFD2, LMAN1, and FVIII, as well as negative control antibodies (anti-myc and anti-TRAP{alpha} (27), a component of the ER translocon that forms a tight complex with TRAP{beta}). Anti-FVIII antibody detected the 280-kDa FVIII primary translation product in cell extracts. Immunoprecipitation of the same amount of cell extracts with either anti-LMAN1 or anti-MCFD2 antibodies precipitated both LMAN1 and MCFD2. A low level of FVIII (<1%) was seen in immunoprecipitations in the absence of DSP treatment. However, the intensity of the FVIII band increased markedly in immunoprecipitations with both the anti-MCFD2 and the anti-LMAN1 antibodies following DSP treatment, with no increase evident with control antibodies (Fig. 3A). We observed 5–20% of the total FVIII to be cross-linked to LMAN1 or MCFD2 under these conditions. These results indicate a specific interaction between FVIII and the LMAN1-MCFD2 complex.

B-domain-deleted FVIII Interacts Poorly with the LMAN1-MCFD2 Complex—COS1 cells were transiently transfected with FVIII or BDD-FVIII, metabolically labeled, and then incubated with or without DSP. Cell lysates were immunoprecipitated with antibodies against LMAN1 and MCFD2, as well as control antibodies. Again, increased co-IP of FVIII with LMAN1 and MCFD2 was observed following DSP treatment (Fig. 4A). In contrast, very little BDD-FVIII co-immunoprecipitated with LMAN1 or MCFD2 following DSP treatment. We next examined Chinese hamster ovary cells stably expressing FVIII or BDD-FVIII using a polyclonal antibody against the rat homolog of LMAN1. Again, treatment with DSP significantly enhanced the co-IP of LMAN1 with FVIII but not BDD-FVIII (Fig. 4B). These results suggest that the BDD-FVIII does not bind or has markedly reduced binding affinity to the LMAN1-MCFD2 complex.

MCFD2 Can Bind FVIII Independent of LMAN1—COS1 cells were co-transfected with FVIII and a myc-tagged MCFD2 carrying the D129E mutation (MCFD2D129E), which abolishes binding to LMAN1 (12). Cells were treated with DSP and analyzed by immunoprecipitation. As expected, MCFD2D129E did not co-immunoprecipitate with LMAN1 (Fig. 5, lanes 9, 11, 13, and 15). Overexpression of MCFD2 increased the amount of FVIII that co-immunoprecipitated with MCFD2 in the absence of DSP (Fig. 5, lanes 2 and 10) but did not increase co-IP of FVIII with LMAN1 (Fig. 5, lanes 3 and 11). However, DSP treatment significantly increased the amount of FVIII that was pulled down by both anti-LMAN1 and anti-MCFD2 antibodies (Fig. 5, lanes 6, 7, 14, and 15). Anti-myc antibody, which reacts with the myc-tagged MCFD2, pulled down FVIII in cells transfected with wild-type MCFD2, as well as cells transfected with MCFD2D129E (Fig. 5, lanes 5 and 13). These results suggest that MCFD2 can bind to FVIII independent of LMAN1.



View larger version (59K):
[in this window]
[in a new window]
  		FIG. 3.
Cross-linking of FVIII to LMAN1 and MCFD2 is specific. COS1 cells were transfected with a FVIII construct in duplicate and then labeled with [35S]methionine/cysteine at 26 h after transfection. Cells were then incubated with or without 1 mM DSP on ice for 30 min, lysed, and immunoprecipitated with the indicated antibodies. The top panel was from an 8% gel, and the bottom panel was from a 15% gel.

 



View larger version (68K):
[in this window]
[in a new window]
  		FIG. 4.
LMAN1 and MCFD2 do not cross-link efficiently with B-domain-deleted FVIII. a, COS1 cells were transfected with FVIII or BDD-FVIII constructs, metabolically labeled, treated with or without DSP, and immunoprecipitated with the indicated antibodies (Ab). Immunoprecipitates were washed three times in Nonidet P-40 buffer with 0.45 M NaCl and 0.1% SDS. The top panel was from an 8% gel, and the bottom panel was from a 15% gel. wt, wild type; M, anti-MCFD2; L, anti-LMAN1; F8, anti-FVIII; S, nonspecific rabbit serum; T, anti-TRAP{alpha}; myc, anti-myc. b, Chinese hamster ovary cell lines that stably express wild-type FVIII or BDD-FVIII were metabolically labeled and then treated with or without DSP and immunoprecipitated with the indicated antibodies. Non-adjacent lanes from the same gel were rearranged for reproduction purpose.

 



View larger version (81K):
[in this window]
[in a new window]
  		FIG. 5.
Cross-linking of FVIII with MCFD2 is independent of the LMAN1-MCFD2 interaction. COS1 cells were co-transfected with FVIII and wild-type (wt) myc-tagged MCFD2 or with FVIII and myc-tagged MCFD2D129E (D129E) and labeled with [35S]methionine/cysteine at 26 h after transfection. Cells were then incubated with or without 1 mM DSP on ice for 30 min, lysed, and immunoprecipitated with the indicated antibodies. The top panel was from an 8% gel, and the bottom panel was from a 15% gel. Lane 17 of the bottom panel contained conditioned medium from wild-type-MCFD2-transfected cells immunoprecipitated with anti-MCFD2 antibody. myc, anti-myc; M, anti-MCFD2; L, anti-LMAN1; F8, anti-FVIII.

 
Overexpressed MCFD2 Is Secreted—In the conditioned medium of COS1 cells transfected with MCFD2, immunoprecipitation with anti-MCFD2 detected a protein with the apparent molecular mass of 28 kDa (Figs. 5, lane 17, and 6). Immunoprecipitation of cell lysates with anti-MCFD2 antibody also detected a small amount of the same 28-kDa band in addition to the endogenous MCFD2 (16 kDa) and the 18-kDa species of myc-tagged MCFD2 (Figs. 5 and 6). The 28-kDa species failed to co-immunoprecipitate with LMAN1 (Fig. 5, lanes 3 and 7). We treated the immunoprecipitates with either N-glycosidase F or O-glycanase (Fig. 6). No mobility shift was observed with N-glycosidase F treatment, whereas upon O-glycanase treatment, the 28-kDa MCFD2 band is lost from the cell lysate, and MCFD2 in the conditioned medium is converted into 10 intermediates between the 28-kDa species and the 18-kDa species. Since mature MCFD2 contains 10 serine and threonine residues, these results could be consistent with O-glycosylation of all available sites in secreted MCFD2. Alternatively, the intermediate bands may represent incomplete digestion products. The 28-kDa species detected in cell lysates is most likely the O-glycosylated form of MCFD2 in the Golgi that is in the process of being secreted. Both the endogenous MCFD2 and the myc-tagged MCFD2 appear larger on the SDS gel than the predicted molecular masses of the mature proteins (14 and 16 kDa). We performed N-terminal amino acid sequencing of immunoprecipitated MCFD2 and myc-tagged MCFD2. The results indicate that the signal peptide of MCFD2 was cleaved at the predicted site, between amino acid residues 26 and 27. The slower migration on the SDS gel is likely due to the amino acid composition of MCFD2.

Cross-linking of FVIII to the LMAN1-MCFD2 Complex Is Inhibited by Calcium Depletion but Not Affected by the Glycosylation State of FVIII—Calcium concentration in the ER lumen is known to be significantly higher than that in the cytosol (28). LMAN1 and MCDF2 interaction is abolished in the presence of a calcium chelator (12). To determine the contributions of Ca2+ in the interaction of FVIII with LMAN1 and MCFD2, FVIII-transfected COS1 cells were treated with thapsigargin, a potent Ca2+-ATPase inhibitor that depletes Ca2+ in the ER lumen (29). To minimize potential complications associated with the ER stress response, cells were treated for 3 h or less before major pathways of the unfolded protein response are fully activated. After cross-linking with DSP, cells were lysed in the presence of EGTA to prevent reassociation of LMAN1 with MCFD2. Thapsigargin treatment disrupted the LMAN1-MCFD2 interaction in the cell, as evidenced by the reduced level of co-IP (Fig. 7, lanes 13–14), as well as reduced amounts of intracellular MCFD2, along with the appearance of the higher molecular weight, O-glycosylated secretion intermediate (Fig. 7, lane 13). The co-IP of FVIII with MCFD2 was nearly abolished (Fig. 7, lane 13), likely due to the low level of MCFD2 in the ER caused by Ca2+ depletion. The co-IP of FVIII with LMAN1 was also markedly reduced (Fig. 7, lanes 14), despite the normal level of LMAN1, suggesting that Ca2+ and/or MCFD2 binding is required for LMAN1 to interact with FVIII. Dissociation of FVIII from LMAN1 observed upon thapsigargin treatment is not likely a nonspecific result of ER stress. Lysis of DSP-treated cells in EGTA-containing buffer alone had no effect on the amount of FVIII cross-linked to LMAN1 or MCFD2 (data not shown).



View larger version (71K):
[in this window]
[in a new window]
  		FIG. 6.
Secreted MCFD2 is O-glycosylated. COS1 cells were transfected with wild-type MCFD2. At 24 h after transfection, cells were labeled with [35S]methionine/cysteine, changed to serum-free medium, and incubated for an additional 12 h. Conditioned medium and cell lysates were separately immunoprecipitated with anti-MCFD2 antibodies. The immunoprecipitates were divided into three aliquots, which were mock-treated (no enzyme) or treated with endoglycosidase F (Endo-F) or O-glycanase. Protein bands were resolved on a 15% gel and visualized by fluorography.

 



View larger version (66K):
[in this window]
[in a new window]
  		FIG. 7.
Cross-linking of FVIII with LMAN1 and MCFD2 is dependent on Ca2+, but independent of the FVIII glycosylation state. COS1 cells were transfected with FVIII. At 26 h after transfection, cells were incubated with thapsigargin (400 nM) or Tm (10 µg/ml) for 3 h before subjecting to 35S labeling and DSP treatment (30). Thapsigargin and tunicamycin were maintained throughout the metabolic labeling process. Cell lysates were immunoprecipitated with the indicated antibodies (Ab). Immunoprecipitates were washed three times in Nonidet P-40 buffer with 0.45 M NaCl and 0.1% SDS. S, nonspecific serum; T, anti-TRAP{alpha}; M, anti-MCFD2; L, anti-LMAN1; F, anti-FVIII.

 
To study whether carbohydrate structures on FVIII are important for the interaction of FVIII with LMAN1-MCFD2, we treated cells for 3 h with tunicamycin (Tm), which blocks the first step in core oligosaccharide assembly and thus inhibits the synthesis of all N-linked structure. As expected, unglycosylated FVIII, as well as TRAP{alpha} and TRAP{beta} from the Tm-treated cells, migrated faster than normal (Fig. 7, lanes 16–19). Tm treatment had no significant effect on the amount of FVIII that cross-linked to LMAN1 or MCFD2 (Fig. 7, lanes 17–18). Co-IP of FVIII was not detected with the control antibody anti-TRAP{alpha} or in cells treated with Tm alone (data not shown), indicating that Tm did not result in random aggregation of FVIII. Treatment with castanospermine or deoxymannojirimycin, which specifically inhibit trimming of N-linked oligosaccharide structures, also had no significant effect on the amount of FVIII that cross-linked with LMAN1 or MCFD2 (data not shown). These results suggest that the interaction of FVIII with LMAN1 and MCFD2 is not dependent on the glycosylation state of FVIII.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The identification of LMAN1 mutations in F5F8D provided the first direct evidence for a cargo-specific sorting receptor in the ER of higher eukaryotes (11). The identification of the soluble protein MCFD2 as another essential component of this pathway (12) distinguishes this mammalian cargo receptor from those identified in yeast. Association of a soluble protein with a transmembrane protein may represent a general feature of transport receptors in higher eukaryotes. The data presented here demonstrate a specific interaction between both MCFD2 and LMAN1 and the FVIII cargo. Our cross-linking data suggested that interactions between the LMAN1-MCFD2 complex and its cargo proteins may be of low affinity or transient in nature. Detection of interactions between LMAN1 with procathepsin Z (30, 31) and between Emp24p and Erv29p and their cargo is also dependent upon chemical cross-linking (8, 9).

Receptor-mediated ER-to-Golgi protein trafficking has been elegantly dissected in yeast. Both Emp24p and Erv29p have homologs in higher eukaryotes. Emp24p is a member of the p24 family of proteins that have homologs in plants and mammals. p24 proteins fall into four subfamilies ({alpha}, {beta}, {gamma}, {delta}) that exhibit different tissue distribution patterns and can form heteromeric complexes. Deletion of all p24 proteins in yeast produces only a mild secretory defect in a limited number of proteins (7). However, specific p24 genes may have diverse functions in other organisms (32–34). Interestingly, clear LMAN1 orthologs exist in invertebrates prior to the appearance of blood coagulation system components (35), including FV and FVIII, suggesting that this transport pathway may be required for a much broader array of protein cargo. Mutations in the Drosophila LMAN1 homolog, rhea, cause a late embryonic recessive lethal phenotype, resulting from somatic wing and muscle developmental defects (36, 37). Disrupting the function of Rhea may cause a secretion defect or a mislocalization of a crucial cargo.

We observed a quantitative association of LMAN1 and MCFD2 in COS1 cells (Fig. 1b), raising the possibility that synthesis of LMAN1 and MCFD2 may be coordinated. However, it remains possible that the ratio of LMAN1/MCFD2 may vary among different cell or tissue types. Interestingly, a recent report presented in vitro evidence that the rat ortholog of MCFD2 may function extracellularly as a stem cell-derived neuronal survival factor (38). This would require MCFD2 to be expressed in excess to LMAN1 and thus secreted in neuronal stem cells. We have shown that overexpressed MCFD2 is secreted in an extensively O-glycosylated form (Fig. 6). Although F5F8D patients with MCFD2 deficiency do not exhibit obvious neurological symptoms, a partially redundant role of MCFD2 in neuronal development cannot be excluded.

The receptor-mediated transport model requires sorting signals to be present on the cargo that are recognized by their receptors. A recent study suggested that three hydrophobic residues on pro-{alpha}-factor may mediate its interaction with Erv29p (10). Our results demonstrated that the interaction of FVIII with both LMAN1 and MCFD2 requires the B domain but appears to be independent of its glycosylation (Fig. 7), suggesting that the lectin activity of LMAN1 is not the primary sorting signal for cargo receptor binding. However, we cannot rule out the possibility of a non-native interaction as a result of incomplete folding of the unglycosylated FVIII. These data were consistent with the observation that in MCFD2-deficient patients, normal level of LMAN1 alone cannot support the efficient secretion of FV and FVIII. A crystal structure of the carbohydrate recognition domain of p58, the rat homolog of LMAN1 (39), suggested potential protein-binding sites in addition to the carbohydrate-binding site. However, our data did not exclude the contribution of carbohydrate binding in further stabilizing the receptor-cargo interaction. Targeting of procathepsin Z appears to require both an oligosaccharide chain and a surface-exposed peptide {beta}-hairpin loop (31). The FVIII B domain contains 970 amino acid residues. Further studies are required to pinpoint elements within the B domain that are important for cargo receptor binding. Secretion of BDD-FVIII is only moderately increased despite a 40-fold increase in mRNA level as compared with the full-length FVIII (20). Previous experiments using a cell line overexpressing an ER exit-deficient LMAN1 observed a secretion defect only for intact FV and FVIII, not the corresponding B-domain-deleted molecules, consistent with an interaction between the B domains and LMAN1 (22). However, a subsequent study suggested that both FVIII and BDD-FVIII can co-immunoprecipitate with LMAN1 (26) and reported a decrease in FVIII co-IP with a mutant LMAN1 that is deficient in carbohydrate recognition. In the latter experiments, ER exit-deficient LMAN1 was overexpressed, which could explain the discrepancy with our experiments. Although we readily observed co-IP of FVIII with antibodies against LMAN1 and MCFD2, only faint or no LMAN1 and MCFD2 bands were detected with anti-FVIII (faint LMAN1 bands in Fig. 3, last lane; Fig. 5, lanes 8 and 16; Fig. 7, lane 10). The poor detection of LMAN1/MCFD2 with anti-FVIII antibody probably resulted from the much longer half-lives of MCFD2 and LMAN1 (~26 h, Fig. 1) as compared with FVIII (~2 h). With the relatively short labeling time of 45 min, the specific radioactivity of FVIII should be much greater than that of LMAN1 or MCFD2, likely accounting for the difference in detection sensitivity. A similar phenomenon has been previously observed for co-IP of FVIII with two long-lived ER chaperones, calreticulin and calnexin (40).

LMAN1 has been shown to bind Ca2+ (41). MCFD2 contains EF-hand domain that presumably also bind Ca2+. We showed that the luminal Ca2+ concentration is critical for the interaction of LMAN1 and MCFD2, as well as the cargo binding activity of this complex. In addition, binding of MCFD2 to FVIII appeared to be independent of its interaction with LMAN1 (Fig. 5). Binding of FVIII to LMAN1 appeared to require Ca2+ or Ca2+-dependent LMAN1-MCFD2 complex formation (Fig. 7). Taken together, our data supported a model in which MCFD2 functions at the point of cargo loading during the ER-to-Golgi transport of FV and FVIII. Ca2+-MCFD2 could function as the initial contact that specifically captures correctly folded FV and FVIII in the ER lumen. The FV/FVIII-MCFD2-LMAN1 ternary complex could then be further stabilized by interactions between LMAN1 and the oligosaccharides and/or amino acid side chains of FVIII. This cargo-containing complex is then packaged into COPII-coated vesicles for budding. An intriguing implication of this model is that the primary cause of F5F8D is defective MCFD2, and failure to retain MCFD2 in the ER is the mechanism by which LMAN1 mutations cause F5F8D. The continued secretion of FV and FVIII at reduced levels in F5F8D patients could be due to residual MCFD2 or LMAN1 transport function in the absence of the other component, with a complete block of FV and FVIII secretion requiring deficiency of both proteins. Alternatively, a second transport pathway such as bulk flow or an alternative cargo receptor could be responsible for the background level of FV/FVIII secretion observed in F5F8D.


   FOOTNOTES
 
* This work was supported in part by by Project Grant PO1 HL057346 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ A recipient of a Career Development Award from the National Hemophilia Foundation. Back

** Investigators of the Howard Hughes Medical Institute. Back

¶¶ To whom correspondence should be addressed: Life Sciences Institute, 210 Washtenaw Ave., Ann Arbor, MI 48109-0650. Tel.: 734-647-4808; Fax: 734-936-2888; E-mail: ginsburg{at}umich.edu.

1 The abbreviations used are: ER, endoplasmic reticulum; ERGIC, ER-Golgi intermediate compartment; FV, factor V; FVIII, factor VIII; co-IP, co-immunoprecipitation; DSP, dithio-bis(succinimidyl propionate); Tm, tunicamycin; BDD, B-domain-deleted; CAPS, 3-(cyclohexylamino)propanesulfonic acid; SNARE, soluble NSF attachment protein receptors; NSF, N-ethylmaleimide-sensitive factor; COPI, coat protein complex I; COPII, coat protein complex II; TRAP, translocon-associated protein complex. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Randy Schekman (University of California, Berkeley) and Hans-Peter Hauri (University of Basel) for critical reading of the manuscript. We thank T. Rutkowski for help in obtaining negative control antibodies.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Schekman, R., and Orci, L. (1996) Science 271,1526 -1533[Abstract]
  2. Martinez-Menarguez, J. A., Geuze, H. J., Slot, J. W., and Klumperman, J. (1999) Cell 98, 81-90[CrossRef][Medline] [Order article via Infotrieve]
  3. Wieland, F. T., Gleason, M. L., Serafini, T. A., and Rothman, J. E. (1987) Cell 50, 289-300[CrossRef][Medline] [Order article via Infotrieve]
  4. Kuehn, M. J., Herrmann, J. M., and Schekman, R. (1998) Nature 391,187 -190[CrossRef][Medline] [Order article via Infotrieve]
  5. Muniz, M., Morsomme, P., and Riezman, H. (2001) Cell 104,313 -320[CrossRef][Medline] [Order article via Infotrieve]
  6. Nehls, S., Snapp, E. L., Cole, N. B., Zaal, K. J., Kenworthy, A. K., Roberts, T. H., Ellenberg, J., Presley, J. F., Siggia, E., and Lippincott-Schwartz, J. (2000) Nat. Cell Biol. 2,288 -295[CrossRef][Medline] [Order article via Infotrieve]
  7. Springer, S., Chen, E., Duden, R., Marzioch, M., Rowley, A., Hamamoto, S., Merchant, S., and Schekman, R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97,4034 -4039[Abstract/Free Full Text]
  8. Muniz, M., Nuoffer, C., Hauri, H.-P., and Riezman, H. (2000) J. Cell Biol. 148,925 -930[Abstract/Free Full Text]
  9. Belden, W. J., and Barlowe, C. (2001) Science 294,1528 -1531[Abstract/Free Full Text]
  10. Otte, S., and Barlowe, C. (2004) Nat. Cell Biol. 6,1189 -1194[CrossRef][Medline] [Order article via Infotrieve]
  11. Nichols, W. C., Seligsohn, U., Zivelin, A., Terry, V. H., Hertel, C. E., Wheatley, M. A., Moussalli, M. J., Hauri, H.-P., Ciavarella, N., Kaufman, R. J., and Ginsburg, D. (1998) Cell 93,61 -70[CrossRef][Medline] [Order article via Infotrieve]
  12. Zhang, B., Cunningham, M. A., Nichols, W. C., Bernat, J. A., Seligsohn, U., Pipe, S. W., Mcvey, J. H., Schulte-Overberg, U., de Bosch, N. B., Ruiz-Saez, A., White, G. C., Tuddenham, E. G. D., Kaufman, R. J., and Ginsburg, D. (2003) Nat. Genet. 34, 220-225[CrossRef][Medline] [Order article via Infotrieve]
  13. Fiedler, K., and Simons, K. (1994) Cell 77,625 -626[CrossRef][Medline] [Order article via Infotrieve]
  14. Arar, C., Carpentier, V., Le Caer, J. P., Monsigny, M., Legrand, A., and Roche, A. C. (1995) J. Biol. Chem. 270,3551 -3553[Abstract/Free Full Text]
  15. Itin, C., Roche, A. C., Monsigny, M., and Hauri, H.-P. (1996) Mol. Biol. Cell 7, 483-493[Abstract]
  16. Kappeler, F., Klopfenstein, D. R., Foguet, M., Paccaud, J. P., and Hauri, H.-P. (1997) J. Biol. Chem. 272,31801 -31808[Abstract/Free Full Text]
  17. Nufer, O., Guldbrandsen, S., Degen, M., Kappeler, F., Paccaud, J. P., Tani, K., and Hauri, H.-P. (2002) J. Cell Sci. 115,619 -628[Abstract/Free Full Text]
  18. Itin, C., Kappeler, F., Linstedt, A. D., and Hauri, H.-P. (1995) EMBO J. 14,2250 -2256[Medline] [Order article via Infotrieve]
  19. Kane, W. H., and Davie, E. W. (1988) Blood 71,539 -555[Free Full Text]
  20. Pipe, S. W. (2004) Semin. Thromb. Hemostasis 30,227 -237[CrossRef][Medline] [Order article via Infotrieve]
  21. Miao, H. Z., Sirachainan, N., Palmer, L., Kucab, P., Cunningham, M. A., Kaufman, R. J., and Pipe, S. W. (2004) Blood 103,3412 -3419[Abstract/Free Full Text]
  22. Moussalli, M., Pipe, S. W., Hauri, H.-P., Nichols, W. C., Ginsburg, D., and Kaufman, R. J. (1999) J. Biol. Chem. 274,32539 -32542[Abstract/Free Full Text]
  23. Kaufman, R. J., Wasley, L. C., Davies, M. V., Wise, R. J., Israel, D. I., and Dorner, A. J. (1989) Mol. Cell. Biol. 9,1233 -1242[Abstract/Free Full Text]
  24. Pittman, D. D., Alderman, E. M., Tomkinson, K. N., Wang, J. H., Giles, A. R., and Kaufman, R. J. (1993) Blood 81,2925 -2935[Abstract/Free Full Text]
  25. Schweizer, A., Fransen, J. A. M., Bachi, T., Ginsel, L., and Hauri, H.-P. (1988) J. Cell Biol. 107,1643 -1653[Abstract/Free Full Text]
  26. Cunningham, M. A., Pipe, S. W., Zhang, B., Hauri, H.-P., Ginsburg, D., and Kaufman, R. J. (2003) J. Thromb. Haemost. 1,2360 -2367[CrossRef][Medline] [Order article via Infotrieve]
  27. Fons, R. D., Bogert, B. A., and Hegde, R. S. (2003) J. Cell Biol. 160,529 -539[Abstract/Free Full Text]
  28. Pezzati, R., Bossi, M., Podini, P., Meldolesi, J., and Grohovaz, F. (1997) Mol. Biol. Cell 8,1501 -1512[Abstract]
  29. Sagara, Y., and Inesi, G. (1991) J. Biol. Chem. 266,13503 -13506[Abstract/Free Full Text]
  30. Appenzeller, C., Andersson, H., Kappeler, F., and Hauri, H.-P. (1999) Nat. Cell Biol. 1, 330-334[CrossRef][Medline] [Order article via Infotrieve]
  31. Appenzeller-Herzog, C., Nyfeler, B., Burkhard, P., Santamaria, I., Lopez-Otin, C., and Hauri, H.-P. (2005) Mol. Biol. Cell
  32. Denzel, A., Otto, F., Girod, A., Pepperkok, R., Watson, R., Rosewell, I., Bergeron, J. J. M., Solari, R. C. E., and Owen, M. J. (2000) Curr. Biol. 10, 55-58[CrossRef][Medline] [Order article via Infotrieve]
  33. Carney, G. E., and Taylor, B. J. (2003) Genetics 164,173 -186[Abstract/Free Full Text]
  34. Bouw, G., Van Huizen, R., Jansen, E. J. R., and Martens, G. J. M. (2004) Mol. Biol. Cell 15,1244 -1253[Abstract/Free Full Text]
  35. Davidson, C. J., Tuddenham, E. G., and Mcvey, J. H. (2003) J. Thromb. Haemost. 1,1487 -1494[CrossRef][Medline] [Order article via Infotrieve]
  36. Martin-Bermudo, M. D., and Brown, N. H. (2000) J. Cell Sci. 113,3715 -3723[Abstract]
  37. Prout, M., Damania, Z., Soong, J., Fristrom, D., and Fristrom, J. W. (1997) Genetics 146,275 -285[Abstract]
  38. Toda, H., Tsuji, M., Nakano, I., Kobuke, K., Hayashi, T., Kasahara, H., Takahashi, J., Mizoguchi, A., Houtani, T., Sugimoto, T., Hashimoto, N., Palmer, T. D., Honjo, T., and Tashiro, K. (2003) J. Biol. Chem. 278,35491 -35500[Abstract/Free Full Text]
  39. Velloso, L. M., Svensson, K., Schneider, G., Pettersson, R. F., and Lindqvist, Y. (2002) J. Biol. Chem.15979 -15984
  40. Pipe, S. W., Morris, J. A., Shah, J., and Kaufman, R. J. (1998) J. Biol. Chem. 273,8537 -8544[Abstract/Free Full Text]
  41. Velloso, L. M., Svensson, K., Pettersson, R. F., and Lindqvist, Y. (2003) J. Mol. Biol. 334,845 -851[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
J. D. Malhotra, H. Miao, K. Zhang, A. Wolfson, S. Pennathur, S. W. Pipe, and R. J. Kaufman
Antioxidants reduce endoplasmic reticulum stress and improve protein secretion
PNAS, November 25, 2008; 105(47): 18525 - 18530.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
B. Zhang, M. Spreafico, C. Zheng, A. Yang, P. Platzer, M. U. Callaghan, Z. Avci, N. Ozbek, J. Mahlangu, T. Haw, et al.
Genotype-phenotype correlation in combined deficiency of factor V and factor VIII
Blood, June 15, 2008; 111(12): 5592 - 5600.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
B. Nyfeler, V. Reiterer, M. W. Wendeler, E. Stefan, B. Zhang, S. W. Michnick, and H.-P. Hauri
Identification of ERGIC-53 as an intracellular transport receptor of {alpha}1-antitrypsin
J. Cell Biol., February 25, 2008; 180(4): 705 - 712.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
B. Nyfeler, Y. Kamiya, F. Boehlen, K. Yamamoto, K. Kato, P. de Moerloose, H.-P. Hauri, and M. Neerman-Arbez
Deletion of 3 residues from the C-terminus of MCFD2 affects binding to ERGIC-53 and causes combined factor V and factor VIII deficiency
Blood, February 1, 2008; 111(3): 1299 - 1301.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Kamiya, D. Kamiya, K. Yamamoto, B. Nyfeler, H.-P. Hauri, and K. Kato
Molecular Basis of Sugar Recognition by the Human L-type Lectins ERGIC-53, VIPL, and VIP36
J. Biol. Chem., January 25, 2008; 283(4): 1857 - 1861.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
D. Yamaguchi, N. Kawasaki, I. Matsuo, K. Totani, H. Tozawa, N. Matsumoto, Y. Ito, and K. Yamamoto
VIPL has sugar-binding activity specific for high-mannose-type N-glycans, and glucosylation of the {alpha}1,2 mannotriosyl branch blocks its binding
Glycobiology, October 1, 2007; 17(10): 1061 - 1069.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Renna, M. G. Caporaso, S. Bonatti, R. J. Kaufman, and P. Remondelli
Regulation of ERGIC-53 Gene Transcription in Response to Endoplasmic Reticulum Stress
J. Biol. Chem., August 3, 2007; 282(31): 22499 - 22512.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
K. R. Viel, D. K. Machiah, D. M. Warren, M. Khachidze, A. Buil, K. Fernstrom, J. C. Souto, J. M. Peralta, T. Smith, J. Blangero, et al.
A sequence variation scan of the coagulation factor VIII (FVIII) structural gene and associations with plasma FVIII activity levels
Blood, May 1, 2007; 109(9): 3713 - 3724.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
V. M. Olkkonen and E. Ikonen
When intracellular logistics fails - genetic defects in membrane trafficking
J. Cell Sci., December 15, 2006; 119(24): 5031 - 5045.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
B. Zhang, B. McGee, J. S. Yamaoka, H. Guglielmone, K. A. Downes, S. Minoldo, G. Jarchum, F. Peyvandi, N. B. de Bosch, A. Ruiz-Saez, et al.
Combined deficiency of factor V and factor VIII is due to mutations in either LMAN1 or MCFD2
Blood, March 1, 2006; 107(5): 1903 - 1907.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/27/25881    most recent
M502160200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, B.
Right arrow Articles by Ginsburg, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, B.
Right arrow Articles by Ginsburg, D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement